International Journal of Food Microbiology 153 (2012) 66–72

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Antifungal activity by vapor contact of essential oils added to amaranth, chitosan, or

starch edible films
Raúl Avila-Sosa a, b, Enrique Palou a, María Teresa Jiménez Munguía a,
Guadalupe Virginia Nevárez-Moorillón c, Addí Rhode Navarro Cruz b, Aurelio López-Malo a,⁎
a
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Pue. 72810, México
b
Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, 14 Sur y Av. San Claudio, Ciudad Universitaria, Puebla, Pue. 72420, México
c
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, P.O. Box 1542-C Chihuahua, Chih., México

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

Article history: Antimicrobial agents can be incorporated into edible films to provide microbiological stability, since films can be
Received 13 April 2011 used as carriers of a variety of additives to extend product shelf life and reduce the risk of microbial growth on
Received in revised form 3 October 2011 food surfaces. Addition of antimicrobial agents to edible films offers advantages such as the use of small antimi-
Accepted 22 October 2011
crobial concentrations and low diffusion rates. The aim of this study was to evaluate inhibition by vapor contact
Available online 30 October 2011
of Aspergillus niger and Penicillium digitatum by selected concentrations of Mexican oregano (Lippia berlandieri
Keywords:
Schauer), cinnamon (Cinnamomum verum) or lemongrass (Cymbopogon citratus) essential oils (EOs) added to
Natural antimicrobials amaranth, chitosan, or starch edible films. Essential oils were characterized by gas chromatography–mass spec-
Edible films trometry (GC/MS) analysis. Amaranth, chitosan and starch edible films were formulated with essential oil con-
Vapor contact centrations of 0.00, 0.25, 0.50, 0.75, 1.00, 2.00, or 4.00%. Antifungal activity was evaluated by determining the
Essential oils mold radial growth on agar media inoculated with A. niger and P. digitatum after exposure to vapors arising
from essential oils added to amaranth, chitosan or starch films using the inverted lid technique. The modified
Gompertz model adequately described mold growth curves (mean coefficient of determination 0.991 ± 0.05).
Chitosan films exhibited better antifungal effectiveness (inhibition of A. niger with 0.25% of Mexican oregano
and cinnamon EO; inhibition of P. digitatum with 0.50% EOs) than amaranth films (2.00 and 4.00% of cinnamon
and Mexican oregano EO were needed to inhibit the studied molds, respectively). For chitosan and amaranth
films a significant increase (pb 0.05) of lag phase was observed among film concentrations while a significant
decrease (pb 0.05) of maximum specific growth was determined. Chitosan edible films incorporating Mexican
oregano or cinnamon essential oil could improve the quality of foods by the action of the volatile compounds
on surface growth of molds.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction mixtures of several components. The inherent aroma and antimicro-

Application of antimicrobial agents could prevent or delay micro-
bial spoilage of food products; nevertheless advances in biological
and chemical analytical methods have raised questions regarding
the safety of some chemical food additives (Avila-Sosa et al., 2010a).
Because of concerns related to the use of chemical additives, con-
sumers prefer natural additives (including antimicrobial agents)
incorporated into food products (Davidson, 2001).
Several research groups are studying the chemical structure
and activity of natural antimicrobials from fruits, vegetables, grains,
herbs, and spices. Efforts have concentrated on the use of natural
extracts, such as those obtained from spices. Essential oils (EOs) are
aromatic oily liquids obtained from plant material and are usually
⁎ Corresponding author. Tel.: + 52 222 229 2126; fax: + 52 222 229 2727.
E-mail address: aurelio.lopezm@udlap.mx (A. López-Malo).
bial activity of EOs are related commonly to the chemical structure
of their components, the concentration in which the components
are present, and the interactions among them affecting their bioactive
properties. Some studies have demonstrated that EOs have greater
antibacterial activity than their major constituents when tested sepa-
rately (Burt, 2004). Since EOs are generally recognized as safe (GRAS)
for use as flavor additives (Tunc et al., 2007), the possibility of utiliz-
ing their antimicrobial effects by adding EOs to the surface of food is
being investigated (Ayala-Zavala et al., 2009; Brul and Coote, 1999;
Holley and Patel, 2005). Despite the high efficacy of the EOs and
their constituents against food-borne pathogens and spoilage micro-
organisms when in vitro tests are conducted, the same effect in food
products is only achieved when higher concentrations of EOs are uti-
lized. When EOs are applied directly on a food surface by dipping,
powdering or spraying, their highly hydrophobic and volatile active
substances are bound by food components, while other components
of the EOs are partitioned through the product according to their

0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2011.10.017
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72
affinity with water. To avoid this problem an alternative is the incor-
poration of essential oil within edible films. Edible films can serve as
carriers releasing antimicrobials onto the food surface; controlling
microbial growth. Other advantages of edible films are the ability to
delay transmission of moisture, oxygen, aroma and solutes, thus
enhancing product shelf life (Cha and Chinnan, 2004; Goni et al.,
2009; Guilbert et al., 2002; Krochta, 1997; Marsh and Bugusu, 2007).
Edible films can reduce antimicrobial diffusion into the product
since the essential oil forms part of the chemical structure of the
film and interacts with the polymer and the plasticizer. Antimicrobial
release from the edible film depends on many factors, including elec-
trostatic interactions between the antimicrobial agent and the poly-
mer chains, osmosis, structural changes induced by the presence of
antimicrobial, and environmental conditions. Compared with direct
application, smaller amounts of antimicrobial agents would be needed
when edible films are used in order to achieve a specific shelf life
due to a gradual release on food surfaces (Ponce et al., 2008; Sebti
et al., 2005).
Many reported data focus on the discovery of new natural antimi-
crobials that can be incorporated into edible films. A number of com-
pounds have been proposed to have antimicrobial activity in foods,
including organic acids, enzymes, bacteriocins, and essential oils
(Cagri et al., 2004). In a previous paper (Avila-Sosa et al., 2010b) we
evaluated by direct contact the antifungal effect of Mexican oregano
essential oil incorporated into different edible films providing evidence
of inhibition of spoilage microorganisms (A. niger and Penicillium spp.).
Some studies have been published regarding inhibition of several
microorganisms by the vapor-phase of EOs (Edris, 2007; Inouye et al.,
2000, 2001, 2006; Inouye, 2003; López et al., 2005, 2007; Nielsen and
Rios, 2000; Suppakul et al., 2003). Until now, no standard assay exists
to evaluate microbial inhibition by vapor contact of EOs. There are
many methods reported by different authors (Kloucek et al., in press)
the most common are: 1) the overlay method (Du et al., 2008, 2009b)
which is just a simple modification of the disk diffusion assay and can
be related to direct contact of the films on food surfaces and 2) the
vapor phase method (López et al., 2007) where EOs and microorgan-
isms are placed separately in some sealed environment and can be
related to microbial inhibition from a distance without direct contact
of the film with the contaminated food.
The aim of this study was to evaluate inhibition of A. niger and
P. digitatum by vapor contact with selected concentrations of Mexican
oregano, cinnamon or lemongrass EOs added to amaranth, chitosan, or
starch edible films.
2. Materials and methods
2.1. Essential oils and chemical characterization
Mexican oregano (L. berlandieri Schauer) essential oil was pro-
vided by CiReNA (Natural Resources Research Center of Salaices,
López, Chihuahua, Mexico). Cinnamon bark (Cinnamomum verum)
and lemongrass (Cymbopogon citratus) were bought at a local market
in Puebla, Mexico. Plant material was authenticated by botanists of
the Department of Biology of Benemerita Universidad Autonoma de
Puebla, Puebla, Mexico by visual inspection and comparison with
the herbarium reference by data base. Lemongrass was washed and
air-dried at room temperature for 3 days and then finely ground
with a mortar and pestle. All EOs were obtained by vapor distillation
for 4 h with a Cleavenger-type apparatus. EOs were analyzed with
a GC Perkin Elmer Turbo Mass Gold MS-Auto system XL™ (Perkin-
Elmer, Norwalk, CT) with a splitless injector and an FID detector,
equipped with an AT-1 capillary column (30 m ∗ 0.25 I.D. ∗ 0.25 μm).
Helium was used as the carrier gas, and the following conditions
were set for the analysis: injector, 100 °C, detector 225 °C; the initial
oven temperature was 55 °C held for 1 min, followed by a ramp-up
of 3 °C/min up to 95 °C, and a second ramp-up of 25 °C/min up to
67
220 °C, and held at the final temperature for 10 min, for a total run
time of 30 min. The obtained spectra were compared with respective
mass spectra of pure compounds, and also with the mass profile of
the same compounds available from the US National Institute of
Standard Technology (NIST) library (Avila-Sosa et al., 2010b).
2.2. Edible film preparation
Studied edible films were made by the casting method, which con-
sists of drying the corresponding film forming solution (FFS) that had
been applied on a support. FFSs were formulated with amaranth flour
(bought at a local market in Puebla, Mexico) with water solution of
4% w/w amaranth flour, pH was adjusted to 10.7 with NaOH (0.1 N)
(Tapia-Blácido et al., 2005). Medium molecular weight (450 kDa) of
chitosan (Aldrich Chemical Co. Milwaukee, WI) was prepared with
1.5% w/w chitosan in 1.5% v/v acetic acid, the solution was stirred
overnight at room temperature. High amylose corn starch (CPI Ingre-
dients, Mexico) was used with 1 g of starch, in previously sterilized
10 ml 0.25 N sodium hydroxide and 10 ml distilled water. FFS were
maintained 60 min under stirring conditions (Bertuzzi et al., 2007).
Amaranth and chitosan solutions were sterilized at 121 °C for
15 min. To enable film formation, glycerol (Aldrich Chemical Co.
Milwaukee, WI) 1.3% v/v and Tween 20 (Aldrich Chemical Co.
Milwaukee, WI) 0.5% v/v were added as plasticizers for amaranth
and chitosan films respectively. Starch FFS was gelatinized in a shaker
water bath at 78–80 °C for 10 min; when the solution was near 40 °C,
glycerol (1.2% v/v) was added (Zivanovic et al., 2005).
FFSs were mixed (IKA High Performance Disperser T18, Chicago,
IL) under aseptic conditions at 20,000 rpm for 1 min at room temper-
ature with the incorporation of EOs at 0.00%, 0.25%, 0.50%, 0.75%,
1.00%, 2.00%, or 4.00% (v/v) final concentration and poured into
60 mm inner diameter sterile Petri dishes covers. Films were prepared
with 7 ml of FFS per Petri dish (1 film), dried under 0.35 kg/cm 2
vacuum at 30 °C for 12 h. Films were kept in sealed Petri dishes at
4 °C until analysis (Avila-Sosa et al., 2010b).
2.3. Thickness
Film thickness (μm) was determined on every film formulation,
reporting means of measurements at 5 points of every film using a
micrometer (Scala, 111-B, Mexico City, Mexico).
2.4. Fungal strains and spore suspensions
A. niger and P. digitatum were obtained from Universidad de las
Américas Puebla Food Microbiology Laboratory. They were cultivated
for 5 days at 25 °C on potato-dextrose agar plates (PDA, Merck,
Mexico) acidified with 10% tartaric acid, then were used to recover
fungal spores by pouring 9 ml of sterile physiological water (0.90% w/v
of NaCl) on the agar plate surface, followed by a gentle scraping
using a sterile rake to remove the maximum quantity of spores. Spore
suspensions were transferred into sterile tubes. The number of spores
present in the suspension was determined using a hemocytometer and
an optical microscope (Zeiss Primo Star, Göttingen, Germany), and
expressed as number of spores per milliliter (spores/ml). Suspensions
were serially diluted to approximately 1×106 spores/ml (Sebti et al.,
2005).
2.5. Vapor contact assay
The inverted lid technique was used (Du et al., 2009a); 10 μl of
spore suspension (1 × 10 6 spores/ml) were placed in the center of
the PDA plate and were dried in a laminar flow hood under aseptic
conditions (Labconco, Kansas City, MO, USA) at room temperature
for 30 min, then Petri dish cover with the corresponding antimicro-
bial film was placed to cover the plate with inoculated agar. Radial
68 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72

growth was measured every 24 h during 8 days of incubation 25 °C. A
growth control was prepared in parallel, in order to ensure that viable
organisms were present. Every test was performed in triplicate.
2.6. Fungal growth modeling and statistical analysis
Growth data were fitted using the modified Gompertz model as
reported by Char et al. (2007):
 
D ified Gompertz model adequately fitted the experimental data (mean
Ln t ¼ A expf− exp½ðυm ⋅e=AÞðλ−t Þ þ 1g
Do
where: Dt (cm) is the average colony diameter at time t (day), and
Do (cm) is the average colony diameter at initial time; A is the maxi-
mum mold growth achieved during the stationary phase, υm is the
maximum specific growth rate (1/day), λ is the lag phase (day) and
e = exp (1).
Statistical analyses were performed with the General Linear Model
procedure in Minitab 15 (LEAD Technologies Inc., NJ). Data were
obtained by triplicate with each replicate value obtained from an in-
dividual experiment. Significantly (p b 0.05) different means were
separated with Tukey's test.
3. Results
Chemical analysis of Mexican oregano EO distinguished several com-
pounds, particularly thymol (2.103 g/ml) and carvacrol (0.533 g/ml),
while others were detected in very low concentrations, such as
p-cymene, 1, 8-cineole, and γ-terpinen. Cinnamon EO chemical compo-
sition presents eugenol (3.402 g/ml), cinnamaldehyde (0.652 g/ml)
and acetoeugenol as major constituents. Finally lemongrass GC-MS
exhibited geranial (2.873 g/ml) and geranial acetate as major compo-
nents of this essential oil.
Figs. 1, 2, and 3 present the change in mold colony diameter under
the effect of amaranth, chitosan, and starch edible films respectively,
with added Mexican oregano, cinnamon or lemongrass EOs. The mod-
ð1Þ
coefficient of determination 0.991 ± 0.05). Gompertz model can be
utilized to describe mold radial growth, despite the fact that this
model was originally proposed for bacterial growth (Char et al.,
2007). Calculated growth parameters were useful to compare anti-
fungal effects among concentrations of the studied EOs in the edible
films tested.
For A. niger, amaranth edible films exhibited the lowest antifungal
effectiveness since only high concentrations of cinnamon (2.00%) and
Mexican oregano (4.00%) EOs exerted antifungal effects (Fig. 1A and
Table 1). Gompertz parameters (Table 1) for amaranth edible films
with cinnamon (1.00%) and Mexican oregano EOs (2.00%) showed
significant differences (p b 0.05) in lag phase values with respect to
amaranth films formulated without EO. In the case of P. digitatum,
amaranth films (Fig. 1B) exhibited growth inhibition at cinnamon
EO concentrations of 2.00%; for Mexican oregano and lemongrass
EOs at higher concentrations, higher maximum specific growth
rates were observed which were statistically different (p b 0.05)

A 2.5 A 2.5
2 2
Ln(Dt/Do)

Ln(Dt/Do)

1.5 1.5

1 1

0.5 0.5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (Days) Time (Days)

B 2.5 B 2.5
2 2
Log(Dt/Do)

Ln(Dt/Do)

1.5 1.5

1 1

0.5 0.5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (Days) Time (Days)
Fig. 1. Effect of amaranth edible films added with essential oils at selected concentra-
tions [0.00% (▲), cinnamon 1.00% (■), Mexican oregano 2.00% (●), Mexican oregano Fig. 2. Effect of chitosan edible films added with essential oils at selected concentra-
4.00% (Δ), or lemongrass 4.00% (□)] on Aspergillus niger (A) and Penicillium digitatum tions [0.00% (▲), cinnamon 0.25% (■), Mexican oregano 0.25% (●), or lemongrass
(B) growth. Dt is the average colony diameter at time t and Do is the average colony di- 4.00% (□)] on Aspergillus niger (A) and Penicillium digitatum (B) growth. Dt is the aver-
ameter at initial time. age colony diameter at time t and Do is the average colony diameter at initial time.
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72 69

A from films without EO (Table 2). The thickness of amaranth films

2.5 (14.333 ± 1.479 μm) was not significantly different (p b 0.05) when
the concentration of the EOs increased.
2 Chitosan films exhibited the greatest inhibition against A. niger
and P. digitatum. Inhibition was achieved at low concentrations of
cinnamon and Mexican oregano EOs, statistical differences (p b 0.05)
Log(Dt/Do)

1.5 were observed among υm and λ (Tables 1 and 2) parameters. Lemon-

grass EO exhibited no inhibition effect against A. niger, however a sig-
1 nificant increase (p b 0.05) in the lag phase (Fig. 2A, Table 2) was
observed when EO concentration increased. Chitosan film thickness
varied from 5.33 ± 0.836 to 13.26 ± 0.414 μm.
0.5
A. niger inhibition was obtained with starch edible films with con-
centrations of 0.50% cinnamon, 2.00% Mexican oregano and 4.00%
0 lemongrass EOs (Table 1). Significant differences (p b 0.05) in the lag
0 1 2 3 4 5 6 7 8 phase (Fig. 3A) among 0.00%, 0.25% of cinnamon, 1.00% of Mexican
Time (Days) oregano, and 0.50% of lemongrass concentrations were observed
(Table 1). A similar effect on P. digitatum growth is seen in Fig. 3B.
B 2.5 Films formulated with Mexican oregano presented inhibition at a
concentration of 0.75%. For starch films when the concentration of
essential oil increased, the film thickness varied from 11.533 ± 0.547
2 to 22.066 ± 0.435 μm.
Ln(Dt/Do)

1.5 4. Discussion

The present study has demonstrated the potential of EOs added

1
to edible films as antifungal agents by vapor contact. Antimicrobial
activities of the different EO vapors could be achieved at lesser amounts
0.5 than when applying the EOs in direct contact with a food surface.
Mexican oregano and cinnamon EO were the most effective, their activ-
0 ity due to their constituents as revealed during chemical characteriza-
0 1 2 3 4 5 6 7 8 tion. Determined concentrations of components of Mexican oregano
Time (Days) EO concur with previous reports (Avila-Sosa et al., 2010b; Dunford
and Silva-Vazquez, 2005; Silva-Vazquez and Dunford, 2005). The main
Fig. 3. Effect of starch edible films added with essential oils at selected concentrations component of Mexican oregano EO is thymol, and its concentration is
[0.00% (▲), cinnamon 0.25% (■), Mexican oregano 0.50% (●), Mexican oregano 1.00% greater when the extracts are obtained from younger plants (Arcila-
(Δ), or lemongrass 2.00% (□)] on Aspergillus niger (A) and Penicillium digitatum (B) Lozano et al., 2004; Avila-Sosa et al., 2010a). In contrast, in essential
growth. Dt is the average colony diameter at time t and Do is the average colony diam-
oils from European oregano the concentration of carvacrol is much
eter at initial time.
higher (70–88%) than that of thymol (2%) (Aligiannis et al., 2001;

Table 1
Modified Gompertz model parameters+ (mean ± standard deviation) for Aspergillus niger growth curves subjected to selected concentrations of Mexican oregano (Lippia berlandieri
Schauer), cinnamon (Cinnamomum verum), or lemongrass (Cymbopogon citratus) essential oils added to amaranth, chitosan, or starch edible films by vapor contact.

Edible film Gompertz parameters

A υm (1/day) λ (day) A υm (1/day) λ (day) A υm (1/day) λ (day)

Amaranth Chitosan Starch

Without essential oil 2.21a ± 0.04 2.46a ± 0.09 0.15a ± 0.01 2.40a ± 0.02 0.85a ± 0.01 0.00a ± 0.01 2.21a ± 0.01 1.59a ± 0.09 0.07a ± 0.03
Cinnamon 0.25% 2.34a ± 0.12 2.52a ± 0.13 0.23a ± 0.05 –⁎ –⁎ > 7.00b,⁎ 1.34b ± 0.18 1.07b ± 0.04 2.12b ± 0.13
Cinnamon 0.50% 2.41a ± 0.18 2.66a ± 0.16 0.31a ± 0.08 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 0.75% 2.38a ± 0.09 2.61a ± 0.11 0.27a ± 0.02 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 1.00% 1.99b ± 0.12 2.30a ± 0.16 2.14b ± 0.02 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 2.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 4.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 0.25% 2.37a ± 0.09 3.16a ± 0.05 0.52a ± 0.01 –⁎ –⁎ > 7.00b,⁎ 1.94a ± 0.03 1.45a ± 0.12 0.12a ± 0.02
Oregano 0.50% 2.28a ± 0.11 2.96a ± 0.08 0.45a ± 0.13 –⁎ –⁎ > 7.00b,⁎ 1.98a ± 0.16 1.32a ± 0.16 0.27a ± 0.08
Oregano 0.75% 2.98a ± 0.07 2.99a ± 0.23 0.33a ± 0.11 –⁎ –⁎ > 7.00b,⁎ 2.04a ± 0.11 1.47a ± 0.01 0.33a ± 0.08
Oregano 1.00% 2.45a ± 0.22 3.10a ± 0.16 0.39a ± 0.08 –⁎ –⁎ > 7.00b,⁎ 1.91a ± 0.20 1.96c ± 0.24 1.13d ± 0.04
Oregano 2.00% 1.96b ± 0.18 1.96b ± 0.25 1.15d ± 0.07 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 4.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Lemongrass 0.25% 2.13a ± 0.04 2.63a ± 0.09 0.40a ± 0.01 2.40a ± 0.02 0.85a ± 0.01 0.00a ± 0.01 1.78a ± 0.21 1.33a ± 0.15 0.17a ± 0.01
Lemongrass 0.50% 2.03a ± 0.02 2.51a ± 0.15 0.44a ± 0.08 2.40a ± 0.03 0.85a ± 0.02 0.00a ± 0.02 1.93a ± 0.14 1.41a ± 0.12 0.22a ± 0.05
Lemongrass 0.75% 2.17a ± 0.07 2.55a ± 0.18 0.41a ± 0.04 2.40a ± 0.04 0.85a ± 0.03 0.00a ± 0.03 2.01a ± 0.11 1.55a ± 0.18 0.21a ± 0.08
Lemongrass 1.00% 2.07a ± 0.01 2.62a ± 0.21 0.38a ± 0.08 2.40a ± 0.05 0.85a ± 0.04 0.00a ± 0.04 2.09a ± 0.07 1.64a ± 0.11 0.19a ± 0.01
Lemongrass 2.00% 2.31a ± 0.18 2.48a ± 0.13 0.23a ± 0.02 2.40a ± 0.06 0.85a ± 0.05 0.00a ± 0.05 2.13a ± 0.13 2.40d ± 0.28 1.14d ± 0.02
Lemongrass 4.00% 1.94b ± 0.05 1.84b ± 0.34 0.12a ± 0.05 2.29a ± 0.02 2.65b ± 0.24 2.16c ± 0.01 – –⁎ >7.00c,⁎

Means followed by a different superscript letter within a column for each studied film (amaranth, chitosan or starch) are significantly different (p b 0.05).
+
A: maximum mold growth in the stationary phase; υm: maximum specific growth rate; λ: lag phase.
⁎ No growth.
70 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72

Table 2
Modified Gompertz model parameters+ (mean ± standard deviation) for Penicillium digitatum growth curves subjected to selected concentrations of Mexican oregano (Lippia
berlandieri Schauer), cinnamon (Cinnamomum verum), or lemongrass (Cymbopogon citratus) essential oils added to amaranth, chitosan, or starch edible films by vapor contact.

Edible film Gompertz parameters

A υm (1/day) λ (day) A υm (1/day) λ (day) A υm (1/day) λ (day)

Amaranth Chitosan Starch

Without essential oil 1.97a ± 0.08 1.10a ± 0.45 0.13a ± 0.03 2.14a ± 0.01 1.91a ± 0.01 0.09a ± 0.01 1.90a ± 0.12 2.42b ± 0.16 0.09a ± 0.02
Cinnamon 0.25% 1.90a ± 0.02 0.96a ± 0.12 0.22a ± 0.07 1.62b ± 0.08 1.46b ± 0.04 0.10a ± 0.05 1.15b ± 0.21 1.67a ± 0.07 2.48b ± 0.03
Cinnamon 0.50% 1.89a ± 0.07 1.05a ± 0.36 0.45a ± 0.18 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 0.75% 1.95a ± 0.04 1.23a ± 0.09 0.34a ± 0.09 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 1.00% 1.82b ± 0.02 3.17b ± 0.29 2.17b ± 0.01 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 2.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 4.00% –⁎ –⁎ >7.00c,⁎ 1.81c ± 0.05 0.98b ± 0.07 0.05a ± 0.05 –⁎ –⁎ >7.00c,⁎
Oregano 0.25% 2.02a ± 0.02 1.32a ± 0.35 0.42a ± 0.06 –⁎ –⁎ >7.00b,⁎ 0.96b ± 0.15 0.98c ± 0.19 2.35b ± 0.05
Oregano 0.50% 1.94a ± 0.09 0.93a ± 0.04 0.54a ± 0.11 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 0.75% 1.97b ± 0.10 1.22a ± 0.17 0.33a ± 0.05 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 1.00% 2.10b ± 0.05 1.17a ± 0.12 0.14 ± 0.02 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 2.00% 2.03b ± 0.01 1.30a ± 0.09 0.49a ± 0.12 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 4.00% 1.71c ± 0.09 2.23c ± 0.23 1.85b ± 0.58 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Lemongrass 0.25% 1.95b ± 0.03 1.23b ± 0.08 0.43a ± 0.12 2.09a ± 0.01 1.99a ± 0.05 0.11a ± 0.08 1.99a ± 0.10 2.53b ± 0.16 0.14a ± 0.11
Lemongrass 0.50% 1.99b ± 0.09 1.37b ± 0.16 0.39a ± 0.07 1.97a ± 0.20 1.83a ± 0.11 0.08a ± 0.04 1.88a ± 0.01 2.61b ± 0.06 0.08a ± 0.01
Lemongrass 0.75% 2.10b ± 0.16 1.06b ± 0.22 0.47a ± 0.11 2.04a ± 0.03 1.96a ± 0.08 0.12a ± 0.03 1.93a ± 0.04 2.32b ± 0.22 0.12a ± 0.02
Lemongrass 1.00% 1.99b ± 0.05 1.29b ± 0.42 0.29a ± 0.16 1.88a ± 0.11 1.97a ± 0.12 0.10a ± 0.06 1.97a ± 0.13 2.39b ± 0.14 0.14a ± 0.04
Lemongrass 2.00% 1.92b ± 0.14 1.33b ± 0.26 0.33a ± 0.19 2.44a ± 0.15 1.91a ± 0.03 0.09a ± 0.02 1.74c ± 0.08 2.48b ± 0.05 1.13d ± 0.03
Lemongrass 4.00% 1.76c ± 0.07 1.91d ± 0.22 1.17d ± 0.01 2.21a ± 0.06 3.20c ± 0.32 0.06a ± 0.05 –⁎ –⁎ >7.00c,⁎

Means followed by a different superscript letter within a column for each studied film (amaranth, chitosan or starch) are significantly different (p b 0.05).
+
A: maximum mold growth in the stationary phase; υm: maximum specific growth rate; λ: lag phase.
⁎ No growth.

Chorianopoulos and Kalpoutzakis, 2004). The antimicrobial activity of
oregano EO is due to the presence of both thymol and carvacrol along
with other minor components (Lambert et al., 2001; Sivropoulou et
al., 1996). Inouye et al. (2001), reported concentrations of cinnamalde-
hyde (63%) in cinnamon bark oil as main component, however the oil
from leaves and bark of some cinnamon bushes has eugenol as the
main component (Ayala-Zavala et al., 2009). Chericoni et al. (2005)
reported a similar chemical composition for cinnamon EO as that
obtained in our study. The main component of lemongrass EO is
geranial with similar concentrations as previously reported (Inouye,
2003; Masuda et al., 2008; Schaneberg and Khan, 2002). Although
major active compounds are known to be responsible for the antimicro-
bial activity displayed by EOs, some studies reported that minor
compounds might also have synergistic or additive effects (Tyagi and
Malik, 2011).
We have not found any other reports of fungal inhibition by vapor
contact of EOs incorporated into edible films, however there are many
studies that demonstrated that EOs have antifungal activity at higher
concentrations than those determined here. Inouye et al. (2001,
2006) and Inouye (2003) evaluated the inhibition of a variety of
molds including Aspergillus fumigatus, and A. niger by cinnamon and
lemongrass EOs and mentioned that EOs affected the three stages of
the life cycle of filamentous fungi. A fungicidal effect of cinnamon
and European oregano EOs in Penicillium islandicum and Aspergillus
flavus with concentrations near 0.50% has been reported (López et
al., 2005, 2007). Mixtures of cinnamon and clove EOs against A. flavus
were tested at ranges of 1.00–2.00% and results indicated that the
antifungal activity of EOs depends on the experimental assay utilized
(López et al., 2005, 2007). The inhibitory effects of EOs were greater
in the vapor phase than in a liquid state (Guynot et al., 2003; Matan
et al., 2006; Tullio et al., 2006). Tunc et al. (2007) observed inhibition
of Penicillium notatum with single and binary combination of aroma
compounds of carvacrol and cinnamaldehyde. The effect of thyme
EO vapor phase (with a similar concentration of thymol as that
found in Mexican oregano EO) strongly suppressed the sporulation
of A. niger during 60 days of exposure at concentrations of 1.00%
(Segvic-Klaric et al., 2006). Similar results were observed in the
decrease of growth rate and an increase of lag phase for A. flavus
(Bluma et al., 2009). Tzortzakis (2009) studied the inhibition of
A. niger with cinnamon EO; fungal sporulation was completely
retarded at concentrations of 0.50%. However, at lower concentra-
tions spore germination was accelerated.
Some investigators incorporated EOs from herbs and spices (such
as cinnamon and lemongrass) in active packaging polypropylene
films on bakery products, inhibiting by vapor contact Penicillium
commune and A. niger at 2.00% of cinnamon EO, thus increasing the
product shelf life more than three times (Gutiérrez et al., 2009).
Recently, Du et al. (2009a, 2009b) incorporated allspice, garlic and
oregano EOs in tomato films and demonstrated the inhibition by
vapor phase of E. coli O157:H7, Salmonella enterica, and Listeria mono-
cytogenes. We studied the inhibition of A. niger and Penicillium sp. by
direct contact of amaranth, chitosan, and starch edible films added
with Mexican oregano EO; finding inhibition at 0.50% on starch and
chitosan films (Avila-Sosa et al., 2010b).
The degree of fungal inhibition by vapor contact of different EOs
incorporated into the three studied edible films depended directly
on the type of polymer used to form the film, as different polymers
retain EOs to different degrees. Amaranth films were least effective
as carriers for the tested EOs. Amaranth contains starch (62%) and
proteins (14%); a combination of these components is ideal for the
formation of edible films (Tapia-Blácido et al., 2005, 2007). Given
the presence of different kinds of components in the edible films,
the main compounds of the tested EOs (thymol, carvacrol, eugenol
and geranial) could be interacting with these components, potentially
affecting the antimicrobial activity, thus no significant differences
(p > 0.05) were observed in maximum growth rate and lag phase
values at low concentrations of EOs. In order to counteract this effect,
higher concentrations of EOs must be added to the film, promoting
saturation of the system and thus allowing the presence of free
molecules. Valencia-Chamorro et al. (2008) established that diffusion
effectiveness of an antimicrobial incorporated to an edible film
depends on the polarity of the molecule, its chemical structure, and
the formation of cross-links between molecules, and this would
apply also to volatile compounds. Cagri et al. (2001) reported that
edible films made with proteins, presented retention of cyclic mole-
cules (such as thymol, eugenol, and carvacrol) while Ponce et al.
(2008) observed that chemical interactions between the amino
groups present in casein edible films and carboxyl groups could
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72
block the active antimicrobial sites. In our case, chitosan and starch
films, which have a single type of polymer, retained volatile com-
pounds less effectively and therefore allowed more molecules into
the vapor phase with resultant increase in antimicrobial activities.
This promoted a fungicidal effect at lower concentrations of cinna-
mon and Mexican EOs with long lag phases. Cagri et al. (2004) and
Rhim et al. (2006) reported that these kinds of edible films are able
to disperse different compounds homogeneously. Components that
form the edible films certainly affect film structure as can be observed
for cinnamon and Mexican oregano EOs. At higher concentrations
film thickness increased, so EOs acted as plasticizers. These effects
could favor the antimicrobial activity of incorporated EO and promote
a relatively fast diffusion to vapor phase. Marin et al. (1998) and
Rasooli et al. (2006) suggested that the mode of action on fungal
mycelium of the active and volatile compounds of EOs is due their
lipophilic nature. In general, incorporation of essential oils into edible
films to enable application by vapor phase requires smaller concen-
trations compared with those required for direct application of
EO to inhibit different types of microorganisms (Cagri et al., 2004;
Ponce et al., 2008; Sebti et al., 2005).
Microbial modeling was useful to evaluate fungal inhibition by
volatile compounds of EOs and compare the capabilities of different
fungal spores to colonize food surfaces. Soylu et al. (2007) reported
that exposure to volatile compounds of EOs resulted in alterations
in the hyphal morphology; such modifications may be related to the
effect of EOs in enzymatic reactions regulating wall synthesis. The
lipophilic properties of oil components may also have aided in the
ability of the EO to penetrate plasma membrane.
Chitosan edible films incorporating Mexican oregano or cinnamon
EO can inhibit A. niger and P. digitatum by vapor contact at lower EO
concentrations than those required for amaranth and starch edible
films. Chitosan edible films improved the release of the antimicrobial
compounds of EOs. Applications of these edible films could improve
the quality of foods by the action of volatile compounds on surface
growth of molds.
Acknowledgments
We acknowledge financial support from the National Council for
Science and Technology of Mexico (CONACyT) for the project Combi-
nación de Factores Físicos y Químicos para la inactivación de Microorga-
nismos Relacionados con Alimentos. Author Avila-Sosa gratefully
acknowledges financial support for his PhD studies from CONACyT
and Universidad de las Américas Puebla.
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