Food Control 36 (2014) 1e7

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Antimicrobial activity of alginate/clay nanocomposite films enriched

with essential oils against three common foodborne pathogens
Mehdi Alboofetileh a, Masoud Rezaei a, *, Hedayat Hosseini b, Mehdi Abdollahi a
a
Department of Seafood processing, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran
b
Department of Food Science and Technology, Vice-Deputy for Research National Nutrition and Food Technology Research Institute,
Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The overall objective of this study was to develop antimicrobial nanocomposite films to control
Received 7 April 2013 the growth of foodborne pathogens. In the first step, the antibacterial effects of clove, coriander,
Received in revised form caraway, marjoram, cinnamon, and cumin essential oils were studied against three important food
15 July 2013
pathogens, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes by application of
Accepted 23 July 2013
agar diffusion assay. The intensity of antimicrobial efficacy was in the following order:
marjoram > clove > cinnamon > coriander > caraway > cumin. In the next study, the three most potent
Keywords:
essential oils were subsequently incorporated into alginate/clay nanocomposite films. The antibacterial
Antimicrobial films
Alginate
effectiveness of the prepared films against E. coli, S. aureus, and L. monocytogenes was studied during 12
Escherichia coli days. The antibacterial activity of the essential oils was maintained when incorporated into the nano-
Staphylococcus aureus composite film. The nature and amount of the essential oils play an important role in the film’s anti-
Listeria monocytogenes microbial activity. In all film matrices, marjoram showed the highest antimicrobial activity. Films with
1.5% marjoram decreased the numbers of L. monocytogenes, E. coli, and S. aureus populations with respect
to the control up to 6.33, 4.52, and 5.80 log, respectively.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Antimicrobial biodegradable films and coatings have received

increasing attention over the past two decades because of their
Food quality and safety are major concerns in the food industry. potential to delay microbial spoilage of food and to reduce the risk
Food manufacturers strive to reduce or eliminate microorganisms of surface contamination of food by pathogenic microorganisms
from food products, because microorganisms surviving in food can (Ouattara, Simard, Piette, Bégin, & Holley, 2000). Among the many
lead to spoilage and deterioration of the quality of food products, or materials that can form edible films, alginate is appealing film-
cause infection and illness. In this regard, has been estimated that forming compound, as it is non-toxic, biodegradable, and biocom-
about one-third of the world’s food production is lost annually on patible (Zactiti & Kieckbusch, 2009). Alginate is a linear water-
account of microbial spoilage. So far, several technologies, including soluble polysaccharide consisting of monomeric units of 1e4
electron beam, thermal processing, acidified sodium chlorite, lactic linked a-D-mannuronate (M) and ß-L-guluronate (G) at different
acid bacteria, antimicrobial ice, freezing, irradiation, and high proportions in the chain, which is isolated from brown algae and
pressure have been investigated to eliminate pathogenic microor- can be synthesized from microorganisms (Olivas & Barbosa-
ganisms on food (Castellano, Belfiore, Fadda, & Vignolo, 2008; Cánovas, 2008; Vauchel, Kaas, Arhaliass, Baron, & Legrand, 2008).
Lakshmanan, Piggott, & Paterson, 2003; Nykänen, Weckman, & In order to control undesirable microorganisms on food sur-
Lapveteläinen, 2000; Ritz et al., 2000). However, some of these faces, natural or synthetic antimicrobial agents can be incorporated
techniques cannot be applied to some food products and some have into edible films and coatings. The use of chemical preservatives is
negative effects on food quality. Therefore, it is essential to find very controversial because they have been shown to cause respi-
novel ways to reduce or eliminate food-related microorganisms ratory or other health problems (Quintavalla & Vicini, 2002).
throughout the shelf life of food products. Furthermore, consumers clamor for natural products. Therefore,
research seeks alternative strategies for reducing the use of
chemical additives in the food industry. In this context, the use of
* Corresponding author. Tel.: þ98 122 6254986; fax: þ98 122 6253499. natural compounds such as essential oils (EOs), with antimicrobial
E-mail address: [email protected] (M. Rezaei). properties appears to be an attractive option (Sanchez-Garcia,

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.07.037
2 M. Alboofetileh et al. / Food Control 36 (2014) 1e7

Lopez-Rubio, & Lagaron, 2010). EOs are volatile oily liquids obtained coriander (Coriandrum sativum) essential oils were acquired from
from different plant parts and widely used as food flavors. However, Magnolia Co. (Tehran, Iran).
a number of studies have demonstrated that EOs also possess
antimicrobial activity (Burt, 2004). Among these, clove, oregano, 2.2. Bacterial strain and maintenance
rosemary, thyme, and sage oils have been found to be the most
effective (Holley & Patel, 2005). Although most EOs are classified as Stock culture of L. monocytogenes (PTCC 1298), S. aureus (PTCC
Generally Recognized as Safe, their use as food preservatives is 25923), and E. coli (PTCC 1330) were obtained from Persian Type
often limited due to flavoring considerations since effective anti- Culture Collection (Tehran, Iran). All strains were stored in brain
microbial doses may exceed sensorily acceptable levels. Incorpo- heart infusion (BHI) broth supplemented with 30% glycerol
ration of EOs in edible films seems rather appealing since, and given at 20  C until used. Subculturing was carried out every 30 days to
the low diffusion rate of the active compounds, smaller amounts maintain bacterial viability. The thawed culture (0.1 mL) was
will be required to accomplish the desired antimicrobial effect transferred to 10 mL of BHI broth and grown in a shaker incubator
(Zinoviadou, Koutsoumanis, & Biliaderis, 2009). at 37  C for 24 h. A second transfer of 0.1 mL of culture into 10 mL of
However, so far the use of edible/biodegradable films for food BHI broth was grown in a shaker incubator at 37  C for 24 h to the
packaging due to their inherent water sensitivity and poor water end of the exponential phase of growth. Subsequently, these
resistance has been limited (Sorrentino, Gorrasi, & Vittoria, 2007). appropriately diluted cultures were used for the inoculation of agar
Currently, one of the most effective alternatives to improve the plates in order to obtain a target inoculum of 102 CFU/cm2.
properties of biopolymer films for potential food application is the
formation of nanocomposites. Owing to the reinforcement provided 2.3. Antimicrobial activity of essential oils
by the nanometer-sized particles dispersed in the biopolymer ma-
terial, polymer nanocomposites, especially natural biopolymer- The antibacterial properties of the six mentioned essential oils
layered silicate nanocomposites, exhibit markedly improved pack- were studied using the agar diffusion method (Gómez-Estaca,
aging properties as compared with pure biopolymer (Petersson & López de Lacey, López-Caballero, Gómez-Guillén, & Montero,
Oksman, 2006). Additionally, biologically active ingredients such 2010). 30 ml of the essential oils were poured into an agar well
EOs can be added to impart desired functional properties to the (5 mm diameter), previously created with a sterile core bore on the
resulting packaging materials. It seems that the use of active nano- agar, after their plates had been seeded with 0.1 ml of inoculum by
composite films for food packaging can be suitable due to their swab containing approximately 106 CFU/ml of the indicated bac-
acceptable structural integrity and barrier properties imparted by teria. All the strains were cultured in Tryptone Soya Agar (Merck,
the nanocomposite matrix, and the antimicrobial/antioxidant Germany). The plates were incubated at 37  C for 48 h in the
properties contributed by the natural antimicrobial/antioxidant suitable incubation chamber. After incubation, the microbial
agents impregnated within (Abdollahi, Rezaei, & Farzi, 2012). In this growth was observed and the degree of inhibition was expressed as
way, nanocomposite can be a vehicle to release active compounds follows: totally inhibited, þþþ; partially inhibited, þþ; slightly
slower when they are used in contact with food products (Sánchez- inhibited, þ; no inhibition, e.
González, Cháfer, Hernández, Chiralt, & González-Martínez, 2011).
Most recently published research has also confirmed that the pres- 2.4. Preparation of nanocomposite film
ence of nanoclay in films can improve retention of aroma com-
pounds (Kurek, Descours, Galic, Voilley, & Debeaufort, 2012). An aqueous solution of sodium alginate was prepared by dis-
Consequently, such nanocomposite films can extend food’s shelf life, solving 10 g of sodium alginate powder in 1000 ml distilled water to
and improve food quality. However, nanocomposite film material obtain a 1% w/v alginate solution, using a magnetic stirring plate at
with an active packaging function is in its infancy. It is now emerging 70  C and 1200 rpm for 30 min. Nanocomposite films were pre-
because of environmental concerns and expectations for high pared according to the methods reported by Xu, Ren, and Hanna
quality food products (Rhim & Ng, 2007). Therefore, a great deal of (2005). A specific amount of MMT (3% w/w on solid sodium algi-
research on the preparation and application of bionanocomposite nate) was dispersed in 50 mL of distilled water and vigorously
packaging with functional properties is expected for these biode- stirred for 24 h at room temperature. Afterward, the alginate so-
gradable nanocomposite films. lution was slowly added into the pretreated clay solution and the
The present study has two goals. First, it tests the antimicrobial mixture was stirred for 4 h. Then, the marjoram (MO), Clove (CO),
properties of six selected essential oils against three common and cinnamon (CI) essential oils, previously mixed with Tween 80
pathogenic foodborne bacteria including Escherichia coli, Staphylo- (0.25 g/g of essential oil) to help create a uniform and stable dis-
coccus aureus, and Listeria monocytogenes, to select the most tribution in the alginate matrix, were incorporated into the film-
effective EOs in the enrichment of alginate/clay nanocomposite forming solution at several concentrations (0.5, 1.0, and 1.5% w/v
films by agar diffusion method. Second, this study assigns the effect on the basis of neat film solution). The final solution was homog-
of essential oil incorporation in the antimicrobial properties of enized with Ultra-Turrax (IKA T25-Digital Ultra-Turrax, Staufen,
alginate/clay films to evaluate the effectiveness of prepared films Germany) at 9000 rpm for 2 min. The resulting mixture was
against the three above-mentioned foodborne pathogens. degassed under vacuum for 30 min in order to remove all bubbles.
Finally, the film-forming solution was cast onto Petri dishes (9 cm
2. Materials and methods in diameter) and dried for 72 h at ambient conditions (T ¼ 25  C and
RH ¼ 55%  2%) to prepare nanocomposite films. The dried films
2.1. Materials were removed from the Petri dishes and preconditioned in desic-
cators containing saturated magnesium nitrate solution at 25  C
Sodium alginate (medium viscosity) was obtained from Sigma- and 52.89% relative humidity prior to testing.
Aldreich Chemical Co., USA and Naþ-montmorillonite (MMT) was
purchased from Southern Clay Products, USA. Glycerol was ob- 2.5. Antimicrobial effectiveness of films
tained from Merck Co., Germany. Clove (Syzgium aromaticum),
cumin (Cuminum cyminum), caraway (Bunium persicum), marjoram The methodology was adapted from Sánchez-González et al.
(Origanum majorana), cinnamon (Cinnamomum zeylanicum), and (2011). Tryptone Soy Agar with 3% NaCl (SigmaeAldrich,
 M. Alboofetileh et al. / Food Control 36 (2014) 1e7 3

Germany) was used as a model solid food system (TSA-NaCl). Ali- Villalobos-Carvajal, & Reyes, 2012; Pranoto, Rakshit, & Salokhe,
quots (20 g) of TSA-NaCl were poured into Petri dishes (8.5 cm 2005). Results indicate that the nanocomposite films containing
diameter). After the culture medium solidified, aliquots (0.1 ml) of EOs were effective in reducing the growth of L. monocytogenes. In
the properly diluted culture were inoculated on the surface of TSA- this study, the nanocomposite containing MO essential oil
NaCl. Then, different test films (containing or without antimicrobial demonstrated the lowest L. monocytogenes population (P < 0.05) in
substances) of the same diameter as the Petri dishes were placed on comparison with other active nanocomposites (containing C and CI
the inoculated surface. Inoculated uncoated TSA-NaCl were used as essential oils at the same concentration) on inoculated TSA-NaCl
control. Plates were then covered with Parafilm to avoid dehydra- plates. Lis-Balchin and Deans (2003) reported the potent antimi-
tion and stored at 10  C for 12 days. Instead of using 4  C, a typical crobial activity of marjoram against the L. monocytogenes strain.
refrigerated storage temperature, a relative high temperature was With the highest concentration of MO (1.5%), a total inhibition of
used so that the efficacy of antimicrobial film against mentioned the pathogen growth occurred during the storage period. When 1%
pathogens could be determined in a relatively shorter period of of the MO essential oil was incorporated into the film matrix,
time. Microbial counts on TSA-NaCl plates were examined imme- nanocomposite films displayed less antimicrobial activity, so that
diately following inoculation and periodically during the storage total inhibition of pathogen growth occurred during the first 7 days
period. To this end, the agar was removed aseptically from the Petri of the storage period. Subsequently, the L. monocytogenes popula-
dishes and placed in a sterile glass container. 100 ml of 0.9% NaCl tion increased up to 3.6 log UFC/cm2 at the end of the storage
solution was added to each container and homogenized for 3 min, period. As can be seen in Fig. 1a, following a storage period of 12
which resulted in a very homogeneous system. Serial dilutions days, films with 1% MO reduced microbial growth with respect to
were made and then poured onto TSA. Plates were incubated at the control to 4.3 log. As expected, the antimicrobial effect is less
37  C for 48 h before colonies were counted. All tests were done in marked with 0.5% MO, where a microbial reduction of approxi-
triplicate. mately 1.63 log, as compared to the control plates, was observed
during the storage period. Fig. 1b showed that SA/clay films con-
2.6. Statistical analysis taining CO essential oil had less effect in controlling
L. monocytogenes growth than SA/clay films containing MO essen-
Tukey’s multiple comparisons were used to determine any sig- tial oil. At the highest concentration of CO (1.5%), a total inhibition
nificant differences in mean log CFU/cm2 among treatments at a of the pathogen growth occurred during the first 7 days of the
95% confidence interval. storage period and this film reduced the population of
L. monocytogenes more than 4.2 log UFC/cm2 compared with the
3. Results and discussion control plates. In the presence of 1% CO, a complete inhibition of
microbial growth was observed during the first 3 days of the stor-
3.1. Antimicrobial activity of the essential oils age period. Similar to 0.5% MO, the 0.5% CI essential oil was not
sufficient to control the microbial growth for the entire storage
The qualitative antimicrobial activity of the essential oils period. SA/clay films containing CI essential oil showed a less
is shown in Table 1. Marjoram essential oil presented the marked antimicrobial activity than SA/clay containing MO and CO
highest inhibitory effect. Cumin essential oil was least effective (Fig. 1c). The highest level of CI oil (1 and 1.5%) led to a complete
against the three studied pathogens. The intensity of inhibition of L. monocytogenes growth for the first 3 days; then the L
antimicrobial efficacy was in the following order: marjoram > - monocytogenes population increased to 5.86 and 4.16 log UFC/cm2
clove > cinnamon > coriander > caraway > cumin. Among the at the end of the storage period, respectively. In the presence of
three tested pathogens, E. coli were the most resistant. Based on 0.5% CI, the L. monocytogenes population increased to 6.83 log CFU/
inhibition zone test results, marjoram, clove and cinnamon EOs cm2 at the end of the storage period. These results are in agreement
were selected for further study. with those of Sánchez-González, González-Martínez, Chiralt, and
Cháfer (2010), when analyzing the antilisterial activities of chito-
3.2. L. monocytogenes san film containing tea tree essential oil against L. monocytogenes in
TSA-NaCl plates. Their results demonstrated that the incorporation
Fig. 1 shows the growth curves of L. monocytogenes on control of tea tree essential oil into chitosan matrix (2% w/w) improves the
TSA-NaCl plates and TSA-NaCl plates covered with EOs enriched antilisterial effect of chitosan.
films. The counts of L. monocytogenes on the control TSA-NaCl
plates increased substantially from the initial inoculation level of 3.3. S. aureus
2e7.86 log CFU/cm2 by the end of the experiment. Similarly, Kristo,
Koutsoumanis, and Biliaderis (2008) reported that Fig. 2 shows the growth curves of S. aureus on control TSA-NaCl
L. monocytogenes grew approximately 8 log on TSA-NaCl plates plates, and on TSA-NaCl plates covered with the antimicrobial SA/
when stored at 10  C for 12 days. No significant differences clay film stored for 12 days at 10  C. No significant differences
(P > 0.05) were observed between growths of L. monocytogenes on (P > 0.05) were observed between growths of S. aureus on control
control TSANaCl plates and plates coated with the SA/clay film TSA-NaCl plates and on plates covered with the EOs-free film
during the storage period. The absence of antimicrobial activity of during storage. These findings indicate that the SA/clay film had no
pure alginate films has been reported in other studies (Benavides, inhibitory effective on the growth of S. aureus (similar to

Table 1
Antibacterial activity of the EOs and MMT over 3 bacterial strains.

Microorganisms/Essential oil Clove Cumin Marjoram Caraway Coriander Cinnamon MMT

Listeria monocytogenes þþ e þþþ þ þþ þþ e

Staphylococcus aureus þ þ þþþ e þ þþ e
Escherichia coli þþ e þþ þ þ þþ e

Totally inhibited: þþþ; partially inhibited: þþ; slightly inhibited: þ; no inhibited: e.

4 M. Alboofetileh et al. / Food Control 36 (2014) 1e7

Fig. 2. Antibacterial activity of different films against Staphylococcus aureus on TSA-

NaCL medium stored at 10  C.

Fig. 1. Antibacterial activity of different films against Listeria monocytogenes on TSA-

NaCL medium stored at 10  C. than films containing CO or CI in reducing the growth of S. aureus
(Fig. 2a). After 12 days of storage, the S. aureus population reached a
value of 7.36 log UFC/cm2 in the control samples, while the use of
L. monocytogenes and E. coli). Regardless of the type of essential oil, films enriched with the highest concentration of MO maintained
incorporating this agent in the film matrix increased the antimi- the population of S. aureus under the initial inoculation level. In
crobial activity of film, and the application of EO-enriched films addition, the use of films containing 1% MO resulted in a significant
resulted in significant inhibition of S. aureus counts after 12 days of reduction of the S. aureus population during the entire storage
storage at 10  C (more than 3 log UFC/cm2). During the whole period (P < 0.05), and a total inhibition of the pathogen growth
storage period, films containing MO were more effective (P < 0.05) occurred during the first 9 days of the storage period. However, low
 M. Alboofetileh et al. / Food Control 36 (2014) 1e7 5

as compared to the control plates, respectively, was observed dur-

ing the storage period. As can be seen in the Fig. 2c, although
nanocomposite films containing CI showed less antimicrobial ac-
tivity in comparison to other active nanocomposites, the highest
level of CI essential oil (1.5%) led to a total inhibition of S. aureus
growth for 6 days, reaching a reduction of approximately 3.3 log at
the end of the storage period. The lower CI concentration levels
(0.5) showed less marked antimicrobial activity so that a pathogen
reduction, as compared to the control plates, of about 0.67 and 1.73
log was reached, respectively, at the end of the storage period.

3.4. E. coli

Fig. 3 shows the growth curves of E. coli on control TSA-NaCl

plates and on TSA-NaCl plates covered with the EOs-enriched
films. The counts of E. coli in the control TSA-NaCl plates
increased substantially from the initial inoculation level of ca. 2 to
5.36 log CFU/cm2 by the end of the experiment. This pathogen was
less affected by the active films than other microorganisms. As can
be seen from the growth curve, no significant differences were
observed among the growths of E. coli on the control TSA-NaCl
plates and on plates covered with the SA/clay film during the
storage period. Similar to data obtained for the L. monocytogenes,
MO was the most effective antimicrobial to control the growth of
E. coli and in the highest concentration of MO (1.5%), a total inhi-
bition of pathogen growth occurred during the storage period
(Fig. 3a). In the presence of 1% MO, a complete inhibition of mi-
crobial growth was observed during the first 6 days of the storage
period, and this film reduced the population of E. coli more than
2.68 log UFC/cm2 compared with the control TSA-NaCl plates at the
end of the study period. CO incorporated into SA/clay films reduced
pathogen growth. Microbial growth was totally inhibited by the
highest concentration of CO (1.5%) during the first 6 days of the
study period although the antimicrobial effectiveness decreased
afterwards. However, at the end of the storage period, a pathogen
reduction, as compared to the control plates, of about 2.26 and 2.8
log was observed for the 1% and 1.5% concentrations of CO,
respectively (Fig. 3b). At the end of the 12-day storage at 10  C,
E. coli reached populations of 4.86 log UFC/cm2 on TSA-NaCl plates
covered with films containing 0.5% CO. Fig. 3c is shown, SA/clay
films containing CI essential oil showed less marked antimicrobial
activity than SA/clay film containing MO and CO. However, the
incorporation of CI in SA/clay films also resulted in significantly
lower (P < 0.05) E. coli counts in comparison to the control TSA-
NaCl plates. The reduction in pathogen growth was consequent to
increasing the CI concentration of the film. CI at the highest con-
centration (1.5%) totally inhibited pathogen growth for the first 6
days of storage. Furthermore, at the end of the storage period, E-coli
counts on TSA-NaCl plates covered with film containing 1% and 1.5%
CI were at 1.8 and 2.49 log UFC/cm2; respectively, considerably
lower than that of the control TSA-NaCl plates (5.36 log CFU/cm2).
0.5% CI was not sufficient for controlling the growth of E. coli, and
Fig. 3. Antibacterial activity of different films against Escherichia coli on TSA-NaCL
the population of pathogens increased to 4.9 UFC/cm2 at the end of
medium stored at 10  C. the study period. However, this value was lower than population of
E. coli on the control TSA-NaCl plates.

antimicrobial action was observed for films containing 0.5% MO 3.5. Discussion
essential oil; application of these films reduced the counts from 2 to
1.84 log UFC/cm2 for the first 3 days, after which the pathogen Results obtained showed that of the 6 essential oils tested in this
population increased. Fig. 2b is shown the incorporation of CO into study, marjoram, clove and cinnamon exhibited strong antibacte-
SA/clay films was less effective than in films enriched with MO. rial activities and among the three tested pathogens, E. coli were the
With the highest concentration of CO (1.5%), a total inhibition of the most resistant. Gómez-Estaca et al. (2010) showed that clove,
pathogen growth occurred during the first 6 days of the storage Lavender, thyme and rosemary had strong and consistent inhibi-
period. The antimicrobial effect was less marked with 0.5% and 1% tory effects against various pathogens and spoilage microorgan-
CO where a microbial reduction of approximately 0.08 and 2.66 log, isms. In addition, Winward, Avery, Stephenson, and Jefferson
6 M. Alboofetileh et al. / Food Control 36 (2014) 1e7

(2008) reported that cinnamon essential oil had a good inhibitory antimicrobial properties. Benavides et al. (2012) showed this effect
effect on pathogenic bacteria such as E. coli and S. aureus. The lower in oreganoealginate-based film against several strains as E. coli, L.
antimicrobial activity against E. coli can be attributed to the fact that monocytogenes, Salmonella enteritidis, and S. aureus. The combina-
gram-negative are in general more resistant due to the external tion essential oilealginate results also effective to control the
lipopolysaccharide wall surrounding the peptidoglycan cell wall growth of pathogens in food products (in vivo). In this sense,
(Burt, 2004). The mechanism of antimicrobial activity of essential Oussalah, Caillet, Salmieri, Saucier, and Lacroix (2006) observed a
oils is related with the attack on the phospholipid present in cell good inhabitation of Salmonella Typhimurium and E. coli O157:H7
membranes, which causes increased permeability and leakage of on whole beef muscle coated with alginate films containing 1% (w/
cytoplasm, or in their interaction with enzymes located on the cell v) of Spanish oregano, Chinese cinnamon, or savory essential oils.
wall (Gómez-Estaca et al., 2010). Thus, the resistance of Gram- Another factor that affects the antimicrobial activity of active films
negative bacteria to the essential oils likely lies in the protective is polymer structure. When montmorillonite (MMT) are incorpo-
role of their cell wall lipopolysaccharides or outer membrane pro- rated into film-forming solution and a polymer/clay nano-
teins. Moreover, the antimicrobial activity of EOs depends on the composite film is formed, the sheet-like clay layers orient in parallel
type of spice or herb, the chemical composition and the content of with the film surface. For this reason, active molecules of EOs have
extracts and essential oils (Castellano et al., 2008). Chemical to take a long way around the impermeable clay layers in nano-
composition of EOs is complex and strongly dependent on the va- composite films (Tunç & Duman, 2011). On the other hand, the
riety of plant, the part of the plant considered (e.g., seed vs. leaves), establishment of hydrogen bonds between carboxyl groups of the
origin, time of harvest, the harvesting season and processing, as alginate and nanoclays would result in a denser polymer matrix
well as storage conditions (Burt, 2004; Tajkarimi, Ibrahim, & Cliver, (Alboofetileh, Rezaei, Hosseini, & Abdollahi, 2013). Therefore, active
2010). The major components in EOs are phenolic substances, component of EO cannot be easy release form film matrix. Finally,
which are thought to be responsible for the antimicrobial proper- incorporating of MMT to film matrix led to increase the film
ties. In this regard, the major components in MO, CI, and CO are thickness and this factor is also effective to slow down the active
Terpinen-4-ol, Cinnamaldehyde, and Eugenol, respectively. Suble- component losses. Therefore, the release of essential oil has been
thal concentrations of Eugenol and Cinnamaldehyde have been reduced; consequently, lower antimicrobial activity was observed
found to inhibit production of amylase and proteases by Bacillus (for a constant EOs concentration). These results confirm that
cereus. Cell wall deterioration and a high degree of cell lysis were incorporation of these MMT can potentially be used to control the
also noted (Hosseini, Razavi, & Mousavi, 2009). In the next step of release of antimicrobial agents from film materials. Some studies
study, in order to form the active nanocomposite films, MO, CI and have reported controlled-release or diffusion of various antimi-
CO essential oils were incorporated into alginate-clay nano- crobial agents including carvacrol (Tunç & Duman, 2011), thymol
composite film at several concentrations (0.5, 1.0, and 1.5% w/v) and (Sánchez-García, Gimenez, Ocio, & Lagaron, 2008) and rosemary
antibacterial activity of them was evaluated against EOs (Abdollahi et al., 2012) from polymer/nanoclay films. In the
L. monocytogenes, E. coli, and S. aureus. When antimicrobial agents present study, during the first days of the storage period, the con-
such EOs are incorporated into food packaging films, these mate- centration of the EOs was a significant factor in the inhibition of
rials diffuse through agar gel and result in a clear zone around the pathogens growth. Nevertheless, at the end of storage period, the
film discs (Hosseini et al., 2009). In this regards, when EOs were tendency changed and the nature of the incorporated EOs became
incorporated into the film matrix, the antimicrobial activity of EOs more significant. Furthermore, it must be highlighted that, as
was maintained but EOs showed less antibacterial activity in film- storage time increased, the film effectiveness decreased. As can be
forming solution in comparison with pure essential oil. The causes seen from the growth curve, in all pathogens, growth was reduced
that would explain this result could be the lower amount of EOs in in the 3 initial days of the storage period, and then the population of
the film solution in comparison with the well test for pure EOs as bacteria increased until the end of the storage period. This phe-
well as partial loss of volatile compounds during film preparation nomenon can be explained by the evaporation of volatile com-
(Abdollahi et al., 2012; Sánchez-González et al., 2010). Fisher & pounds responsible for the antimicrobial activity and/or by the
Phillips, 2008 noted that essential oils contain around 85e99% migration of EOs components into the agar medium (Sánchez-
volatile and 1e15% non-volatile components. According to González et al., 2011). On other hand, after the films were placed
Sánchez-González et al., (2011), various factors affect the antimi- on the inoculated surface of TSA-NaCl, the SA/clay hydrophilic
crobial action of film, such as the nature of EOs, type of bacteria, matrices absorbed water, which induced changes in the film
characteristics of the film matrix, method and manufacturing structure. This condition facilitates the evaporation and liberaliza-
conditions of films. Like the disk test result, the antimicrobial ac- tion of EOs components from the polymer matrix. Consequently,
tivity of alginate-clay nanocomposite film films varied distinctively the effectiveness of the films tends to decrease during the storage
depending on the nature and amount of the essential oils and mi- period.
croorganisms tested. In this sense, a nanocomposite film containing
MO was most effective in controlling the growth of the pathogens. 4. Conclusion
The reasons for the generally higher efficacy of marjoram over the
other investigated EOs are mainly attributed to marjoram’s high The antimicrobial effects of pure EOs of clove (CO), coriander,
content of phenolic compounds as well as good interaction be- caraway, marjoram (MO), cinnamon (CI), and cumin against E. coli,
tween the constituents of the marjoram with the polymer matrix S. aureus, and L. monocytogenes were determined by an agar
(Burt, 2004; Papadokostaki, Amarantos, & Petropoulos, 1998). As diffusion test. Marjoram essential oils showed the highest antimi-
above mentioned, concerning the type of bacteria, given their extra crobial effect, followed by clove and cinnamon, respectively. Next,
protective outer membrane, gram-negative bacteria usually are the antimicrobial films were prepared by incorporating different
considerably more resistant to antibacterial agents than their concentrations of MO, CO, and CI into alginate/clay films. During
gram-positive counterparts. So the active nanocomposite films the storage period in this study, the concentration of the EOs, rather
showed lower antimicrobial activity against E. coli in comparison than the type of EOs, was the most relevant parameter in the in-
with the gram-positive bacteria (L. monocytogenes, S. aureus). hibition of all 3 pathogens’ growth. The results also revealed that
Several authors observed in in vitro studies that the incorporation the films with EOs incorporated were more effective against gram-
of different essential oil into alginate films improved its positive bacteria (S. aureus and L. monocytogenes) than gram-
 M. Alboofetileh et al. / Food Control 36 (2014) 1e7 7

negative bacteria (E. coli). Furthermore, complete growth inhibition films prepared with chitosan. International Journal of Food Microbiology, 62(1),
139e148.
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