Food Research International 44 (2011) 198–203

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Development of edible bioactive coating based on modified chitosan for increasing

the shelf life of strawberries
K.D. Vu a, R.G. Hollingsworth b, E. Leroux a, S. Salmieri a, M. Lacroix a,⁎
a
Institut National de la Recherche Scientifique – Institut Armand-Frappier, Research Laboratories in Sciences Applied to Food, 531, Bd des Prairies, Laval, Québec, H7V 1B7, Canada
b
US Pacific Basin Agricultural Research Center, USDA-ARS, PO Box 4459, Hilo, Hawaii

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

Article history: For increasing the shelf life of strawberries during storage, bioactive coatings were applied using modified
Received 23 August 2010 polysaccharides of chitosan. First, antimicrobial tests were performed with selected essential oils to evaluate
Accepted 22 October 2010 their antimicrobial capacities against moulds and total flora isolated from strawberries. Red thyme (RT) and
oregano extract (OR) were found as strong bioactive agents against moulds and total flora isolated from
Keywords:
strawberries, whereas limonene (LIM) and peppermint (PM) had lower antimicrobial properties. These
Bioactive coating
Chitosan
essential oils were also used as bioactive compounds which were sprayed onto strawberries and evaluated for
Essential oils their potential to increase shelf life during storage at 4 °C. RT, PM and LIM were found to be more efficient
Limonene preservative agents for strawberries during 14 days of storage. Finally, chitosan was functionalized by
Strawberries acylation with palmitoyl chloride to increase its hydrophobicity, to ensure a controlled release and improve its
Shelf life stability and adhesion to the fruit product. LIM and PM were incorporated into the modified chitosan to create
bioactive edible coatings and these were tested for their ability to extend the shelf life of fresh strawberries
during storage. Formulations based on modified chitosan containing LIM and Tween®80 were shown to
perform better than other formulations.
© 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Strawberries are especially perishable fruits, being susceptible to
mechanical injury, desiccation, decay and physiological disorders
during storage. Tournas and Katsoudas (2005) have examined mould
and yeast growth in strawberries in order to isolate and identify the
predominant species infecting fruits following surface disinfection
and incubation at room temperature over 14 days. Botrytis cinerea and
Rhizopus stolonifer were the two major storage pathogens observed in
strawberries. Their high level of contamination, as compared to other
type of fruits, was mainly due to a lower pH, an optimal water activity
for fungal growth, high levels of sugars and other nutrients, and a soft
skin that can be easily ruptured, favoring microorganism proliferation.
B. cinerea (grey mould rot) is a ubiquitous pathogen which causes
severe damage in many fruits, vegetables and ornamental crops both
pre- and post-harvest. The pathogen infects leaves, stems, flowers and
fruits (Bouchra, Achouri, Idrissi Hassani & Hmamouchi, 2003) and is a
major obstacle to long-distance transport and storage (Xu et al.,
2007). R. stolonifer is the causal agent of Rhizopus rot disease of various
fruits and vegetables. This fungus primarily infects ripe fruit only after
harvest unless the fruit in the field has major injuries (Zhang, Zheng,
⁎ Corresponding author. Tel.: + 1 450 687 5010; fax: + 1 450 686 5501.
E-mail address: monique.lacroix@iaf.inrs.ca (M. Lacroix).
Wang, Li & Liu, 2007). Therefore, finding suitable methods for
preserving the quality of strawberries during storage is important.
The use of edible coatings represents one of the important
methods being used for preserving quality. Edible coatings have
been traditionally used to improve food appearance and maintain
quality because they are considered environmentally friendly
(Khwaldia, Perez, Banon, Desobry & Hardy, 2004). Coating films can
act as barriers to moisture and oxygen during processing, handling
and storage (Xu et al., 2007). Moreover, they can retard food
deterioration by inhibiting the growth of microorganisms, due to
their natural intrinsic activity or to the incorporation of antimicrobial
compounds (Cha & Chinnan, 2004). Normally, edible films are made of
proteins or polysaccharides that can also help to maintain moisture,
thereby improving shelf life. However, the hydrophilic nature of these
compounds limits their ability to provide desired edible film
functions. Current approaches to extend functional and mechanical
properties of these films include (i) incorporation of hydrophobic
compounds such as lipids to improve their resistance to water
(McHugh & Krochta, 1994), (ii) optimization of the interaction
between polymers (protein-protein interactions, charge–charge
electrostatic complexes between proteins or polysaccharides) and
(iii) cross-linking or functionalization through physical, chemical, or
enzymatic treatments (Pierro et al., 2006).
Furthermore, it has been demonstrated that bioactive compounds
such as essential oils (EOs) can be added to such coatings in order to
lengthen shelf life, to prevent microorganism growth and to preserve

0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.10.037
 K.D. Vu et al. / Food Research International 44 (2011) 198–203
nutritional values of foods (Salmieri & Lacroix, 2006; Vargas, Albors,
Chiralt & González-Martínez, 2009). EOs have been found to exhibit
antimicrobial and antifungal properties, making them as natural
alternatives to combat foodborne pathogens and normal food decay
caused by bacterial and mould growth (Lacroix, 2007). The immobili-
zation of the active compounds in polymer can maintain high
concentrations of the active compounds on the surface of foods in
order to achieve a longer storage time (Ouattara, Sabato & Lacroix, 2001).
Chitosan is a cationic polysaccharide obtained from partial deace-
tylation of chitin, the main constituent of the crustacean skeleton
(Rinaudo & Domard, 1989). This polymer is non-toxic, biodegradable
and biocompatible (Shapiro & Cohen, 1997), and is also easily modified
by physical or chemical methods (Le Tien, Lacroix, Ispas-Szabo &
Mateescu, 2003). It is widely used in encapsulation applications due to
its ability to form gels in the presence of certain divalent cations such as
calcium, barium and strontium by ionotropic gelation (Braccini & Perez,
2001; Ramadas, Paul, Dileep, Anitha & Sharma, 2000). A new
formulation based on acylation with fatty acid derivatives was
elaborated on chitosan to enhance the hydrophobic properties of the
polymers (Han, Guenier, Salmieri & Lacroix, 2008). Consequently, the
encapsulated substances in chitosan-based beads could be protected
against moisture and the controlled release mechanism was improved
(Han et al., 2008; Le Tien et al., 2003).
Thus, the objective of this study was to develop an active edible
coating formulation in order to increase the shelf life of fresh food
products. Strawberries were used as a model for the application of edible
coatings. First, an inhibitory in vitro test (antimicrobial assay) was
performed with selected antifungal natural compounds in order to
evaluate their antimicrobial capacities against moulds and total flora
isolated from strawberries. Second, antimicrobial compounds were
evaluated as emulsion-based coatings for their specific antifungal
properties on fresh strawberries. Finally, functionalized polymer of
chitosan was synthesized. Then, the antimicrobial coatings using
functionalized chitosan, containing limonene and peppermint in
presence of an emulsifier was developed to increase the shelf life of
strawberries.
2. Materials and methods
2.1. Materials
Fresh strawberries were bought from a local supermarket (IGA,
Laval, Quebec, Canada) and were inspected for no sign of disease and
visual decay.
Limonene (LIM) was from Sigma-Aldrich Ltd. (Oakville, Ontario,
Canada). Oregano (OR; Oreganum compactum) essential oil was from
Robert & Fils (Montreal, QC, Canada). Red thyme (RT; Thymus
vulgaris), peppermint (PM; Mentha piperita) and lemongrass (LG;
Cymbopogon citrates) essential oils were from BSA (Montreal, QC,
Canada).
Chitosan (Kitomer™, Mw 1600 kDa, 85–89% deacetylation degree)
was obtained from Marinard Biotech Inc. (Rivière-aux-Renards, QC,
Canada). Palmitoyl chloride was from Fluka Biochemika (Buchs,
Switzerland). The other reagents were purchased from Laboratoire
MAT (Beauport, QC, Canada) and used without further purification.
2.2. Evaluation of growth inhibition of antimicrobial compounds on total
flora and moulds by antimicrobial assay
An antimicrobial assay was performed on selected antifungal
agents to evaluate their effect on the growth of total flora and moulds
isolated from strawberries.
2.2.1. Isolation of total flora and moulds
Isolation of total moulds: in sterile conditions, 25 g of fresh
strawberries (no sign of disease and visual decay) was placed in a
199
bag containing 225 ml of sterile peptoned water and homogenized for
2 min using a Lab blender 400 stomacher (Laboratory Equipment,
London, UK). The obtained mixture was serially diluted, then plated
on acidified Potato Dextrose Agar (PDA, pH 3.5) and incubated at
25 °C for 72 h. Mould colonies were isolated and inoculated in a flask
containing 36 ml of sterilized Potato Dextrose Broth (PDB), then, the
flask was incubated at 25 °C for 48 h. At the end of incubation, sterile
glycerol was added into the flask to obtain a final glycerol
concentration of 20% (w/v) and aliquots of 1 ml of the mixture were
divided into cryovials and frozen at -80 °C for future utilization.
Isolation of total flora: in sterile conditions, 25 g of fresh
strawberries (no sign of disease and visual decay) was placed in a
bag containing 225 ml of sterile peptoned water and homogenized for
6 min using a stomacher. A volume of 1 ml of homogenized mixture
was inoculated in a flask containing 36 ml of sterilized Tryptic Soy
Broth (TSB) and incubated at 37 °C for 24 h. At the end of incubation,
sterile glycerol was added into the flask to achieve a final glycerol
concentration of 20% (w/v) and aliquots of 1 ml of the mixture were
divided into cryovials and frozen at − 80 °C for future utilization.
Before each experiment, stock cultures were propagated through 2
consecutive growth cycles: (i) in PDB for total moulds at 25 °C for 72 h
and (ii) in TSB for total flora at 37 °C for 24 h. The fermented broths
were centrifuged at 1500×g for 10 min at 4 °C. The obtained pellets
were washed twice with sterile saline water (0.85%, w/v) and then
diluted to have working cultures with approximately 108 CFU/ml.
2.2.2. Antimicrobial assay
LIM, OR, RT, PM, and LG were used to evaluate their inhibition
potential against the growth of total flora and total moulds by paper
disk method (Bona da Silva, Guterres, Weisheimer & Schapoval,
2008). Tryptic soy Agar (TSA) and acidified PDA plates were
thoroughly inoculated with the working cultures of total flora and
total moulds. A paper disk was placed in the middle of the plate except
for the control plates which received no treatment. A volume of 5 μl of
antifungal agent (0.02%, w/v) was then added to the paper disk and
incubated at 37 °C for 24 in case of TSA plates and at 25 °C for 48 h in
case of acidified PDA plates. The test was conducted in triplicate for
each sample. The test ended when the control plates were fully
covered by bacteria or moulds and the inhibition radius of paper disk
was measured and expressed as mm of inhibition.
2.3. Evaluation of antimicrobial compounds on the shelf life of strawberries
A shelf life study was conducted to verify the effect of selected
antifungal compounds (LIM, LG, PM, RT and OR) on the growth of
moulds (R. Stolonifer and B. cinerea) on strawberries during storage at
4 °C. Prior to use, the essential oils were passed through a 0.2 μm filter
to ensure sterility. Concentrations of 0.2% essential oil with 0.4%
Tween®80 were used to prepare coating emulsions in aqueous phase
under vigorous stirring using an IKA® T25 digital Ultra-Turrax
disperser (IKA® Works Inc.) for 10 min at room temperature to
obtain a final concentration of 0.02% antifungal compounds.
The strawberries were separated into 6 groups with 20 strawberries
per group: (i) control (uncoated); (ii) coated with LIM; (iii) coated with
LG; (iv) coated with OR; (v) coated with PM; and (vi) coated with RT.
These groups were placed on sterile aluminum foil-covered trays. The
coatings suspensions were sprayed onto fresh strawberries in sterile
conditions (under a biological containment hood) at room temperature.
Each side of each strawberry was sprayed twice (fully cover by coating
suspensions). After spraying, strawberries were allowed to dry for
15 min on sterile aluminum sheets. Then, the strawberries were covered
using a second sterile aluminum sheet and were stored at 4 °C. Visual
decay of strawberries was evaluated for 14 days (day 0 corresponded to
the day of the treatment) and the mould contamination percentage
(defined as the percentage of strawberries that showed the presence of
200 K.D. Vu et al. / Food Research International 44 (2011) 198–203
one or more white colonies (R. stolonifer) or dark brown multi-celled
colonies (B. cinerea)) was calculated during storage.
2.4. Evaluation of polymer bioactive coatings on the shelf life of strawberries
2.4.1. Synthesis of modified chitosan
Polymer modification (N-acylation of chitosan) was prepared
according to a method developed in our laboratory. An amount of 5 g
of chitosan was dissolved in 600 ml of aqueous acetic acid (0.12 M, pH
4), respectively, at room temperature. The mixture was stirred
overnight to ensure a complete solubility. Thereafter, the pH of solutions
was adjusted to 7.5 by slow addition of 0.1 M NaOH and the volumes
were adjusted to about 800 ml. The acylation reactions were carried out
by adding palmitoyl chloride (d = 0.907 g/ml) to polymer solution in a
ratio of 1:2 (w/w), maintaining the pH at 7.5 with NaOH 0.5 M, and
temperature at 55 °C. After 1 h, the reaction mixture was neutralized
(pH 6.8–7.0) and precipitated with acetone. The precipitate, collected by
filtration, was washed at 50 °C with an excess of methanol and decanted.
The washing was repeated twice to eliminate free fatty acids. Finally, the
modified product was dried with pure acetone to obtain the
corresponding functionalized polymer powder (N-acylated chitosan)
(Han et al., 2008).
2.4.2. Shelf life study of strawberries coatings using modified chitosan-based
films
A shelf life study of strawberries by bioactive coatings was
conducted where limonene (LIM) and peppermint (PM) were
compared as antimicrobial agents. First, the polymer powder of MC
was autoclaved at 121 °C for 40 min to ensure sterility, then; the
aqueous suspension was prepared by solubilizing 2% of polymer into
3% acetic acid solution under vigorous stirring for 2 h at room
temperature. The bioactive coating suspensions were prepared by
dispersing 0.2% (w/v) bioactive compounds (LIM or PM) in the
presence of 0.4% Tween®80 under vigorous stirring for 10 min at
room temperature to obtain a final concentration of bioactive
compounds at 0.02 %, w/v. For visual appearance improvements of
coatings, polymer suspension MC were adjusted to pH 3.5 (pH of
strawberries) by 10% acetic acid solution before spraying.
The strawberries were separated into 3 groups with 20 strawberries
per group: (i) control (uncoated) and (ii) coated with MC+ LIM; (iii)
coated with MC + PM. Coating of strawberries by spraying suspensions
on strawberries was conducted using the same method mentioned
above. During storage at 4 °C, visual decay of strawberries was evaluated
and the mould contamination percentage was calculated.
2.6. Statistical analysis
For each measurement, three replicates were tested. For each
replicate, 20 strawberries were used. Analysis of variance and
Duncan's multiple-range tests were used to perform statistical
analysis on all results, using SPSS Base 10.0 software (Stat-Packets
statistical analysis software, SPSS Inc., Chicago, IL). Differences
between means were considered to be significant when p ≤ 0.05.
3. Results and discussion
3.1. Antimicrobial activity of selected essential oils against total flora and
moulds isolated from strawberries
3.1.1. Antimicrobial activity against total flora
The inhibition diameter (mm) of selected EOs in response to total
flora isolated from strawberries is presented in Table 1. RT and OR can
be classified as strong antimicrobial with respective inhibition
diameters of 43.3 and 32.3 mm whereas LG and PM can be considered
as medium antimicrobials (20.5 and 19.5 mm in inhibition diameter,
Table 1
Antimicrobial activity against total flora and moulds isolated from strawberries.
Flora Essential oils
Limonene Lemongrass Oregano Peppermint Red thyme
(LIM) (LG) (OR) (PM) (RT)
Diameter of inhibition zone (mm)a
Total 0.0e 20.5 ± 1.2d 32.3 ± 1.5c 19.5 ± 1.4d 43.3 ± 2.4b
flora
Moulds 15.5 ± 0.7e 46.3 ± 2.1c 56.0 ± 2.0b 22.3 ± 2.0d 57.5 ± 2.2b
a
The presented values are mean ± standard deviation. In each row, mean followed by
the different lower case letter between different essential oils are significantly different
(p ≤ 0.05).
respectively).However, LIM exhibited no antimicrobial activity to
total flora (Table 1).
In fact, it has been demonstrated that RT, OR, LG, and PM possess
antimicrobial activity against different microbial targets; however, this
is the first published report on their potential as antimicrobials against
total flora isolated from strawberries. For example, Skandamis, Tsigarida
and Nychas (2000) found that OR at a concentration of 0.03% (w/v)
causes growth inhibition against Salmonella typhimurium in liquid
culture and in a gel matrix system (gelatine gel). Importantly, it was
found that OR in liquid culture is more efficient than gel matrix
(Skandamis et al., 2000). Mahboubi and Haghi (2008) analyzed the
components of the essential oil extract from Mentha pulegium L. (a near
species of peppermint M. piperita L.) and they found the presence of
piperitone (38.0%), piperitenone (33.0%), terpineol (4.7%), and pule-
gone (2.3%) as the major components. The results showed a significant
activity against microorganisms, especially Gram-positive bacteria, with
inhibition zones and minimal inhibitory concentration values in the
range of 8–21 mm and 0.25–4 μl/ml, respectively; Their study found
that the least susceptible microorganisms were Gram-negative bacteria
especially Escherichia coli. Moreover, instead of using PM essential oil,
other authors have used different extracts (by different solvents) of the
leaves of M. piperita L. (peppermint) and they also observed that these
extracts have strong antibacterial activity against a range of pathogenic
bacteria using in vitro agar well diffusion method (Bupesh et al., 2007).
Among different leaf extracts of M. piperita, ethyl acetate extracts
showed greater inhibition than leaf extracts by chloroform, petroleum
ether and water, when tested against Bacillus subtilis, Pseudomonas
aerogenosa than Streptococcus aureus, Pseudomonas aureus and Serratia
marcesens (Bupesh et al., 2007).
In case of LG, this essential oil has been confirmed to have stronger
antibacterial activity against respiratory tract pathogens such as
Haemophilus influenzae, Steptococcus pneumoniae, Streptococcus pyop-
genes and Staphylococcus aureus as compared to limonene (Inouye,
Takizawa & Yamaguchi, 2001). In this research, the authors found that
a high concentration of LIM is required (from 200 to 800 mg/L air) to
have antibacterial activity against the target bacteria (Inouye et al.,
2001).
It should also be mentioned that our results which confirmed that
RT is more effective than PM in terms of antimicrobial activity
(Table 1) are in accordance with the research of Janssen, Chin, Scheffer
and Baerheim Svendsen (1986). For example, the authors tested 53
EOs against 5 microorganisms and they found that thyme oil was
better than peppermint oil in terms of growth inhibition against
bacteria and yeast such as E. coli (19.3 N 9.7 mm); P. aeruginosai
(8.7 N 0 mm); C. albicans (40.7 N 10 mm); B. subtilis (33.2 N 14.7 mm)
and S. aureus (33.7 N 19.0 mm) (Janssen et al., 1986).
3.1.2. Antifungal activity against total moulds
The diameter of inhibition zone (mm) of selected EOs in response
to total moulds isolated from strawberries is presented in Table 1. RT,
OR and LG can be classified as strong antifungal agents with inhibition
diameters of 57.5, 56.0 and 46.3 mm, respectively. In comparison, PM
 K.D. Vu et al. / Food Research International 44 (2011) 198–203
and LIM presented a medium inhibition diameter against moulds with
only 22.3 and 15.5 mm of inhibition, respectively. There have been
many studies on the antifungal activity of EOs and the results of
different studies are difficult to compare due to: (i) different methods
of antifungal assay (disk diffusion technique, semisolid agar antifun-
gal susceptibility technique); (2) different concentrations of EOs used
in assays and (iii) different types of fungi tested and (iv) the type and
consistency of the medium which may affect the degree and rate of
diffusion of the essential oil from the disk. However, the results
obtained in our research are in general agreement with the results of
other researchers in terms of antifungal activity of RT, OR, LG, PM and
LIM (Bouchra et al., 2003; Curtis, Shetty, Cassagnol & Peleg, 1996; Kim,
Marshall & Wei, 1995; Reddy, Angers, Gosselin & Arul, 1998). For
example, RT has already been used successfully to inhibit both B.
cinerea and R. stolonifer in strawberry fruits (Reddy et al., 1998).
Reddy et al. (1998) showed that thyme essential oil (EO) from T.
vulgaris at a concentration of 200 ppm exhibited strong antifungal
activity against B. cinerea and R. stolonifer, with respective inhibition
values of 90% and 66% by measuring the percentage of radial growth
inhibition of moulds in PDA medium. The major constituents of thyme
oil are p-cymene (20.8%), thymol (18.1%), linalool (13.3%) and
carvacrol (8.9%) and it has already been suggested that p-cymene
and linalool may synergize the antimicrobial effect of thymol and
carvacrol (Curtis et al., 1996; Kim et al., 1995). In case of oregano
essential oil (OR), it has been confirmed as effective against B. cinerea,
exhibiting 100% inhibition activity at 100 ppm (Bouchra et al., 2003).
Using a disk diffusion assay, Bona da Silva et al. (2008) found that
applying 8 μl of LG per disk (6 mm in diameter) resulted in a
significant inhibition zone against different species of Candida such as
C. glabrata (N 30 mm), C. krusei (19.6 mm), C. parapsilosis (28.6 mm),
C. tropicalis (29.5 mm) and 4 strains of C. albicans (N 35 mm). Also, LIM
used at a concentration of 75 ppm resulted in an antifungal activity of
75% against Fusarium verticillioides MRC 826 (a plant pathogen)
compared to the control, using the semisolid agar antifungal
susceptibility technique (Dambolena et al., 2008). It is interesting
that LIM at a concentration of 500 ppm can totally inhibit the growth
of Aspergillus flavus, a carcinogenic fungus producing aflatoxin (Singh
et al., 2010). In the case of PM, it was demonstrated that PM at a dose
of 30 μl/400 ml air space can inhibit totally the growth of R. stolonifer,
Mucor sp. and Sclerotinia sclerotiorum (Edris & Farrag, 2003).
Table 1 also shows that LIM, LG, OR, PM and RT are more active
against moulds than total flora. The results are in accordance with
other researchers who also demonstrated that some EOs are more
active against fungi than Gram positive and Gram negative bacteria
(Chao, Young & Oberg, 2000; Janssen et al., 1986).
3.2. Evaluation of antimicrobial compounds on the shelf life of strawberries
Based on the above results of antimicrobial activity of EOs against
moulds, it is necessary to know if these compounds can increase the
shelf live of storage strawberries by prevention of mould growth or
not. Thus, these EOs were used as bioactive compounds for coating
strawberries in a shelf life study. The effect of antifungal compound
applications on the percentage of decay in strawberries during
14 days of storage at 4 °C is presented in Fig. 1.
From day 1 to day 6, the decay levels (%) of strawberries treated with
LIM, OR, PM and RT were significantly less than that of control sample
(20%) (p ≤ 0.05); however, at day 8 the decay level of strawberries
treated with LG (65%) was higher than that of control (25%) (p ≤ 0.05)
(Fig. 1). At day 12, the decay levels (%) of strawberries treated with LIM
(40%), PM (30%) and RT (30%) were still significantly less than that of
control (50%) (p ≤ 0.05); however, the decay levels of strawberries
treated with LG (80%) and OR (60%) were higher than that of control
(50%) (p ≤ 0.05) (Fig. 1). It can be concluded that LIM, PM and RT were
efficient for preserving the quality of strawberries for 12 days. RT can be
considered the most efficient antifungal since it had a lower decay level
201
Fig. 1. Evolution of the decay level (%) in antimicrobial coated strawberries during
storage at 4 °C.
as compared to the control and other treatments. The higher decay level
observed for OR and LG treated strawberries suggests that these
essential oils had detrimental effects on the quality of strawberries after
6 and 8 days of storage, respectively (Fig. 1). It is also supposed that the
components that constitute LG and OR essential oils are more volatile
than that of other essential oils (LIM, PM and RT) which may cause LG
and QR to lose their antifungal activity against B. cinerea and R. stolonifer.
Moreover, it is interesting to note that regardless of the applied
treatments the rate of decay increased significantly from day 12 in all
groups (Fig. 1).
RT demonstrated high antifungal activity both in the antimicrobial
assay and in the shelf life test. However, in the case of the other
essential oils, the results of shelf life study are different to some extent
as compared to those of the antimicrobial assay. For example, PM and
LIM were found to be relatively ineffective as antifungal or
antimicrobial agents against total flora and moulds isolated from
strawberries in the antimicrobial assay; however, they were slightly
effective when applied as coating materials of strawberries in the shelf
life study.
Furthermore, it should be mentioned that a sensory evaluation
was done on EO sprayed strawberries on the same day of treatment
using Hedonic test with 20 panelists who tested the odor and taste
parameters. The answers were based on a 9-point hedonic scale, 9
being “like very much” and 1 “dislike very much” (Larmond, 1979). It
was found that only LIM and PM coated strawberries have acceptable
taste (6.3 and 7.2 points, respectively) and odor (6.0 and 6.5 points,
respectively) while RT coated strawberries obtained low points in
taste (2.5 points) and odor (3.0 points). Therefore, we decided to
choose only LIM and PM for further research in which these EOs will
be incorporated into modified chitosan as coating materials of
strawberries for increasing the shelf life of strawberries during
storage.
3.3. Evaluation of bioactive coatings on the shelf life of strawberries
After the modified chitosan (MC) was first synthesized, the
functionalization of the MC was characterized by FTIR structural analysis
(data not shown). It demonstrated that MC exhibited the changes in
band intensities that were correlated to a chemical modification in the
presence of palmitoyl chains linked to the polymers by acylation. The
results are also in accordance with our previous publication (Han et al.,
2008).
202 K.D. Vu et al. / Food Research International 44 (2011) 198–203

3.3.1. Shelf life study with modified chitosan-based coating

The effect of a MC-based bioactive coating containing LIM and PM on
the decay percentage of strawberries during 14 days of storage at 4 °C is
presented in Fig. 2. The coatings were developed using 0.4% Tween®80
as an emulsifier to improve the stability of the coating suspension.
Coatings were sprayed without neutralization (pH~3.5–4.0) to ensure
that MC would remain dissolved during drying. Generally the percentage
of strawberries showing decay was lower in the MC+LIM-based
coatings at most time points during the experiment in comparison
with MC or MC+PM-based coatings and uncoated strawberries. On Day
8 for example, 45% of fruits had some level of decay in the MC+LIM
treatment while the percentage was 65, 75 and 90% of decay for
MC-based coating, MC+PM-based coating and uncoated strawberries,
respectively. The MC+LIM-based coating can be considered as a more
effective coating since its related percentage of decay was lower than the
control and other coatings on days 8, 10 and 12. Moreover, it is
interesting to note that the coating did not cause phytotoxicity and did
not affect the appearance of strawberries, probably due to the presence
of an emulsifier and to pH conditions (pH~3.5–4.0) that improved the
solubilization of modified chitosan. Furthermore, pictures of three
Fig. 3. Appearance of strawberries coated with modified chitosan-based formulation
representative fruits from each treatment (uncoated strawberries at containing limonene and emulsifier.
day 10 of storage and coated strawberries by MC+LIM formulation at
day 16 and day 21 of storage) were taken. The pictured fruits are
presented in Fig. 3. It can be easily observed that uncoated strawberries
are dehydrated and are largely contaminated by moulds at day 10
Administration as a GRAS (Generally Recognized as Safe) compound
whereas coated strawberries kept a good hydrated, red-colored
when used as a food additive or flavoring (USEPA, 1994). LIM is also
appearance even at day 21, which implies a promising attribute for
known to have fungicidal properties, including activity against Botrytis,
consumption.
Aspergilus niger, and Candida albicans (Ernestina et al., 2003; Jirovetz et al.,
It is not known why in PM incorporated into MC for coating
2003; Sharma & Tripathi, 2008; Wilson et al., 1997). Therefore, it is
strawberries, there was no effect on increasing the shelf life of
reasonable that MC+LIM-based coatings have significant impact on
strawberries as compared to control. It is possible that there might be
increasing the shelf life of storage strawberries.
a reaction that occurred between MC and PM during the preparation
of coating suspension which caused the loss of antifungal activity of
PM. Future research on this problem needs to be done to find out the 4. Conclusion
suitable reasons.
In fact, chitosan-based coatings are known to possess a protective Different selected essential oils were evaluated for their antimi-
effect against mould growth on fresh fruits and vegetables. According crobial capacities against moulds and total flora isolated from
to Mazur and Waksmundzka (2001), the application of chitosan strawberries. RT and OR were found as strong bioactive agents against
aqueous solutions on strawberries had a protective effect and reduces moulds and total flora isolated from strawberries, whereas LIM and
the decay of strawberry fruits by 15%. However, the study on coating PM had lower antimicrobial properties. RT, PM and LIM were found to
of strawberries by MC + LIM for increasing the shelf life of storage be the most efficient preservative agents in strawberries during
strawberries is not yet mentioned elsewhere. storage. Formulations based on modified chitosan containing LIM in
Furthermore, it should be mentioned that LIM has been widely used in the presence of Tween®80 provided a better preservation than other
a variety of foods and beverages and is classified by the U.S. Food and Drug formulations. Therefore, the functionalized chitosan-based edible
coating could become a promising method to carry specific antifungal
agents without detrimental effects on strawberries or other fruits. In
this context, future research will focus on developing a new coating
formulation which consists of MC, lipid (fatty acids, acetylated
monoglycerides, etc.), plasticizers (polyols) and reinforcement agents
(nanocellulose) to improve the functional properties of coatings
applied on strawberries and increase the shelf life of strawberries
during storage.

Acknowledgments

The authors are grateful to Ministère du Développement Économique,

de l'Innovation et de l'Exportation, Programme de Soutien à la Recherche,
Soutien à des Initiatives Internationales de Recherche et d'Innovation for
the financial support. Asmaâ Imzilne is thanked for her excellent technical
support.

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