Food Control 17 (2006) 336–341

www.elsevier.com/locate/foodcont

Microbiological evaluation of an edible antimicrobial coating

on minimally processed carrots
a,b a,* a
A.M. Durango , N.F.F Soares , N.J. Andrade
a
Food Science Department, Universidade Federal de Viçosa, 36570-000 Viçosa-MG, Brazil
b
Food Engineering Department, Universidad de Córdoba, Monterı́a, Colombia

Received 3 February 2004; received in revised form 23 October 2004; accepted 25 October 2004

Abstract

This work aimed to develop an edible antimicrobial coating based on a starch–chitosan matrix to evaluate its effect on minimally
processed carrot by means of microbiological analyses. Coatings based on 4% yam starch (w/w) + 2% glycerol (w/w) and coatings
based on 4% yam starch (w/w) + 2% glycerol (w/w) + chitosan in 0.5% and 1.5% concentrations were prepared. Samples of mini-
mally processed carrot slices were immersed into these coatings. All the samples were placed in expanded polystyrene trays, wrapped
in polyvinylchloride film and stored at 10
C/15 days. During storage, all the samples had counting <100 CFU/g for Staphylococcus
aureus and <3 MPN/g for Escherichia coli. Starch + 0.5% chitosan coating controlled the growth of mesophilic aerobes, yeasts and
molds and psychrotrophs during the first five days of storage, ultimately presenting reductions of only 0.64, 0.11 and 0.16 log cycles,
respectively, compared to the control. Starch + 1.5% chitosan coated samples showed reductions in mesophilic aerobes, mold and
yeast and psychrotrophic counting of 1.34, 2.50 and 1.30 log cycles, respectively, compared to the control. The presence of 1.5%
chitosan in the coatings inhibited the growth of total coliforms and lactic acid bacteria throughout the storage period. The use
of edible antimicrobial yam starch and chitosan coating is a viable alternative for controlling microbiological growth in minimally
processed carrot.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Edible antimicrobial coating; Chitosan; Starch

1. Introduction to reduce, inhibit or delay the growth of microorganisms

The greatest losses in food are due to microbiological
alterations. Many chemical and physical processes have
been developed to preserve food quality. Among such
processes, adequate packaging is a fundamental factor
in the conservation and marketing phases. Thus, pack-
aging plays a prominent role in maintaining food quality
(Debeaufort, Quezada-Gallo, & Voilley, 1998).
Antimicrobial films and coatings have innovated the
concept of active packaging and have been developed
*
Corresponding author. Address: Food Science Department,
Universidade Federal de Viçosa, 36570-000 Viçosa-MG, Brazil.
E-mail address: nfsoares@ufv.br (N.F.F Soares).
on the surface of foods in contact with the packaged
product (Appendini & Hotchkiss, 2002).
In most fresh or processed foods, microbial contam-
ination occurs at a higher intensity on the food surface,
thus requiring an effective microbial growth control
(Padgett, Han, & Dawson, 1998). Traditionally, antimi-
crobial agents are added directly to the foods, but their
activity may be inhibited by many substances in the food
itself, diminishing their efficiency. In such cases, the use
of antimicrobial films or coatings can be more efficient
than adding antimicrobial agents directly to the food
since these may selectively and gradually migrate from
the package onto the surface of the food, thereby high
concentrations being maintained when most necessary
(Ouattara, Simard, Piette, Bégin, & Holley, 2000).

0956-7135/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2004.10.024
 A.M. Durango et al. / Food Control 17 (2006) 336–341
Edible antimicrobial films and coatings have shown
to be an efficient alternative in controlling food con-
tamination. The growth of both deteriorating and
pathogenic microorganisms may be prevented by incor-
porating antimicrobial agents into edible films or coat-
ings (Debeaufort et al., 1998).
The antimicrobial agents most commonly utilized in
edible coatings are: sorbic acid, propionic acid, potas-
sium sorbate, benzoic acid, sodium benzoate and citric
acid (Quintavalla & Vicini, 2002). Bacteriocins, such as
nisin and pediocin (Sebti & Coma, 2002); enzymes, such
as peroxidase and lysozyme (Padgett et al., 1998); and
polysaccharides displaying natural antimicrobial prop-
erties, such as chitosan, (Debeaufort et al., 1998) are
also being used as antimicrobial agents.
Chitosan, is a polysaccharide obtained by deacetyla-
tion chitin, originated from crustacean exoskeleton
and fungal cell walls. Chitosan has widely been used in
antimicrobial films and coatings due to its property of
inhibiting the growth of many pathogenic bacteria and
fungi (Romanazzi, Nigro, Ippolito, Di Venere, & Sal-
erno, 2002). In some fungi, chitosan can produce altera-
tions of membrane functions, by interaction with the
strongly electronegative microbial surface leading to
changes in permeability, metabolic disturbances, and
eventually death (Fang, Li, & Shih, 1994).
According to Muzzarelli et al. (1990) chitosan antimi-
crobial activity against bacteria, could be due to the
polycationic nature of its molecule, which allows inter-
action and formation of polyelectrolyte complexes with
acid polymers produced at the bacteria cell surface (lipo-
polysaccharides, teichoic and teichuronic acids or capsu-
lar polysaccharides). Chitosan based films and coatings
tested on Listeria monocytogenes were found to inhibit
the growth of this organism (Coma et al., 2002). El Gha-
outh, Ponnampalam, Castaigne, and Arul (1992)
showed that coatings based on 1% and 2% chitosan re-
duced the incidence of tomato deterioration, mainly that
caused by Botrytis cinerea. Studies have shown that
chitosan based coatings have the potential to increase
the shelf life of fresh fruits and vegetables, inhibiting
the growth of microorganisms, reduced ethylene pro-
duction, increased internal carbonic gas and decreased
oxygen levels (Lazaridou & Biliaderis, 2002).
Among the polysaccharides used in the production of
edible packaging, starch is the natural biopolymer most
commonly used. Starch can be an interesting alternative
for edible films and coatings because this polymer is
cheap, abundant, biodegradable, edible and easy of
use (Mali, Grossmann, Garcı́a, Martino, & Zaritzky,
2002).
Studies carried out by Lawton (1996), show that
starch based films and coatings exhibit different proper-
ties, attributed to the amylose content in the starch.
Yam (Dioscorea sp) could be a good source of starch
for the production of edible films and coatings, since
337
its starch contains about 30% of amylose, and amylose
is responsible for the film forming capacity of starches
(Mali et al., 2002).
Carrot (Daucus carota L.) is one of the most popu-
larly consumed vegetables, but marketing is limited by
its fast deterioration during storage, due to physiologi-
cal changes that reduce its shelf life (Peiyin & Barth,
1998). The product loses its firmness and develops
odors characteristic of anaerobic catabolism, due to
the high respiration rate and microbiological deteriora-
tion (Barry-Ryan, Pacussi, & OÕBeirne, 2000).
Minimally processed carrot quickly lose their bright
orange color during storage, developing a whitish
appearance or white blush on its surface (Bolin & Hux-
soll, 1991), thereby reducing consumersÕ acceptability.
Both microbial proliferation and white blush on the sur-
face of the product can be controlled by application of
biopolymer based edible coatings (Cisneros-Zevallos,
Saltveit, & Krochta, 1997).
The objective of this work was to develop a yam
starch and chitosan based edible antimicrobial coating
to evaluate its effect on the microbiota normally present
in minimally-processed carrot.
2. Materials and methods
The experiment was conducted at the Packaging Lab-
oratory of the Universidade Federal de Viçosa-MG,
Brazil. The experimental design was arranged in a ran-
domized complete design using a split plot scheme, with
the variable treatments being coatings 1, 2, 3 and 4 in
the plot and the variable times being 0, 5, 10 and 15 days
in the split plot, with two repetitions in triplicate.
2.1. Edible coating
The coatings were prepared using yam starch (Dios-
corea sp) and chitosan. Yam starch was isolated accord-
ing to Cruz and El Dash methodology (1984). Chitosan
(85% deacetylated) was acquired from Padetec (Fort-
aleza Ceará–Brazil).
The coatings were prepared by heat-gelatinization
using suspensions of 4% yam starch (w/w) and 2% glyc-
erol (w/w) (Mali et al., 2002); chitosan was then added in
concentrations of 0.5% and 1.5% (w/w) previously dis-
solved in 0.4% glacial acetic acid (w/w). An agitator (Ul-
tra Turrax T 18 basic) was used at 10.000 rpm for 5 s to
homogenize the suspensions. Starch coatings without
chitosan were also prepared (Table 1).
2.2. Sample preparation and coating application
Carrot (Daucus carota L.) from an unknown cultivar
was purchased at the local market, washed, manually
peeled and cut into slices (5 mm thick) using a vegetable
338 A.M. Durango et al. / Food Control 17 (2006) 336–341

Table 1
Composition of edible antimicrobial coatings used in minimally
processed carrots
Treatment Starch (%) Glycerol (%)
Coating 1 (control) – –
Coating 2 4.0 2.0
Coating 3 4.0 2.0
Coating 4 4.0 2.0
processor (Robout Coup). The carrot slices were placed
in sanitized nylon bags (200 mg/L active chlorine) and
rinsed (3 mg/L active chlorine), at 5
C, for 10 min and
centrifuged at 800g for 10 min. Samples containing
80 g of sliced carrots were submerged in different coat-
ings for 3 min and air flow dried at 20
C for 3 h. The
control (noncoated carrots) was submerged in sterile dis-
tilled water under similar conditions. All the samples
were placed in expanded polystyrene trays, wrapped in
polyvinylchloride (PVC) film and stored at 10
C for
15 days.
2.3. Microbiological analyses
To evaluate the microbiological efficiency of the anti-
microbial coatings on the carrot microbiota, microbio-
logical analyses of mesophilic aerobes, total and fecal
coliforms, Escherichia coli, Staphylococcus aureus, yeasts
and molds, lactic acid bacteria and psychrotrophs were
carried out in the coated and noncoated carrot samples
at 0, 5, 10 and 15 storage days, according to the method-
ology described by the Compendium of Methods for the
Microbiological Examination of Foods (Vanderzant &
Splittstoesser, 1992). At time zero, the coated carrots
were analyzed after the treatment.
2.4. Statistical analyses
The computer program Statistical Analysis System
(SAS) was used in all the statistical analyses. Variance
analysis (ANOVA) tests were applied to the split plots
and regression analysis was performed at a level of 5%
significance.
3. Results
During storage, all the samples showed absence of
Chitosan (%) S. aureus and E. coli with counting of <100 CFU/g
and <3 MPN/g, respectively. Coating 4 inhibited the
– growth of lactic acid bacteria and total coliforms,
0.5
1.5
throughout the storage time presenting counting <10
CFU/g and <3 MPN/g, respectively. Compared to
coating 1 (control), coating 4 showed a reduction of 4,
18 and 2.56 log cycles in the growth of lactic acid bacte-
ria and total coliforms, respectively. (Fig. 1). During
storage time, the samples with coatings 1, 2 and 3
showed similar acid lactic bacteria and total coliform
counts.
According to ANOVA analysis of the mesophilic aer-
obes, yeasts and molds and psychrotrophs count in min-
imally processed carrots, submitted to four coatings
during 15 storage days at 10
C, the coatings and coat-
ing-time interaction showed significant differences at
5% probability (Table 2).
The starch–1.5% chitosan coated sample (coating 4)
showed the lowest counting with 3.88 log CFU/g and
a reduction of 1.34 log cycles compared to the con-
trol, at the end of the experiment (Fig. 2). The starch
coated sample (coating 2) had the highest mesophilic
aerobes count during the first 10 days of storage.
Although presenting a reduced mesophilic aerobes
count when evaluated at 5 storage days, coating 3 failed
to control the development of this microbial group,
afterwards. At the end of the storage time, coating 3
showed a reduction of only 0.64 log cycles, compared
to the control.
The antimicrobial activity of the coatings on the
yeasts and molds was most efficient in coating 4, con-
taining 1.5% chitosan (Fig. 3). At this concentration,
chitosan reduced 2.5 log cycles the counting of this
microbiological group in the carrot, after 15 storage
days. Coating 3, at a concentration of 0.5% chitosan,
controlled the development of yeasts and molds in min-
imally processed carrot, during the first 5 days of stor-
age. After this time, these samples had counting
similar to the control.

Coating 1 Coating 2 Coating 1 Coating 2

6 Coating 3 Coating 4 4
Coating 3 Coating 4
5
3
Log MPN/g
Log CFU/g

3 2

2
1
1

0 0
0 5 10 15 20 0 5 10 15 20
(A) Time (days) (B) Time (days)

Fig. 1. Effect of coatings on lactic acid bacteria (A) and total coliforms (B) in minimally processed carrot stored at 10
C/15 days.
 A.M. Durango et al. / Food Control 17 (2006) 336–341 339

Table 2
Summary of variance analysis of mesophilic aerobes, yeasts and molds and psychrotrophic bacteria in minimally processed carrots, submitted to four
coatings during 15 days of storage at 10
C
FV GL QM Mesophilic aerobes QM Yeasts and molds QM Psychrotrophs
Coating 3 1.6987** 6.0143** 2.6090**
Residue (a) 4 0.0003 0.0009 0.0067
Time 3 11.1527** 9.1639** 13.6104**
Coating · time 9 0.3210** 1.5102** 0.1697**
Residue (b) 12 0.0047 0.0018 0.0013
**Significant at 5% probability (P < 0.05).

6 7

5 6

5
4

Log CFU/g
Log CFU/g

4
3

3
2 Coating 1 = 2,1258 + 0,2787X – 0,0055 X2 R2 = 0, 93
Coating 1 = 2,3140 + 0,3928X – 0,0118 X2 R2= 0,99
= 2,5695 + 0,4804X – 0,0224 X2 R2 = 0, 94 2
Coating 2
Coating 2 = 3,1265 + 0,4248X – 0,0170 X2 R2= 0,93
1 Coating 3 = 1,5950 + 0,3700X – 0,0110 X2 R2 = 0, 96 = 1,9985 + 0,4582X – 0,0154 X2 R2= 0,99
1 Coating 3
Coating 4 = 1,7213 + 0,3548X – 0,0142 X2 R2 = 0, 99 = 1,9887 + 0,3262X – 0,0114 X2 R2= 0,99
Coating 4
0
0
0 5 10 15
Time (days) 0 5 10 15
Time (days)
Fig. 2. Effect of coating on the growth of mesophyll aerobes in
minimally processed carrot stored during 15 days at 10
C. Fig. 4. Psychrotrophic counting in minimally processed carrot sub-
mitted to four coatings during 15 days at 10
C.

Coating 1 = 1,7840 + 0,1613X – 0,0023 X2 R2= 0,84

6 4. Discussion
Coating 2 = 2,0525 + 0,1615X – 0,0047 X2 R2= 0,95

Coating 3 = 0,6462 + 0,2802X – 0,0005 X2 R2= 0,81

5
Coating 4 = 1,0500 + 0,0900X – 0,0100 X2 R2= 0,93 Coating 4, consisting of 1.5% chitosan added—starch
was the most efficient in controlling the microorganisms
4
evaluated, normally present in minimally processed car-
Log CFU/g

rot, stored at 10
C for 15 days. At this concentration,

3
2 bacteria and total coliforms, during storage time. Lactic
1 tion (Zagory, 1999). According to Carlin, Nguyen-the,
0
0 5
10
Time (days)
Fig. 3. Effect of coatings on the growth of filamentous fungi and yeast
in minimally processed carrot stored at 10
C for 15 days.
The effect of the coatings on minimally processed
carrotÕs psychrotrophic microbiota was greater for coat-
ing 4 (Fig. 4), which presented the lowest psychrotrophic
counting, 4.3 log CFU/g, at the end of the storage peri-
od. Starch coated samples (coating 2) presented the
highest psychrotrophic counting throughout the storage
period, starting at 3.0 log CFU/g, and reaching
5.8 log CFU/g after 15 days of storage. Coating 3 did
not inhibit the growth of this microbiological group,
compared to coating 1 (control), with a reduction of
0.16 log cycle at the end of 15 storage days.
the coating totally inhibited the growth of lactic acid
acid bacteria have been associated to carrot deteriora-
Cudennes, and Reich (1989), deterioration of minimally
processed carrot after 14 days storage at 10
C was asso-
15
ciated to Leuconostoc mesenteroides populations due to
the process of lactic fermentation. Marchetti, Casadei,
and Guerzoni (1992), found lactic acid bacteria counting
of 3.16 · 105 CFU/g and 108 CFU/g in carrot salad after
7 day storage at 5
C. Coating 4 also had a satisfactory
performance in controlling mesophilic aerobes, with a
reduction of 1.34 log cycles at the end of the storage per-
iod. Such reduction is considerable, when compared to
other methods applied to reduce the microbial load in
foods. For instance, the sanitation method used in sliced
carrot showed a reduction in the number of mesophilic
aerobic bacteria of, approximately, one log cycle, using
200 mg/L of chlorine during the process (Silva, 2003). A
lower efficiency of this process was found by Amanati-
dou, Slump, Gorris, and Smid (2000), in sliced carrot
with a reduction of only 0.4 log cycle in the microbial
340 A.M. Durango et al. / Food Control 17 (2006) 336–341
load of mesophilic aerobes. The number of mesophilic
aerobes found by Chervin and Boisseau (1992), in min-
imally processed carrots was 6.7 log CFU/g. Buick and
Damoglou (1987) showed that 70% of the initial micro-
biota in minimally processed sliced carrot consists of
Erwinia ssp, which attacks the carrot, causing the so-
called bacterial wet rot (Müeller, 1981). In this work,
the coating containing 0.5% chitosan did not present a
good performance in controlling deteriorating bacteria,
despite the initial low number of mesophilic aerobes,
lactic acid bacteria and psychrotrophs found in mini-
mally processed carrot.
In 1993, carrot was associated to an infection out-
break caused by enterotoxigenic E. coli in the US. In
Lebanon, the prevalence of S. aureus in raw carrot has
been 14.3% (Beuchat, 2002). Zheng and Zhu (2003)
showed that 1% chitosan at a degree of deacetylation
of 88.76% and molecular weight of 48.5 kDa, has an
antimicrobial effect on E. coli and S. aureus at an inhibi-
tion rate of 100% on these microorganisms growth.
Concentration of 1% chitosan presented reductions be-
tween 1 and 2 log cycles in the total counting of bacteria
in small meat pies; concentrations of 0.2% and 0.5% did
not have an inhibiting effect on the deteriorating micro-
flora, but the number of bacteria before starting the
experiment was high >107 CFU/g, and chitosan is likely
to be more effective in low microbial populations (Dar-
madji & Izumimoto, 1994).
Regard to the action of the coatings on yeasts and
molds, coating 4 presented the highest fungicidal action.
Studies have shown that the effect of chitosan on some
fungi is mainly due to alterations in the functions of
the cellular membrane (Fang et al., 1994). The yeasts
most commonly isolated from vegetables belong to the
genera Cryptococcus, Rhodotorula and Candida; among
the moulds are Fusarium, Mucor, Rhizopus and Penicil-
lium (Francis, Thomas, & OÕBeirne, 1999). The main
disease caused by fungus in carrots is Sclerotinia rot.
Cheah, Page, and Shepherd (1997) showed that chitosan
applied as a 2% or 4% solution inhibits Sclerotinia stor-
age rot on carrots, and that the effect was due, at least in
part, to inhibition of fungal growth.
In this work, coating 4 containing 1.5% chitosan was
the most efficient on the psychrothrophic flora in mini-
mally processed carrot. According to Garg, Churey,
and Splittstoesser (1990) the shelf life of refrigerated
vegetables is especially affected by the psychrotrophic
population, with Pseudomonas sp. being the main psy-
chrotrophic agent present in these foods. Several
authors have found psychrotrophic counting in mini-
mally processed carrot sold at supermarkets ranging be-
tween 106 and 109 UFC/mL (Rosa, 2002). Listeria
monocytogenes is one of the pathogens most frequently
associated with ready-to-use vegetables (Francis et al.,
1999). Studies conducted by Coma et al. (2002) show
that chitosan coatings have a bactericidal effect on Lis-
teria monocytogenes and that such activity was probably
due to the positive charges of chitosan which interfere
with the negatively charged residues of macromolecules
at the Listeria cell surface, presumably by competing
with calcium for electronegative sites on the membrane.
Chitosan has been reported to inhibit various spoilage
bacteria, through its capacities to both bind water and
inactivate various enzymes, and through its ability to
absorb nutrients normally used by bacteria (Ouattara
et al., 2000).
Coating 2, which contained only starch, favored the
development of mesophilic and psychrotrophic bacteria,
indicating that these microorganisms may have utilized
this carbohydrate as a source of energy.
5. Conclusions
The results of this experiment showed that the use of
an antimicrobial coating consisting of chitosan-added
yam starch is a viable alternative in controlling the mic-
robiota present in minimally processed carrot, since the
growth of lactic acid bacteria, total coliforms, psychro-
trophs, yeasts and molds and mesophilic aerobes, was
substantially inhibited by the application of 1.5% chito-
san coating.
Based on the concept of protection barrier technol-
ogy, the use of such coating may contribute to improve
safety in minimally processed carrot thereby prolonging
its shelf life. Coating may be applied on minimally pro-
cessed fruits and vegetables, combined to other types of
controls, such as quality raw material, hygienic process-
ing conditions and storage temperatures. The combina-
tion of these treatments as barrier offers a greater
potential for shelf-life extension of minimally processed
vegetables.
Acknowledgements
The authors thank CNPq and FAPEMIG for the
financial support.
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