Food Microbiology 32 (2012) 345e353

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Efficacy of the application of a coating composed of chitosan and Origanum

vulgare L. essential oil to control Rhizopus stolonifer and Aspergillus niger in grapes
(Vitis labrusca L.)
Nereide Serafim Timóteo dos Santos a, Ana Júlia Alves Athayde Aguiar a,
Carlos Eduardo Vasconcelos de Oliveira a, Camila Veríssimo de Sales a, Silvanda de Melo e Silva b,
Rosana Sousa da Silva b, Thayza Christina Montenegro Stamford c, Evandro Leite de Souza a, *
a
Laboratory of Food Microbiology, Department of Nutrition, Health Science Center, Federal University of Paraíba, João Pessoa, Brazil
b
Laboratório of Biology and Post-harvest Technology, Agricultural Sciences Center, Federal University of Paraíba, Areia, Brazil
c
Laboratory of Microbiology, Department of Physiology and Pathology, Health Science Center, Federal University of Paraíba, João Pessoa, Brazil

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

Article history: This study evaluated the efficacy of the combined application of chitosan (CHI) and Origanum vulgare L.
Received 23 April 2012 essential oil (OV) in the inhibition of Rhizopus stolonifer URM 3728 and Aspergillus niger URM 5842 on
Received in revised form laboratory media and on grapes (Vitis labrusca L.) and its influence on the physical, physicochemical and
8 June 2012
sensory characteristics of the fruits during storage (25
C, 12 days and 12
C, 24 days). The application of
Accepted 27 July 2012
Available online 7 August 2012
mixtures of different CHI and OV concentrations (Minimum Inhibitory Concentration e MIC, 1/2 MIC and
1/4 MIC) inhibited the mycelial growth of the test fungi. The application of CHI and OV at sub-inhibitory
concentrations (CHI 1/2 MIC þ OV 1/4 MIC; CHI 1/2 MIC þ OV 1/2 MIC) inhibited spore germination and
Keywords:
Essential oil
caused morphological changes in fungal spores and mycelia, in addition to inhibiting the growth of the
Chitosan coating assayed fungi strains in artificially infected grapes as well as the autochthonous mycoflora of grapes
Grapes stored at both room and cold temperature. In general, the application of a coating composed of CHI and
Post-harvest decay OV at sub-inhibitory concentrations preserved the quality of grapes as measured by their physical and
physicochemical attributes, while some of their sensory attributes improved throughout the assessed
storage time. These results demonstrate the potential of the combination of CHI and OV at sub-inhibitory
concentrations to control post-harvest pathogenic fungi in fruits, in particular, R. stolonifer and A. niger in
grapes.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Chitosan, a linear polysaccharide derived from chitin deacety-

The grape is a highly perishable non-climacteric fruit with
reduced shelf-life due to a loss of firmness, berry drop, stem
discoloration, desiccation and fungal rot (Meng et al., 2008). The
reduction of losses resulting from fungal rot is a major goal of post-
harvest technology for table grapes, which seeks to use safe and
effective methods to control contamination and the growth of
spoiling fungi (Martínez-Romero et al., 2008).
* Corresponding author. Universidade Federal da Paraíba, Centro de Ciências da
Saúde, Departamento de Nutrição, Campus I, Cidade Universitária, João Pessoa,
Paraíba 58051-900, Brazil. Tel.: þ55 83 3216 7807; fax: þ55 83 3216 7094.
E-mail address: evandroleitesouza@ccs.ufpb.br (E.L. de Souza).
lation, is considered a non-toxic, biodegradable, functional,
biocompatible product with antimicrobial properties (Sathivel
et al., 2007; Badawy and Rabea, 2009). Coatings obtained from
a chitosan dispersion mixed with other active substances that are
applied as a coating material have shown promising results in
inhibiting the growth and survival of microorganisms on foods
(Chen et al., 2002).
Some studies have demonstrated an interesting inhibitory effect
of essential oils and their constituents on food-related spoiling and
pathogenic microorganisms (Souza et al., 2007; Azêredo et al.,
2011; Sousa et al., 2012). Among these, Origanum vulgare L. essen-
tial oil (oregano) has emerged as a growth inhibitor of different
fungal species, including post-harvest pathogens (Camele et al.,
2010; Mitchell et al., 2010). The antimicrobial properties of
O. vulgare essential oil have been attributed to the presence of

0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fm.2012.07.014
346 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353
phenolic compounds, in particular, thymol and carvacrol (Oliveira
et al., 2009).
The addition of essential oils to edible coating can extend the
shelf-life of foods due to increased and prolonged antimicrobial
activity because essential oil compounds are continuously released
over time on the product surface while maintaining a suitable
concentration of antimicrobial components during the storage
period (Outtara et al., 2000). However, the preparation of coating
containing essential oils requires the use of emulsifying agents to
ensure dispersion, casting and formation of a homogeneous coating
(Bonilla et al., 2012).
The incorporation of essential oils in chitosan coating is possible
because chitosan possesses emulsifying properties, which permit
the homogeneous distribution of essential oil droplets in the
system, often enabling the formation of a thin and translucent
coating (Sánchez-Gonzáles et al., 2010a). However, increasing
concentrations of added essential oil in the dispersion reduce the
apparent viscosity of the coating solution by decreasing the
oilewater interaction necessary for emulsion formation, necessi-
tating the addition of plasticizers to disperse the essential oil
molecules and increase the apparent viscosity of the coating solu-
tion (Sánchez-Gonzáles et al., 2010b). The use of glycerol as
a plasticizer enables the formation of coating solutions with satis-
factory viscosity and the formation of flexible coatings from the
emulsion, increasing their potential for application as a coating to
the surface of foods (Pereda et al., 2012).
The present study evaluated the efficacy of the combined
application of chitosan and O. vulgare essential oil to inhibit the
post-harvest fungal pathogens Rhizopus stolonifer and Aspergillus
niger in laboratory media and in the table grape cultivar Isabella.
The effect of the combined application of the compounds tested on
the physical, physicochemical and sensory characteristics of the
fruits during storage at room and cold temperatures was also
assessed.
2. Materials and methods
2.1. Materials
Chitosan (CHI) of medium molecular weight (deacetylation
degree 75e85%, batch 03318AJ) was obtained from Sigma-Aldrich
Co. (St. Louis, USA). O. vulgare essential oil [OV (Batch OREORG01,
density at 20
C: 0.90, refractive index at 20
C: 1.47)] was supplied
by Aromalândia Ind. Com. Ltda. (Minas Gerais, Brazil) and its quality
parameters were described in a technical report issued by the
supplier.
Table grapes cv. Isabella (Vitis labrusca L.) in commercial matu-
ration stage were obtained from EMPASA (Supplies and Services
Company of Paraiba, João Pessoa, Brazil); grapes without signs of
mechanical damage and deterioration were selected and stan-
dardized in small bunches of grapes showing homogeneous size,
color and form.
A. niger URM 5162 and R. stolonifer URM 3482 were obtained
from Culture Collection URM (Center for Biological Sciences, Federal
University of Pernambuco, Recife, Brazil) for use as test microor-
ganisms. For antifungal assays, stock cultures were subcultured in
Sabouraud agar (Himedia, India) at 25e28
C for seven 7 for suffi-
cient sporulation. The fungal spores were collected by adding sterile
saline solution (0.85 g/100 mL NaCl) in fungal growth medium and
filtering the resulting suspension through a sterile triple-gauze
layer to retain hyphal fragments. The number of spores present in
the suspension was determined with a hemocytometer. The spore
concentration obtained was adjusted with sterile saline solution to
provide a fungal inoculum of approximately 106 spores/mL (Rasooli
and Abyaneh, 2004; Rasooli and Owlia, 2005).
2.2. Production of CHI and OV solutions and coating for application
on grapes
CHI solutions were obtained by dissolving the polymer (20 mg/
mL) in acetic acid (1 mL/100 mL) for 24 h at room temperature with
stirring (120 rpm) (Sánchez-Gonzáles et al., 2011). Successive serial
dilutions (1:1) were performed in Sabouraud broth (Himedia,
India) to obtain solutions of different concentrations (10, 5, 2.5, 1.25,
0.06 mg/mL). OV solutions were obtained by dissolving the
substance (80 mL/mL) in Sabouraud broth containing bacteriological
agar [0.15 g/100 mL (Himedia, India)] as a stabilizing agent (Bennis
et al., 2004), with successive dilutions (1:1) in the same broth to
obtain solutions of different concentrations (40, 20, 10, 5, 2.5, 1.25,
0.06 mL/mL).
For the combined application of CHI and OV, CHI (10 or 5 mg/
mL) was initially diluted in acetic acid (1 mL/100 mL) with constant
stirring (120 rpm) for 6 h at room temperature. Then, different OV
concentrations were added (5, 2.5, 1.25 mL/mL), followed by stirring
for an additional 18 h at room temperature. To assay the application
of the solution formed by combining CHI and OV as coating in fruits,
glycerol was added (2 mL/100 mL) as a plasticizer as OV was
incorporated into the coating solution (Ojagh et al., 2010).
2.3. Determination of the minimum inhibitory concentration (MIC)
The MIC of CHI and OV was determined by the macrodilution in
broth technique. Initially, 1 mL of fungal suspension was inoculated
in 4 mL of Sabouraud broth (with concentration adjusted to 10 mL)
and 5 mL of the solutions containing different concentrations of CHI
or OV (with concentration adjusted to 10 mL) were added. The
system was incubated at 25e28
C for 7 days, and at the end of the
incubation period, the lowest CHI and OV concentration (highest
dilution) that exhibited no visible fungal growth was considered
the MIC (Sharma and Tripathi, 2008).
Based on the MIC, different CHI and OV concentrations were
chosen (CHI 10 mg/mL þ OV 5 mL/mL; CHI 10 mg/mL þ OV 2.5 mL/
mL; CHI 10 mg/mL þ OV 1.25 mL/mL; CHI 5 mg/mL þ OV 5 mL/mL;
CHI 5 mg/mL þ OV 2.5 mL/mL; CHI 5 mg/mL þ OV 1.25 mL/mL) for
use in combination in mycelial mass quantification and spore
germination assays.
2.4. Effects on fungal mycelial growth
Inhibition of fungal mycelial growth (dry mass weight) was
determined with the poisoned growth substrate technique (dilu-
tion in broth) by inoculating 10 mL of each fungal suspension
(approximately 106 spores/mL) in 90 mL of Sabouraud broth sup-
plemented with different CHI and OV concentrations and incubated
at 25e28
C. After 3, 5 and 7 days of exposure, the mycelial mass
obtained was dried in an oven at 60
C for 6 h and kept at 40
C for
an additional 18 h (Rasooli and Abyaneh, 2004; Rasooli et al., 2006).
In the control experiment, the fungal suspension was inoculated in
Sabouraud broth without CHI and OV. By comparing the weights of
the dry mycelial masses from the suspension treated with CHI and
OV and the test control, the percentage inhibition of the fungal
mycelial mass at the different time intervals was obtained.
2.5. Effects on fungal spore germination
Aliquots of 0.1 mL of each solution containing different CHI and
OV concentrations were mixed with 0.1 mL of each fungal spore
suspension (approximately 106 spores/mL) obtained from cultures
grown for 10 days on Sabouraud agar at 25e28
C. Then, 0.1 mL of the
system was placed at the center of a sterile glass slide and incubated
in a moist chamber at 25e28
C for 24 h. After this period, each slide
 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353
was fixed with lactophenol cotton blue stain, and spore germination
was observed by light microscopy. Approximately 200 spores were
counted on each slide. The efficacy of spore germination inhibition
was evaluated by comparing the number of germinated spores in
the media supplemented with CHI and OV with those obtained in
the control assay, in which the solutions of CHI and OV were
replaced by Sabouraud broth (Feng and Zeng, 2007).
The results of the mycelial mass quantification and spore
germination assays were used to select the different CHI and
OV concentrations used in the fungal morphogenesis tests
(CHI 5 mg/mL þ OV 5 mL/mL, CHI 5 mg/mL þ OV 2.5 mL/mL, CHI
5 mg/mL þ OV 1.25 mL/mL) and for application on grapes
(CHIOV5  CHI 5 mg/mL þ OV 5 mL/mL þ glycerol 2 mL/100 mL; and
CHIOV2.5  CHI 5 mg/mL þ OV 2.5 mL/mL þ glycerol 2 mL/100 mL).
2.6. Effects on the morphogenesis of fungal strains
To analyze morphological changes, samples of mycelia and
spores were taken from test strains grown on Sabouraud broth
(48 h, 25
C) and Sabouraud agar (7 days, 25
C), respectively, in the
presence of different CHI and OV concentrations (Rasooli and
Owlia, 2005; Rasooli et al., 2006). The samples were washed in
buffered saline solution (pH 7.2) three times for 10 min each and
fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at
28
C. After fixation, the samples were washed with 0.1 M caco-
dylate buffer three times for 10 min each, post-fixed with 2%
osmium in 0.1 M cacodylate buffer, washed twice for 10 min each
with 0.1 M cacodylate buffer and washed once with water for
10 min. The samples were dehydrated with increasing percentages
of ethanol (30%, 50%, 70%, 90%, 15 min for each wash) up to 100%
(three times, 15 min for each wash), dried at the critical point for 2 h
and mounted in Stubbs (carbon ribbon and silver ink) for 1 h.
Subsequently, the samples were coated with gold and observed
with a Quanta 200 FEG (FEI, Hillsboro, OR, USA) scanning electron
microscope (Battistelli et al., 2005; Tyagi and Malik, 2010). Mycelia
and spores not exposed to CHI and OV were fixed and observed
similarly as a control.
2.7. Effects on fungal growth on grapes
Grapes were immersed in sodium hypochlorite solution (1% v/v)
for 15 min, washed with potable water and left for 2 h in a safe
cabinet to dry. The grapes were first immersed in 500 mL of an
inoculum solution (approximately 106 spores/mL) of the test fungal
strain for 1 min with gentle stirring using a sterile glass stem. Then,
the fruits were immersed in 500 mL of the coating solution con-
taining different CHI and OV concentrations (CHIOV5 and
CHIOV2.5) with gently shaking with a glass stem for 1 min. The
fruits were dried on a nylon filter to drain the excess liquid and
packed in a polyethylene container with lid. Then, one group of
fruits was stored at room temperature (25
C), while the other was
stored at a cold temperature (12
C). In addition, a control experi-
ment was performed in which CHI and OV solutions were replaced
by sterile distilled water containing glycerol (2 g/100 mL). Each
treatment included 40 fruits; at different storage time intervals
(room temperature 0, 2, 4, 6, 8, 10 and 12 days; cold temperature 0,
3, 6, 9, 12, 15, 18, 21 and 24 days), the fruits were examined for the
presence of visible fungal infection, and the results were expressed
as the percentage of fruit infected at different time intervals (Feng
and Zeng, 2007; Liu et al., 2007).
2.8. Effects on fruit decay
Grapes were first immersed in sodium hypochlorite solution
(1% v/v) for 15 min, washed with potable water and left for 2 h in
347
contact with air to dry. Then, the fruits were immersed for three
minutes in the coating solutions containing different combinations
of CHI and OV concentrations (CHIOV5 and CHIOV2.5) and gently
shaken with a glass stem for 1 min. The fruits were dried on a nylon
filter to drain the excess liquid and packed in a polyethylene
container with a lid. After this procedure, one group of fruits was
stored at room temperature (25
C), while the other group was
stored at a cold temperature (12
C). In addition, a control experi-
ment was performed in which the CHI and OV solutions were
replaced by sterile distilled water containing glycerol (2 g/100 mL).
Each treatment included 40 fruits and different storage times (room
temperature 0, 2, 4, 6, 8, 10 and 12 days; cold temperature 0, 3, 6, 9,
12, 15, 18, 21 and 24 days). Fruits were examined for the presence of
visible fungal infection, and the results were expressed as the
percentage of fruit infected at different times (Feng and Zeng, 2007;
Liu et al., 2007).
2.9. Physical and physicochemical analyses
The fruits were evaluated for weight loss and general quality
aspects such as color, firmness, soluble solids, titratable acidity and
anthocyanin at the same observation intervals as the examination
for fungal infection. The soluble solids (SS) content was determined
with a digital refractometer (Model HI 96801, Hanna Instruments,
São Paulo, Brazil), and the results were expressed as
Brix (Meng
et al., 2008). The titratable acidity (TA) was determined with
phenolphthalein as an indicator with 0.1 N NaOH, and the results
were expressed as mmol Hþ/100 g of fruit (Meng et al., 2008). The
SS/TA ratio was performed based on the relationship between
soluble solids content and titratable acidity (Meng et al., 2008). The
weight loss of fresh table grapes in each treatment during storage
was measured by monitoring the weight of the fruit at different
intervals. Weight loss was calculated as the percentage loss of
initial weight (Meng et al., 2008). The skin color was measured at
three different equatorial positions of the fruit with the CIELab
system (L* a* b*). Hue angle (h*ab) and chroma (C*ab) were
measured in a Minolta colorimeter Model CR-300 (Osaka, Japan), as
prescribed by the International Commission on Illumination (CIE,
1986). The firmness determination was performed with a 3-mm-
diameter probe (1/8) coupled to a TA-XT2 Texture Analyzer (Stable
Micro Systems, Haslemere, UK), and the results were expressed as
N/mm (Chien et al., 2007). The anthocyanin content was deter-
mined according to a standard procedure with a spectrophotom-
eter (SP220 e Bioespectro, São Paulo, Brazil) at 530 nm, and the
results were expressed as mg/100 g of grapes (Pirie and Mullins,
1976).
2.10. Sensory analysis
Sensory analysis was performed after approval by an Ethics
Research Committee (protocol 098/2011) using affective accep-
tance tests, purchase intent and preference tests with 50 untrained
tasters. The analysis was performed under controlled temperature
and lighting conditions in individual booths. Each panelist received
three units of grapes submitted to the different treatments, which
were served on disposable white plates coded with a random
three-digit number. The 3-grape samples were served simulta-
neously with a blind method of random sequence immediately
after removal from cold storage. Tasters were asked to eat a salty
biscuit and drink water between samples to avoid an aftertaste. For
the preference test, tasters were asked to choose the most and least
appreciated sample based on their overall evaluation. The purchase
intention was assessed with a five-point structured hedonic scale
ranging from one (certainly would not buy) to five (certainly would
buy). For the acceptability of appearance, color, flavor, texture,
348 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353

aftertaste and overall assessment, a nine-point structured hedonic Table 2

scale was used, ranging from one (dislike very much) to nine (liked Inhibition of spore germination of Rhizopus stolonifer URM 3728 and Aspergillus
niger URM 5842 when exposed to different combinations of chitosan and Origanum
very much). vulgare L. essential oil.

Combinations Inhibition (%)

2.11. Statistical analysis
Rhizopus stolonifer URM 3728
CHI (5 mg/mL) þ OV (5 mL/mL) 100%
All analyses were performed in triplicate (one grape from the CHI (5 mg/mL) þ OV (2.5 mL/mL) 97%
apical portion, one from the middle portion and one from the CHI (5 mg/mL) þ OV (1.25 mL/mL) 92%
bottom of each cluster) in three replicates, and the results were Aspergillus niger URM 5842
CHI (5 mg/mL) þ OV (5 mL/mL) 100%
expressed as the mean of the data obtained in each replicate.
CHI (5 mg/mL) þ OV (2.5 mL/mL) 100%
Statistical analyses were performed with descriptive statistics CHI (5 mg/mL) þ OV (1.25 mL/mL) 98%
(mean and standard deviation) and inferential tests (ANOVA fol-
Results expressed as percent inhibition of spores germination in relation to the
lowed by Tukey test) to determine statistically significant differ-
control assay (CHI 0 mg/mL þ OV 0 mL/mL); CHI: chitosan; OV: essential oil from
ences (P < 0.05) between treatments with Sigma Stat software 2.03. O. vulgare L.

3. Results structure, with increased wilting and deepening of typical ridges.

Similarly, the application of the combinations of CHI and OV caused
3.1. In vitro antifungal effects sharp morphological changes in spores of A. niger and disrupted
their structure (Fig. 1Fe1H). When not exposed to the test
CHI and OV exhibited MIC values of 10 mg/mL and 10 mL/mL, compounds, spores of R. stolonifer exhibited the normal globular,
respectively, against both R. stolonifer URM 3728 and A. niger URM elliptical and angular appearance; spores of A. niger also exhibited
5842. Based on these MIC values, CHI and OV were applied in typical characteristics, with a broad and radial head and spherical
combination at different sub-inhibitory and inhibitory concentra- and rough spores (Fig. 1A, E).
tions in tests that measured the effects on mycelial growth. Over 7 Structural changes resulting from the combined CHI and OV
days of incubation, the combined CHI and OV concentrations application were also observed in the mycelia of R. stolonifer and
strongly inhibited (88e100%) the growth of the test fungal strains A. niger (Fig. 2Ae2H). The mycelia of R. stolonifer exposed to CHI and
when compared to the control assays (Table 1). OV exhibited morphological changes, including hyphal thinning
Given the strong inhibition of mycelial growth, CHI and OV were (Fig. 2B), surface wrinkling and the loss of cytoplasmic material
assayed in combinations of sub-inhibitory amounts to evaluate characterized by empty hyphae (Fig. 2C and D). The mycelia of
fungal spore germination, fungal morphogenesis and application A. niger exhibited a rough and irregular surface around the hyphae
on grapes. The CHI concentration was set at 5 mg/mL because it was with an extracellular coating layer (Fig. 2H), distortion and hyphal
the lowest tested concentration (10e1.25 mg/mL) that was capable thinning and destruction (Fig. 2Fe2G). In the absence of CHI and
of forming a viscous solution with coating features to permit its OV, the mycelia of R. stolonifer exhibited homogeneous and regular
application as a coating for grapes. hyphae and a smooth and long outer surface (Fig. 2A), while
The application of CHI and OV in the different tested combina- mycelia of A. niger exhibited a regular morphology, with elongated
tions strongly inhibited spore germination of R. stolonifer and structure, similar diameter and smooth outer surface (Fig. 2E).
A. niger (Table 2). The spore germination inhibition of the test
strains in all tested combinations was greater than 90%, compared
3.2. Antifungal effects on fruits
to the number of spores germinated in the control experiment. SEM
analyses of spores of R. stolonifer exposed to different CHI and OV
When different CHI and OV combinations (CHIOV5 and
concentrations (Fig. 1Be1D) revealed changes in form and
CHIOV2.5) were applied to cover grapes artificially contaminated
with spores of R. stolonifer and A. niger, the rate of fungal growth
Table 1 was delayed relative to the control experiment throughout the
Growth inhibition (mycelial dry mass) of Rhizopus stolonifer URM 3728 and Asper- assessed storage period at both room (12 days) and cold tempera-
gillus niger 5842 URM in liquid medium throughout seven days of exposure to
tures (24 days). Fruits coated with CHIOV5 and CHIOV2.5, stored at
different combinations of chitosan and Origanum vulgare L. essential oil.
low temperature and contaminated with R. stolonifer showed no
Inhibition (%) visible fungal growth throughout the storage period. Grapes stored
Combinations Days of exposure at room temperature (25
C) and coated with CHIOV2.5 showed
3 5 7
visible growth of R. stolonifer only after 12 days of storage (25%
infected fruits). CHIOV5 was capable of inhibiting fungal growth
Rhizopus stolonifer URM 3728
CHI (10 mg/mL) þ OV (5 mL/mL) 97% 98% 99% throughout the storage period at both room and cold temperatures.
CHI (10 mg/mL) þ OV (2.5 mL/mL) 95% 96% 98% The control treatment demonstrated fungal growth from the 2nd
CHI (10 mg/mL) þ OV (1.25 mL/mL) 94% 96% 97% and 9th days of storage at room and cold temperatures, respec-
CHI (5 mg/mL) þ OV (5 mL/mL) 92% 94% 96% tively; 80% of fruits were infected at the end of the storage period at
CHI (5 mg/mL) þ OV (2.5 mL/mL) 90% 92% 94%
CHI (5 mg/mL) þ OV (1.25 mL/mL) 89% 91% 92%
room temperature, and 100% were infected at cold temperature.
Aspergillus niger URM 5842 The growth of A. niger on fruits coated with CHIOV5 and
CHI (10 mg/mL) þ OV (5 mL/mL) 99% 99% 100% CHIOV2.5 and stored at cold temperature was visible from the 10th
CHI (10 mg/mL) þ OV (2.5 mL/mL) 97% 98% 99% day of storage (25% infected fruits), and 33% of fruits were infected
CHI (10 mg/mL) þ OV (1.25 mL/mL) 95% 97% 99%
at the 12th day of storage. The growth of A. niger on fruits coated
CHI (5 mg/mL) þ OV (5 mL/mL) 92% 96% 98%
CHI (5 mg/mL) þ OV (2.5 mL/mL) 89% 94% 97% with CHIOV2.5 and stored at room temperature was visible from
CHI (5 mg/mL) þ OV (1.25 mL/mL) 88% 90% 95% the 4th day of storage (25% infected fruits) and reached 35% of fruits
Results expressed as percent inhibition of the mycelial growth (dry mass) in relation
on the 12th day of storage. The infection rate by A. niger in fruits not
to the control assay (CHI 0 mg/mL þ OV 0 mL/mL); CHI: chitosan; OV: essential oil coated with the tested combinations of CHI and OV was greater
from O. vulgare L. than 65% at the end of the storage period.
 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353 349

Fig. 1. Scanning electron microscopy of spores of Rhizopus stolonifer URM 3728 (AeD) and Aspergillus niger URM 5842 (EeH) exposed and not exposed to different combinations of
chitosan (CHI) and Origanum vulgare L. (OV) essential oil at sub-inhibitory concentrations. Control: CHI 0 mg/mL þ OV 0 mL/mL e (A and E); CHI 5 mg/mL þ OV 5 mL/mL (B and F);
CHI 5 mg/mL þ OV 2.5 mL/mL (C and G); and CHI: 5 mg/mL þ OV 1.25 mL/mL (D and H). Different magnifications were used for improved visualization.

The application of CHIOV2.5 and CHIOV5 inhibited the native
fungal population of fruits because there were no visible signs of
infection throughout the assessed storage intervals at both tested
temperatures. Fruits not coated with CHI and OV combinations
showed visible signs of fungal infection from the 2nd day of storage
at room temperature (41% infected fruits) and from the 10th day of
storage at cold temperature (36% infected fruits).
3.3. Effects on physical and physicochemical characteristics of
grapes
The physical and physicochemical changes in grapes uncoated or
coated with CHIOV5 or CHIOV2.5 were evaluated during storage at
room (25
C) and cold temperatures (12
C) (Tables 3 and 4). Grapes
treated or not treated with combinations of CHI and OV did not differ
(P < 0.05) in weight at any of the assessed storage intervals at both
tested storage temperatures. At most of the evaluated storage
intervals, no differences in the firmness of grapes stored under cold
or room temperature (P > 0.05) were found. The levels of titratable
acidity of grapes coated with CHIOV5 and CHIOV2.5 stored under
cold temperature were equal to (P > 0.05) or lower (P < 0.05) than
the values found for the control experiment. Grapes submitted to
different treatments and stored at room temperature did not differ
(P > 0.05) in titratable acidity values, except at the 10th day of
storage, in which the highest values were observed in the control
samples and in those treated with CHIOV2.5.

Fig. 2. Scanning electron microscopy of mycelia of Rhizopus stolonifer URM 3728 (AeD) and Aspergillus niger URM 5842 (EeH) exposed and not exposed to different combinations of
chitosan (CHI) and Origanum vulgare L. (OV) essential oil at sub-inhibitory concentrations. Control: CHI 0 mg/mL þ OV 0 mL/mL e (A and E); CHI 5 mg/mL þ OV 5 mL/mL (B and F);
CHI 5 mg/mL þ OV 2.5 mL/mL (C and G); and CHI: 5 mg/mL þ OV 1.25 mL/mL (D and H). Different magnifications were used for improved visualization.
350 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353

Table 3
Mean values of physical and physicochemical quality in grapes cultivar Isabella coated with combinations of chitosan and Origanum vulgare L. essential oil at sub-inhibitory
concentrations and stored at room temperature (25
C) for 12 days.

Treatments Days of storage

0 2 4 6 8 10 12
Total anthocyanin (mg/100 g of fruit)
CO 873.53 (91)a 1332.71 (84)ab 1639.63 (49)a 900.44 (85)a 506.29 (30)a 697.29 (92)ab 1071.99 (48)a
CHIOV5 1407.42 (83)b 1389.59 (91)a 1173.53 (58)ab 955.98 (46)ab 692.78 (59)ab 489.26 (61)a 157.66 (73)b
CHIOV2.5 1291.54 (98)ab 1259.38 (75)b 954.24 (61)b 1323.73 (39)b 969.86 (71)b 994.67 (53)b 879.71 (89)ab
Firmness (N/mm)
a
CO 3.2 (0.41) 3.11 (0.3)a 3.05 (0.21)a 3.01 (0.2)a 2.85 (0.67)a 2.87 (0.6)a 2.73 (0.29)a
CHIOV5 2.84 (0.8)a 2.81 (0.21)a 2.89 (0.45)a 2.92 (0.51)a 2.89 (0.43)a 2.99 (0.34)a 3.02 (0.42)a
CHIOV2.5 2.96 (0.5)a 2.92 (0.42)a 2.94 (0.6)a 2.97 (0.5)b 2.91 (0.6)a 2.93 (0.29)a 2.98 (0.5)a
Titrable acidity (mmol Hþ/100 g of fruit)
CO 3.33 (0.7)a 2.26 (0.3)a 2.23 (0.1)a 1.70 (0.8)a 1.63 (0.3)a 3.06 (0.5)a 3.1 (0.2)a
CHIOV5 2.9 (0.1)a 2.46 (0.2)a 2.10 (0.3)a 1.96 (0.4)a 1.6 (0.5)a 1.6 (0.1)b 1.7 (0.5)a
a
CHIOV2.5 2.67 (0.3) 2.36 (0.4)a 2.36 (0.5)a 2.50 (0.2)a 2.46 (0.7)a 2.33 (0.5)ab 2.1 (0.7)a


Soluble solids ( Brix)
a
CO 12.0 (0.4) 15.6 (0.1)a 14.0 (0.2)a 12.0 (0.2)ab 15.3 (0.3)a 11.0 (0.2)a 14.6 (0.5)a
CHIOV5 15.3 (0.1)a 15.3 (0.2)a 17.0 (0.1)b 14.0 (0.1)a 11.3 (0.1)b 10.3 (0.5)a 11.6 (0.5)b
CHIOV2.5 14.3 (0.2)a 16.0 (0.1)a 16.3 (0.3)ab 10.0 (0.2)b 10.6 (0.3)b 11.3 (0.1)a 11.3 (0.5)b

CO: control (CHI 0 mg/mL þ OV: 0 mL/mL); CHIOV5 (CHI 5 mg/mL þ OV 5 mL/mL; CHIOV2.5: CHI 5 mg/mL þ OV 2.5 mL/mL). aec For each trial, different superscript lowercase
letters in the same column denote differences (P < 0.05) between the mean values according to Tukey’s test.

Grapes submitted to different treatments did not differ (P > 0.05)
in soluble solids content at the 3rd, 6th, 12th and 24th days of
storage at cold temperature, while at the other intervals (9th, 15th
and 21st days), the lowest soluble solids contents were observed in
grapes coated with CHIOV5, except at the 9th day of storage. When
stored at room temperature, grapes coated and uncoated with CHI
and OV yielded similar values (P > 0.05) of soluble solids at days
zero, 2, 4 and 10 of storage, while at the 8th and 12th days of storage,
the highest values (P < 0.05) were found in fruits from the control
group. At days zero, 2, 10 and 12 of storage at room temperature, the
SS/TA ratios for grapes coated with CHIOV5 or CHIOV2.5 were equal
to (P > 0.05) or higher (P < 0.05) than that of the control experiment,
whereas at the other assessed storage intervals, fruits coated with
CHIOV2.5 exhibited the lowest values (P < 0.05) of this parameter.
Grapes coated with CHIOV2.5 and stored at cold temperature had
higher values (P < 0.05) of the SS/TA ratio at days zero and 3 of
storage; however, at later storage intervals, the highest values
(P > 0.05) were found in fruits coated with CHIOV5.
The levels of anthocyanin in grapes stored at room temperature
and submitted to treatment with CHIOV2.5 were higher (P < 0.05)
at the 6th, 8th and 10th days of storage relative to the control
experiment, while fruits submitted to treatment with CHIOV5
showed higher values (P < 0.05) at days zero, 2 and 4 of storage.
Fruits stored at cold temperature and coated with either CHIOV2.5
or CHIOV5 exhibited different levels of anthocyanin (P < 0.05) only
at the 3rd, 21st and 24th days of storage. At most of the assessed
storage times, fruits not coated with CHIOV5 and CHIOV2.5 and
stored at cold temperature had higher levels (P < 0.05) of antho-
cyanin. The color of grapes coated with CHIOV2.5 or CHIOV5 was
predominantly purple at the different storage intervals at both
tested temperatures because the values of a* and b* (positive values
close to zero) did not differ (P > 0.05) between fruits coated and
uncoated with CHIOV5 and CHIOV2.5. Furthermore, in all treat-
ments, there was a decrease (P < 0.05) in h* values and a concom-
itant increase in the C* value, revealing a color change from blue to
purple in fruits and maintenance of the fruit brightness, which was

Table 4
Mean values of physical and physicochemical quality in grapes cultivar Isabella coated with combinations of chitosan and Origanum vulgare L. essential oil at sub-inhibitory
concentrations and stored at cold temperature (12
C) for 12 days.

Treatments Days of storage

0 3 6 9 12 15 18 21 24
Total anthocyanin (mg/100 g of fruit)
CO 866.16 (42)a 1312.24 (54)a 1086.45 (72)a 914.15 (92)a 902.66 (72)a 853.21 (61)a 735.33 (29)b 650.93 (69)a 799.58 (43)a
CHIOV5 1423.28 (64)b 1350.4 (46)ab 690.67 (84)b 821.37 (43)ab 811.33 (56)ab 779.81 (82)ab 657.33 (52)a 523.4 (57)b 510.08 (35)a
ab
CHIOV2.5 1257.46 (101) 1574. 87 (81)b 767.42 (93)ab 713.88 (68)b 701.66 (35)b 727.03 (58)b 725.66 (78)ab 724.14 (45)c 759.55 (71)a
Firmness (N/mm)
CO 5.1 (0.3)a 5.09 (0.3)a 4.98 (0.2)a 5.2 (0.2)a 4.92 (0.6)a 4.37 (0.6)a 4.83 (0.2)a 5.1 (0.3)a 5.09 (0.3)a
CHIOV5 5.04 (0.8)a 4.3 (0.2)a 4.89 (0.4)a 5.2 (0.8)a 5.1 (0.4)a 4.99 (0.3)a 4.63 (0.4)a 5.04 (0.8)a 4.3 (0.2)a
CHIOV2.5 4.86 (0.5)a 4.33 (0.4)a 5.1 (0.6)a 4.8 (0.5)b 4.39 (0.6)a 4.23 (0.3)a 4.98 (0.5)a 4.86 (0.5)a 4.33 (0.4)a
Titrable acidity (mmol Hþ/100 g of fruit)
CO 2.7 (0.2)ab 2.6 (0.3)a 3.3 (0.5)a 3.2 (0.2)a 3.0 (0.1)a 3.0 (0.2)a 3.0 (0.2)a 3.7 (0.2)a 3.5 (0.4)a
CHIOV5 3.3 (0.1)b 2.9 (0.2)a 2.9 (0.5)a 3.0 (0.3)a 2.7 (0.1)a 2.7 (0.1)a 2.5 (0.2)a 2.2 (0.3)a 2.1 (0.3)a
a
CHIOV2.5 3.8 (0.4) 2.9 (0.1)a 2.9 (0.4)a 3.0 (0.2)a 2.6 (0.2)a 2.6 (0.2)a 2.6 (0.1)a 2.7 (0.1)a 2.5 (0.5)a
Soluble solids (
Brix)
CO 15.0 (0.5)a 14.0 (0.3)a 12.16 (0.2)a 16.0 (0.8)ab 15.0 (0.3)a 15.0 (0.6)a 15.0 (0.2)ab 14.0 (0.4)a 14.0 (0.4)a
CHIOV5 12.0 (0.3)b 12.0 (0.2)a 12.0 (0.5)a 13.0 (0.4)bc 11.0 (0.5)b 11.0 (0.5)b 11.0 (0.5)b 15.0 (0.5)ab 14.0 (0.3)a
CHIOV2.5 12.0 (0.5)b 14.0 (0.4)a 11.68 (0.3)a 12.0 (0.2)c 15.0 (0.7)a 15.0 (0.3)a 16.0 (0.7)b 15.1 (0.7)b 16.0 (0.5)a

CO: control (CHI 0 mg/mL þ OV: 0 mL/mL); CHIOV5 (CHI 5 mg/mL þ OV 5 mL/mL; CHIOV2.5: CHI 5 mg/mL þ OV 2.5 mL/mL). aec For each trial, different superscript lowercase
letters in the same column denote differences (P < 0.05) between the mean values according to Tukey’s test.
 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353 351

identified from the reading in L*; there was no difference between coated with CHIOV2.5 and CHIOV5 increased (P < 0.05), with
samples (P > 0.05) at cold and room temperatures. a change from “disliked moderately” to “liked slightly”.
When asked to report about the intent to purchase, the panelists
3.4. Effects on the sensory characteristics of grapes reported differences between fruits uncoated and coated with
CHIOV2.5 and CHIOV5. To fruits uncoated with CHI and OV the
Grapes coated with coating composed of CHIOV2.5 or CHIOV5 responses fell between “purchase”, while for fruits coated with
and those not exposed to CHI and OV treatments (control) were CHIOV2.5 and CHIOV5 the responses fell “maybe would purchase/
submitted to acceptance and preference tests throughout the 12 maybe would not purchase” at all the assessed storage periods. The
days of storage at cold temperature (the ensure the microbiological results of the preference test showed that the control experiment
safety of samples offered to the team of panelists). was the most accepted, followed by CHIOV5 and CHIOV2.5, and no
Fruits not coated with CHI and OV coatings received the highest difference (P > 0.05) in preference was observed between fruits
scores for all analyzed attributes when compared to fruits coated coated with the different combinations of CHI and OV.
with CHIOV2.5 and CHIOV5, with an increase (P < 0.05) in scores for
aroma, flavor, aftertaste and firmness throughout the analyzed 4. Discussion
storage period (Table 5). In all assessed periods, samples submitted
to the control treatment received scores corresponding to “liked The combined application of CHI and OV at different concen-
moderately”. trations inhibited fungal growth in liquid media and caused
Scores related to the appearance of grapes treated with changes in the morphology (and germination) of spores and
CHIOV2.5 and CHIOV5 did not differ throughout the storage period mycelia of R. stolonifer and A. niger. The strong, rapid and consistent
(P > 0.05), corresponding to “liked moderately” at all assessed antifungal effect resulting from the combined application of CHI
storage intervals. Scores related to the color of grapes coated with and OV at sub-inhibitory concentrations suggests an enhanced
CHIOV5 did not differ (P > 0.05) throughout the storage period, antifungal effect resulting from the combined application of these
while the scores of fruits coated with CHIOV2.5 decreased compounds.
(P < 0.05); however, the scores always corresponded to “liked Previous studies have reported the efficacy of CHI in the inhi-
moderately”. For the attributes aroma, flavor, aftertaste, firmness bition of the mycelial growth and spore germination of post-
and overall evaluation, scores on the 6th day of storage decreased harvest pathogenic fungi (Hernández-Lauzardo et al., 2008;
(P < 0.05) compared to time zero for fruits treated with CHIOV2.5 or Li et al., 2009), although the intensity of the antifungal effect
CHIOV5; however, the scores for CHIOV5 increased (P < 0.05) on depended on the type of CHI dispersion (solution or coating)
the 12th day of storage, except for firmness, compared to fruits and tested fungal species. Ziani et al. (2009) observed greater
coated with CHIOV2.5. Scores related to flavor, aftertaste and inhibition (>60%) of the mycelial growth of A. niger when exposed
overall evaluation corresponded to “dislike moderately” at the 6th to chitosan (1 g/100 mL) dispersed in solution, whereas the inhi-
day of storage, although at the 12th day of storage, scores corre- bition of Alternaria alternata (97%) and Rhizopus oryzae (92%) was
sponded to “neither liked nor disliked”, revealing an improvement higher when chitosan (1 g/100 mL) was applied as a coating.
in the evaluation of these attributes during the assessed storage Sánchez-Gonzáles et al. (2010a) reported that chitosan coatings
period. At the same interval, scores related to the firmness of fruits (1 g/100 mL) were not effective in the inhibition of Penicillium
italicum, while the combined application of chitosan (1 g/100 mL)
and bergamot essential oil (0.5e3 mL/100 mL) as coating was
Table 5
Mean sensory scores for grapes cultivar Isabella coated with combinations of chi-
effective in inhibiting the growth of this fungus throughout 12 days
tosan and Origanum vulgare L. essential oil at sub-inhibitory concentrations and of storage at 20
C. In general, studies have shown that the CHI
stored at cold temperature (12
C). molecule exhibits more consistent antimicrobial activity against
Attributes Days of CO CHIOV5 CHIOV2.5
bacteria, while the effect on filamentous fungi and yeast has been
storage variable (Tharanathan and Kittur, 2003; Sánchez-Gonzáles et al.,
Appearance 0 7.87 (0.37)aA 7.5 (0.24)bA 6.94 (0.16)bA 2010a).
6 7.51 (0.5)aA 7.28 (0.22)aA 7.0 (0.5)aA Some essential oils significantly inhibit the growth of post-
12 7.78 (0.14)aA 7.26 (0.14)bA 7.12 (0.22)bA harvest pathogenic fungi (Xing et al., 2012; Omidbeygi et al.,
Color 0 7.83 (0.07)aA 7.46 (0.06)bA 7.06 (0.12)cA 2007). Carmo et al. (2008) and Mitchell et al. (2010) reported the
6 7.8 (0.11)aA 7.48 (0.13)bA 7.26 (0.12)cB
12 7.72 (0.1)aA 7.58 (0.15)bA 6.84 (0.1)cC
inhibition of Aspergillus species (Aspergillus parasiticus, A. terreus,
Flavor 0 7.59 (0.11)aA 5.9 (0.1)bA 5.32 (0.1)aC Aspergillus flavus and A. ochraceus) by OV (0.6e80 mL/mL) for 14
6 7.54 (0.16)aA 5.26 (0.1)bB 5.24 (0.14)aB days at 25
C. Freire et al. (2012) observed a strong inhibitory effect
12 7.16 (0.07)aB 5.62 (0.2)bC 5.64 (0.1)bB on the growth of various post-harvest pathogenic fungi (A. flavus, A.
Taste 0 8.21 (0.03)aA 4.44 (0.56)bA 3.5 (0.5)cAB
parasiticus, A. niger, A. ochraceus, Colletrichum gloesporioides and
6 7.72 (0.1)aB 3.84 (0.08)bB 3.54 (0.12)cA
12 7.52 (0.1)aB 5.06 (0.14)bA 5.32 (0.1)bB C. musae) by Mentha piperita essential oil (0.1 and 0.2 mL/100 mL);
Aftertaste 0 7.52 (0.04)aA 4.54 (0.12)bA 3.58 (0.12)cA menthone, neo-menthol and carvone were identified as the major
6 7.22 (0.1)aB 3.7 (0.1)bB 3.32 (0.08)cB fungitoxic compounds of this essential oil.
12 7.24 (0.06)aB 5.16 (0.05)bC 5.18 (0.03)bC The combination of CHI and OV at sub-inhibitory concentrations
Firmness 0 7.42 (0.1)aA 6.12 (0.08)bA 5.88 (0.1)cA
6 7.62 (0.12)aB 5.88 (0.11)bB 5.98 (0.1)bA
caused significant morphological changes in the spores (wilting,
12 7.68 (0.13)aB 6.38 (0.09)bC 6.66 (0.06)cB disruption, loss of cellular material, deepening of ridges) and
Overall 0 7.85 (0.17)aA 5.24 (0.12)bA 4.38 (0.12)cA mycelia (thinning, wrinkling and loss of cytoplasmic material) of
evaluation 6 7.87 (0.11)aA 4.16 (0.14)bB 4.14 (0.15)bB R. stolonifer and A. niger. These alterations suggest that the mech-
12 7.74 (0.17)aA 5.5 (0.23)bC 5.72 (0.19)bC
anism by which CHI and OV inhibit germination results from their
CO: control (CHI 0 mg/mL þ OV: 0 mL/mL); CHIOV5 (CHI 5 mg/mL þ OV 5 mL/mL; interaction with the cell wall of spores (Plascencia-Jatomea et al.,
CHIOV2.5: CHI 5 mg/mL þ OV 2.5 mL/mL). aec For each trial, different superscript 2003; Sharma and Tripathi, 2008). The spore is as an important
lowercase letters within a row denote differences (P < 0.05) between the mean
values according to Tukey’s test. AeC Different superscript capital letters in the
structure for the survival and spread of fungi (Rabea et al., 2009),
same column denote differences (P < 0.05) between the mean values according to and the application of agents that inhibit spore germination or
Tukey’s test. cause the destruction of these structures are potential candidates
352 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353
for the control of spore populations and spreading on various
substrates.
The application of Citrus sinensis essential oil (0.5 mg/mL, 1 mg/mL
and 2 mg/mL) caused loss of cytoplasm, emerging of hyphae tips,
cell wall thinning and distortion and disruption of the cell wall of
A. niger mycelia (Sharma and Tripathi, 2008). The use of CHI (0.01,
0.05, 0.1 g/100 mL) caused a loss of cellular material, texture
modifications and the deposition of extracellular material in hyphae
of T. harzianum and S. sapinea (Vesentini et al., 2007). The exposure
of A. flavus to Hyptus suaveolens essential oil (20 mL/mL) triggered
several morphological abnormalities such as swollen and hetero-
geneous mycelial structure, lack of sporulation, loss of cytoplasmic
content and distortion of hyphae (Moreira et al., 2010). According to
the authors, these changes suggest that the antifungal activity
of essential oils could include attack on the cell wall and retraction
of the hypha cytoplasm, resulting in mycelium death. In addition,
such changes have been associated with the influence of essential
oil components on enzymatic reactions for the synthesis of cell
wall components, affecting morphogenesis and fungal growth.
A previous study detected carvacrol (66.9%) as the major
compound of the OV batch used in this study (Azêredo et al., 2011).
Phenolic compounds (such as carvacrol), which are found in the
hydrophobic fractions of essential oils, may be dissolved in the
hydrophobic domain of the cytoplasmic membrane, causing its
disintegration and loss of adenosine triphosphate, among other
intracellular components, resulting in cell death (Hassani et al.,
2012). The antifungal effect of CHI has been associated with the
interaction and formation of polyelectrolytic complexes because its
protonated amine groups could bind to the cell surface, affecting
membrane permeability and causing loss of intracellular compo-
nents (Yadav and Bhise, 2004).
The CHI and OV mixture also possessed antifungal activity when
it was applied in the form of a coating on grapes artificially
contaminated with A. stolonifer and A. niger, as well as against the
native fungal microbiota of grapes throughout storage at room and
cold temperatures. Similar to the results of this study, some studies
have found that the antifungal activity achieved by the isolated or
combined application of CHI and some essential oils to fruits
increases with decreased storage temperature (Sánchez-Gonzáles
et al., 2011; Xing et al., 2012).
The use of coatings composed of CHI (1 g/100 mL) and
C. zeylanicum essential oil (0.25 g/100 mL) was effective in reducing
the microbial deterioration of sweet peppers throughout 35 days of
storage at room temperature (Xing et al., 2012). Similarly, the use of
a coating composed of chitosan (1 g/100 mL) and bergamot
essential oil (2 mL/100 mL) inhibited the growth of mesophilic
microorganisms, fungi and yeasts on table grapes throughout 24
days of storage at 2
C (Sánchez-Gonzáles et al., 2011).
It has been suggested that the use of essential oils as antifungal
agents on fruits and vegetables is more effective when they are
incorporated into coatings or gels applied to the surface of the
products than when they are used in the form of an aerosol, solu-
tion or directly spread over the product (Sebti and Coma, 2002;
Kristo et al., 2008; Sánchez-Gonzáles et al., 2010a).
In agreement with the results obtained in this study, other
studies have reported that the application of CHI and essential oils
alone or in combination as a coating material preserved or
improved the physical and physicochemical quality of fruits (Xiao
et al., 2010; Sánchez-Gonzáles et al., 2011; Hassani et al., 2012) and
improved the sensory aspects of appearance, color, firmness and
overall acceptability throughout the low-temperature storage
period (Abdolahi et al., 2010; Xiao et al., 2010; Xing et al., 2011).
These data show that the combined use of CHI and essential oils at
sub-inhibitory concentrations could be considered an alternative
to the sensory impact, particularly flavor and odor, resulting from
the application of essential oils alone at higher concentrations on
fruits.
To the best of our knowledge the results of this study demon-
strate by first time that the application of CHI and OV in combi-
nation at different sub-inhibitory concentrations can significantly
inhibit the post-harvest pathogenic fungi R. stolonifer and A. niger as
well as the native fungal microbiota on table grapes stored at room
and cold temperatures. Furthermore, the combined application of
the tested compounds did not negatively affect the physical and
physicochemical aspects of the grapes, and their sensory charac-
teristics improved during the storage period. These findings reveal
the potential of the combined application of CHI and OV at sub-
inhibitory concentrations to control the growth and survival of
pathogenic fungi in fruits, particularly R. stolonifer and A. niger in
grapes, which may be an alternative to synthetic antifungal agents
that are currently applied to reduce post-harvest losses from these
biological contaminants.
Acknowledgments
The authors thank the National Council for Scientific and
Technological Development e CNPq (Brazil) and Coordination for
the Improvement of Higher Education Personnel e CAPES (Brazil)
for financial support for the development of the project.
References
Abdolahi, A., Hassani, A., Ghosta, Y., Bernousi, I., Meshkatalsadat, M.H., 2010. Study
of the potential use of essential oils for decay control and quality preservation
of Tabarzeh table grape. Journal of Plant Protection Research 50, 45e52.
Azêredo, G.A. de., Stamford, T.L.M., Nunes, P.C., Gomes Neto, N.J., Oliveira, M.E. G. de,
Souza, E. L. de, 2011. Combined application of essential oils from Origanum
vulgare L. and Rosmarinus officinalis L. to inhibit bacteria and autochthonous
microflora associated with minimally processed vegetables. Food Research
International 44, 1541e1548.
Badawy, M.E.I., Rabea, E.I., 2009. Potential of the biopolymer chitosan with different
molecular weights to control postharvest gray mold of tomato fruit. Postharvest
Biology and Technology 51, 110e117.
Battistelli, M.R., De Sanctis, R., De Bellis, L., Cucchiarini, M., Golbi, P., 2005. Rhodiola
rosea as antioxidant in red bloods cells: ultrastructural and hemolytic behavior.
European Journal of Histochemistry 49, 243e254.
Bennis, S., Chami, F., Chami, N., Bouchikhi, T., Remmal, A., 2004. Surface alteration of
Saccharomyces cerevisae induced by thymol and eugenol. Letters in Applied
Microbiology 28, 454e458.
Bonilla, J., Atarés, L., Vargas, M., Chiralt, A., 2012. Effect of essential oils and
homogenization conditions on properties of chitosan-based films. Food
Hydrocolloids 26, 9e16.
Camele, I., De Feo, V., Altieru, L., Mancini, E., De Martino, L., Rana, G.L., 2010. An
attempt of postharvest orange fruit rot control using essential oils from
Mediterranean plants. Journal of Medicinal Food 13, 1515e1523.
Carmo, E.S., Lima, E.de O., Souza, E.L.de, 2008. The potential of Origanum vulgare L.
(Lamiaceae) essential oil in inhibit the growth of some food-related Aspergillus
species. Brazilian Journal of Food Microbiology 39, 362e367.
Chen, X.G., Zheng, L., Wang, Z., Lee, C.Y., Park, H.J., 2002. Molecular affinity and
permeability of different molecular weight chitosan membranes. Journal of
Agricultural and Food Chemistry 50, 5915e5918.
Chien, P.-J., Sheu, F., Lin, H.-R., 2007. Coating citrus (Murcott tangor) fruit with low
molecular weight chitosan increases postharvest quality and shelf life. Food
Chemistry 100, 1160e1164.
CIE e Commission Internationale de l’Éclairage, 1986. Colourimetry, second ed. CIE
Publication, Vienna.
Feng, W., Zeng, X., 2007. Essential oils to control Alternaria alternata in vitro and
in vivo. Food Control 18, 1126e1130.
Freire, M.M., Jham, G.N., Dhingra, O.D., Jardim, C.M., Barcelos, R.C., Valente, V.M.M.,
2012. Composition, antifungal activity and main fungitoxic components of the
essential oil Mentha piperita L. Journal of Food Safety 32, 29e36.
Hassani, A., Fathi, Z., Ghosta, Y., Abdollahi, A., Meshkatalsadat, M.H., Marandi, R.J.,
2012. Evaluation of plant essential oils for control of postharvest brown and
gray mold rots on apricot. Journal of Food Safety 32, 94e101.
Hernández-Lauzardo, A.N., Bautista-Baños, S., Valle, V. Del, Méndez-
Montealvo, M.G., Sánchez-Rivera, M.M., Bello-Pérez, L.A., 2008. Antifungal
effects of chitosan with different molecular weights on in vitro development of
Rhizopus stolonifer (Ehren.:Fr.) Vuill. Carbohydrate Polymers 73, 541e547.
Kristo, E., Koutsoumanis, K.P., Biliaderis, C.G., 2008. Thermal, mechanical and water
vapor barrier properties of sodium caseinate films containing antimicrobials
 N.S.T. dos Santos et al. / Food Microbiology 32 (2012) 345e353
and their inhibitory action on Listeria monocytogenes. Journal of Food Hydro-
colloids 22, 373e386.
Li, Y.-C., Sun, X.-J., Bi, Y., Ge, Y.-H., Wang, Y., 2009. Antifungal activity of chitosan on
Fusarium sulphureum in relation to dry rot of potato tuber. Agricultural Sciences
in China 8, 597e604.
Liu, J., Tian, S., Meng, X., Xu, Y., 2007. Effects of chitosan on control of postharvest
diseases and physiological responses of tomato fruit. Postharvest Biology and
Technology 44, 300e306.
Martínez-Romero, D., Guillén, F., Valverde, J.M., Bailén, G., Zapata, P., Serrano, M.,
Castillo, S., Valero, D., 2008. Influence of carvacrol on survival of Botrytis cinerea
inoculated in table grapes. International Journal of Food Microbiology 115,
144e148.
Meng, X., Li, B., Liu, J., Tian, S., 2008. Physiological responses and quality attributes
of table grape fruit to chitosan prehavest spray and postharvest coating during
storage. Food Chemistry 106, 501e508.
Mitchell, T.C., Stamford, T.L.M., Souza, E. L. de, Lima, E. de O., Carmo, E.S., 2010.
Origanum vulgare L. essential oil as inhibitor of potentially toxigenic Aspergilli.
Ciência e Tecnologia de Alimentos 30, 755e760.
Moreira, A.C.P., Lima, E. de O., Wanderley, P.A., Carmo, E.S., Souza, E. L. de, 2010.
Chemical composition and antifungal activity of Hyptis suaveolens (L.) poit
leaves essential oil against Aspergillus species. Brazilian Journal of Microbiology
41, 28e33.
Ojagh, S.M., Rezaei, M., Razavi, S.H., Hosseini, S.M.H., 2010. Effect of chitosan
coatings enriched with cinnamon oil on the quality of refrigerated rainbow
trout. Food Chemistry 120, 193e198.
Oliveira, C.E. V. de., Stamford, T.L.M., Gomes Neto, N.J., Souza, E. L. de, 2009. Inhi-
bition of Staphylococcus aureus in broth and meat broth using synergies of
phenolics and organic acids. International Journal of Food Microbiology 137,
312e316.
Omidbeygi, M., Barzegar, M., Hamidi, Z., Naghdibadi, H., 2007. Antifungal activity of
thyme, summer savory and cloves essential oil against Aspergillus flavus in
liquid medium and tomate paste. Food Control 18, 1518e1523.
Outtara, B., Simard, R.E., Piette, G., Begin, A., Holley, R.A., 2000. Diffusion of acetic
and propionic acids from chitosan-based antimicrobial packaging films. Journal
of Food Science 65, 768e773.
Pereda, M., Amica, G., Marcovich, N.E., 2012. Development and characterization of
edible chitosan/oil emulsion films. Carbohydrate Polymers 87, 1318e1325.
Pirie, A., Mullins, M.G., 1976. Changes in anthocyanin and phenolics content of
grapevine leaf and fruit tissues treated with sucrose, nitrate, and abscisic acid.
Plant Physiology 58, 468e472.
Plascencia-Jatomea, M., Viniegra, G., Olayo, R., Castillo-Ortega, M.M., Shirai, K., 2003.
Effect of chitosan and temperature no spore germination of Aspergillus niger.
Macromolecular Bioscience 3, 582e586.
Rabea, I.E., Badawy, M.E.I., Steurbaut, W., Stevens, C.V., 2009. In vitro assessment of
N-(benzyl) chitosan derivate against some plant pathogenic bacteria and fungi.
European Polymer Journal 45, 237e245.
Rasooli, I., Abyaneh, M.R., 2004. Inhibitory effect of thyme oils on growth and
aflatoxin production by Aspergillus parasiticus. Food Control 15, 479e483.
Rasooli, I., Owlia, P., 2005. Chemoprevention by thyme oils of Aspergillus parasiticus
growth and aflatoxin production. Phytochemistry 66, 2851e2856.
353
Rasooli, I., Rezaei, M.B., Allameh, A., 2006. Growth inhibition and morphological
alterations of Aspergillus niger by essential oil from Thymus eriocalyx and
Thymus x-porlock. Food Control 17, 359e364.
Sánchez-Gonzáles, L., Chafer, M., Chiralt, A., González-Martínez, C., 2010a. Physical
properties of edible chitosan films containing bergamot essential oil and their
inhibitory action on Penicillium italicum. Carbohydrate Polymers 82, 277e283.
Sánchez-Gonzáles, L., Gonzáles-Martínez, C., Chiralt, A., Cháfer, M., 2010b. Physical
and antimicrobial properties of chitosan-tea tree essential oil composite films.
Journal of Food Engineering 98, 443e452.
Sánchez-Gonzáles, L., Pastor, P., Vargas, M., Chiralt, A., González-Martínez,
Cháfer, M., 2011. Effect of hydroxypropylmethylcellulose and chitosan coatings
with and without bergamot essential oil on quality and safety of cold-stored
grapes. Postharvest Biology and Technology 60, 57e63.
Sathivel, S., Liu, Q., Huang, J., Prinyawiwatkul, W., 2007. The influence of chitosan
glazing on the quality of skinless pink salmon (Oncorhynchus gorbuscha) fillets
during frozen storage. Journal of Food Engineering 83, 366e373.
Sebti, I., Coma, V., 2002. Active edible polysaccharide coating and interactions
between solution coating compounds. Carbohydrate Polymers 49, 139e144.
Sharma, N., Tripathi, A., 2008. Effects of Citrus sinensis (L.) Osbeck epicarp essential
oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem.
Microbiological Research 163, 337e344.
Sousa, J.P., Azêredo, G.A., Vasconcelos, M.A.S., Torres, R.A., Conceição, M.L. da.,
Souza, E.L., 2012. Synergies of carvacrol and 1,8-cineole to inhibit bacteria
associated with minimally processed vegetables. International Journal of Food
Microbiology 154, 145e151.
Souza, E.L., Stamford, T.L.M., Lima, E.O., Trajano, V.N., 2007. Effectiveness of Orig-
anum vulgare L. essential oil to inhibit the growth of food spoiling yeasts. Food
Control 18, 409e413.
Tharanathan, R.N., Kittur, F.S., 2003. Chitin e the undisputed biomolecule of great
potential. Critical Review in Food Science and Nutrition 43, 61e87.
Tyagi, A.K., Malik, A., 2010. Antimicrobial action of essential oil vapours and
negative air ions against Pseudomonas fluorescens. International Journal of Food
Microbiology 143, 205e210.
Vesentini, D., Steward, D., Singhi, A.P., Ball, R., Daniel, G., Franich, R., 2007. Chitosan-
mediated changes in cell wall composition, morphology and ultrastructure in
two wood-inhabiting fungi. Mycological Research 111, 875e890.
Xiao, C., Zhu, L., Luo, W., Song, X., Deng, Y., 2010. Combined action of pure oxygen
pretreatment and chitosan coating incorporated with rosemary extracts on the
quality of fresh e cut pears. Food Chemistry 121, 1003e1009.
Xing, Y., Li, X., Xu, Q., Yun, J., Lu, Y., Tang, Y., 2011. Effects of chitosan enriched with
cinnamon oil on qualitative properties of sweet pepper (Capsicum annuum L.).
Food Chemistry 124, 1443e1450.
Xing, Y., Xu, Q., Li, X., Che, Z., Yun, J., 2012. Antifungal activities of clove oil against
Rhizopus nigricans, Aspergillus flavus and Penicillium citrinum in vitro and in
wounded fruit test. Journal of Food Safety 32, 84e93.
Yadav, A.V., Bhise, S.B., 2004. Chitosan: a potential biomaterial effective against
typhoid. Current Science 87, 1176e1178.
Ziani, K., Fernández-Pan, I., Royo, M., Maté, J.I., 2009. Antifungal activity of films and
solutions based on chitosan against typical seed fungi. Food Hydrocolloids 23,
2309e2314.