Food Control 27 (2012) 37e44

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Food Control
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Growth potential of Escherichia coli O157:H7 on fresh-cut fruits (melon and

pineapple) and vegetables (carrot and escarole) stored under different conditions
Maribel Abadias a, *, Isabel Alegre b, Marcia Oliveira b, Rosa Altisent a, Inmaculada Vias b
a
b

IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198 Lleida, Catalonia, Spain

University of Lleida, XaRTA-Postharvest, 191 Rovira Roure, 25198 Lleida, Catalonia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 8 July 2011
Received in revised form
14 February 2012
Accepted 28 February 2012

Minimally processed fruits and vegetables are ready-to-eat and do not require further treatment at
home. These foods are usually stored in a modied atmosphere and should be maintained at refrigerated
conditions until consumption. These fruits and vegetables can become contaminated by foodborne
pathogens such as Escherichia coli O157:H7, Salmonella and Listeria monocytogenes, and it has been
demonstrated that current industrial sanitising washing treatments do not guarantee the total elimination of the pathogen when present. Thus, it is very important to elucidate whether pathogens are able
to grow or survive during storage at different conditions. This study was conducted to determine the
effect of the type of produce (escarole, carrot, pineapple or melon), package gas composition (air or
modied atmosphere) and temperature (5 or 25
C) on the population dynamics of a strain of E. coli
O157:H7. For vegetables, the growth in two lms, which created different O2 and CO2 concentrations, and
air were compared. At 25
C, growth of E. coli O157:H7 was higher in fresh-cut carrots than in endive,
reaching populations between 7.0e8.4 log cfu g1 and 5.2e6.3 log cfu g1 after 3 days of storage,
respectively. In fruits, E. coli O157:H7 grew well in fresh-cut melon regardless of the atmospheric
conditions in the package, reaching populations of 8.5 and 8.9 log cfu g1 after 1 day of storage in
modied atmosphere packaging (MAP) or under air conditions, respectively. No growth was observed in
the fresh-cut pineapple. At 5
C, E. coli O157:H7 did not grow but survived throughout the studied period
in all tested commodities. This work emphasises the importance of strict temperature control from
processing to consumption, including transportation, distribution, storage and handling in supermarkets
and by consumers.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Minimally processed fruits and vegetables
Survival
Population dynamics
Foodborne pathogens
Modied atmosphere packaging
Storage conditions

1. Introduction
Minimally processed fruits and vegetables are widely available
and generally considered safe to eat by consumers. However, the
majority of these products require no further treatment and are
eaten raw, posing a potential safety problem. They may be
contaminated with foodborne pathogens such as Escherichia coli
O157:H7, Salmonella and Listeria monocytogenes. Unfortunately, it
has been demonstrated that current industrial sanitising washing
treatments do not guarantee the total elimination of the pathogen
when present (Abadias, Alegre, Usall, Torres, & Vias, 2010;
Abadias, Usall, Oliveira, Alegre, & Vias, 2008; Beuchat, 1996; Parish
et al., 2003). Outbreaks of E. coli O157:H7 infection have been linked
to the consumption of fresh fruits and vegetables such as alfalfa
and radish sprouts, different lettuce varieties, carrots, spinach,

* Corresponding author. Tel.: 34 973 032 850x1502; fax: 34 973 238 301.
E-mail address: isabel.abadias@irta.cat (M. Abadias).
0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2012.02.032

unpasteurised apple cider, berries and melon (Beuchat, 1996;

De Roever, 1998; Ethelberg et al., 2010; Friesema et al., 2008; Sodha
et al., 2010). In 1994, an outbreak of non-O157:H7 E. coli (O11:H7) in
the US was linked to pineapple consumption (Sivapalasingam,
Friedman, Cohen, & Tauxe, 2004). Recently, there was a large
outbreak of Haemolytic Uraemic Syndrome (HUS), caused by Shiga
toxin-producing E. coli O104 in Germany. Figures updated by the
European Centre for Disease Control and Prevention (ECDC) on 27
July 2011 reported almost 4000 people infected in Europe and 46
people having died from STEC, 45 of them in Germany (ECDC, 2011).
German ofcials initially suspected cucumbers from Spain as the
source of contamination, but further tests showed that those
vegetables did not contain the E. coli O104:H4 strain. Epidemiological evidence suggested that STEC-contaminated sprouts were
the vehicle of infection (EFSA, 2011), but this case still remains
unsolved (CDC, 2011; Frank et al., 2011).
Several studies have demonstrated that E. coli O157:H7 could
survive and/or grow in a range of minimally processed fruits and
vegetables, such as shredded lettuce (Abdul-Raouf, Beuchat, &

38

M. Abadias et al. / Food Control 27 (2012) 37e44

Ammar, 1993; Francis & OBeirne, 2001; Oliveira et al., 2010), sliced
cucumber (Abdul-Raouf et al., 1993), shredded carrot (Abdul-Raouf
et al., 1993), dry coleslaw mix (Francis & OBeirne, 2001), soybean
sprouts (Francis & OBeirne, 2001), packaged fresh-cut salad (Luo,
He, & McEvoy, 2010), apples (Alegre, Abadias, Anguera, Oliveira, &
Vias, 2010; Dingman, 2000), honeydew melon (Leverentz et al.,
2001, 2003) and peaches (Alegre, Abadias, Anguera, & Vias,
2010) at abuse temperatures.
Modied atmosphere packaging (MAP) has been successfully
and widely used in combination with refrigeration for whole and
minimally processed fruits and vegetables as a packaging strategy
to maintain product safety and to extend the shelf-life of these
foods (Werner & Hotchkiss, 2006). MAP systems generally utilise an
internal package atmosphere of something other than air in
a hermetically sealed package of suitable permeability, O2, CO2 and
N2 being the most commonly employed. O2 levels are commonly
reduced below, and CO2 increased above, atmospheric levels.
Vegetable respiration alone will decrease O2 and increase CO2
levels inside the package, thereby passively modifying the in-pack
atmosphere. This leads to a reduction of the produce respiration
rate, which retards ripening and senescence. Moreover, MAP
technology suppresses the growth of most indigenous aerobic ora.
However, under certain conditions, the growth of some anaerobic
or microaerophilic psychrotrophic microorganisms, such as
L. monocytogenes and Clostridium spp., might be allowed or even
stimulated. Moreover, extending the shelf-life of minimally processed produce increases the time available for pathogens, if
present, to grow.
The aim of this study was to determine the effect of type of
produce, package gas composition and temperature on the growth
of a strain of E. coli O157:H7. One leafy vegetable (escarole), grated
root (carrot) and two fruits with different pH values (pineapple and
melon) were used.
2. Material and methods
2.1. Microorganism and preparation of inoculums
Strain NCTC 12900 of E. coli O157:H7 (E. coli) was used in this
study. This strain is non-pathogenic and devoid of the ability to
produce verotoxins but phenotypically similar to the toxigenic
strain of E. coli O157:H7. The strain was adapted to grow on Tryptone Soy Agar (TSA, Oxoid, CM0131) supplemented with
100 mg mL1 of streptomycin (TSAS) and maintained at 5  1
C on
TSAS. When required, the strain was subcultured for 24  2 h at
37  1
C on TSAS, inoculated in 50 mL of Tryptone Soy Broth (TSB,
Oxoid CM0129) supplemented with 100 mg mL1 streptomycin
(TSBS) and incubated at 150 rpm for 18e20 h at 37  1
C. Afterwards, the culture was centrifuged at 9820  g for 10 min, and the
resultant pellets were resuspended in saline peptone (SP, 8.5 g L1
NaCl and 1 g L1 peptone). Bacterial concentrations were estimated
by comparing the suspension transmittance at 420 nm with
a previously determined standard curve. Suspensions of about 107
and 105 cfu mL1 were prepared to inoculate vegetables and fruits,
respectively. The concentration applied was conrmed by plating
0.1 mL of an appropriately diluted culture on TSAS.
2.2. Fruits and vegetables
E. coli growth was studied in different vegetal matrices: pineapple (Ananas sativus L., Del Monte Gold), melon (Cucumis melo L.
var. Piel de sapo), curly endive or escarole (Cichorium endivia L. var.
crispa) and carrot (Daucus carota L.), which were purchased from
a local supermarket the day before the experiment and stored at
4  1
C.

2.3. Preparation and inoculation of samples

Carrots were washed in tap water, topped, tailed, hand-peeled
and grated in a vegetable processing machine (model CL50, Robot
Coupe, France) equipped with a 2 mm grating disk. Escarole outer
leaves were discarded, and the inner leaves were hand-cut, washed
in tap water and spin-dried in a handheld household spinner for
approximately 1 min to remove excess water. Grated carrot and cut
endive were inoculated by being dipped into 2 L of a bacterial
suspension (105 cfu mL1) for 2 min at 150 rpm. Inoculated vegetables were dried in a laminar ow biosafety cabinet. Samples
(w15 g) of fresh-cut carrot or endive were packaged under three
different atmospheric conditions: air and two passive modied
atmospheres created by means of using different 35 mm-oriented
polypropylene (OPP) plastic lms (Amcor Flexibles, Ledbury,
Herefordshire, UK). FILM I (35PA60) had an O2 and CO2 permeability of 3500 cm3 lm1 day1 atm1 at 23
C and water steam
permeability of 0.9 g m2 day1 at 25
C and 75% relative humidity.
FILM II (35 PAPlain, lower permeability than FILM I) had an O2 and
CO2 permeability of 1100 cm3 m2 day1 atm1 at 23
C and water
steam permeability of 0.9 g m2 day1 at 25
C and 75% relative
humidity. Finally, air conditions (Air) were obtained by manually
perforating FILM I with an aseptic needle. Five holes per bag were
made. Bags (120  120 mm) were preformed and sealed using
a heating bar. Bags were stored at 5 and 25
C.
Before use, whole fruits were washed with tap water, surfacesterilised with 70% ethanol and cut into 1-cm slices. For experiments with air conditions, 12-mm diameter fruit tissue plugs
(w1 g) were taken using a cork borer, and the plugs were randomly
placed in glass test tubes. Plugs were inoculated with 15 mL of the
107 cfu mL1 suspension of E. coli, and tubes were incubated at 5
and 25
C for different periods of time. For experiments with freshcut fruit packaged in a modied atmosphere, melon and pineapple
slices were sliced through the equator with a sterile knife, and 10mm-thick rings were cut. Each ring was cut into 8 pieces. A small
cavity (well) was made in each piece to place the inoculums
(Conway, Leverentz, & Saftner, 2000; Leverentz et al., 2001), and the
pieces were subsequently inoculated with 15 mL of the 107 cfu mL1
suspension of E. coli. Fruit pieces were placed in polypropylene
trays, sealed in a thermo-sealing machine with microperforated
lm (40,000 cc m2 day1, FFP Packaging Solutions Ltd., Northampton, UK) and stored at 5 and at 25
C.
Three replications per temperature (three fruit plugs, three bags
or three trays) were performed, and the experiment was conducted
twice.
2.4. Microbial count
Populations of E. coli in inoculum suspensions and in dips were
enumerated by surface plating duplicate samples (0.1 mL), which
were serially diluted in SP, on TSAS plates followed by incubation at
37  1
C for 24  2 h.
The initial concentration of E. coli in the fresh-cut fruit and
vegetable samples was determined in triplicate within 1e2 h of
application (0 h) and after regular intervals throughout the storage
period depending on the storage temperature. Microbial determinations were performed until samples displayed visual symptoms
of decay. To recover E. coli from fresh-cut fruit, each plug was put in
an 80-mL lter bag with 9 mL of SP, homogenised in a stomacher
blender for 2 min at high speed (Bagmixer 100 Minimix, Interscience), ten-fold diluted in SP, plated in duplicate on TSAS and
incubated at 37  1
C for 24  2 h. Colonies were then counted, and
the results were expressed as cfu per gram. For vegetables, 10-g
samples were combined with 90 mL of SP in a sterile polyethylene bag and pummelled with a Stomacher at 150 rpm for

M. Abadias et al. / Food Control 27 (2012) 37e44

2 min. Wash uid was serially diluted, surface plated in duplicate

on TSAS and incubated at 37  1
C for 24  2 h, as described before.
Randomly selected presumptive E. coli O157:H7 colonies were
conrmed using an E. coli O157 Latex Test Kit (Oxoid Ltd., Cambridge, UK).

3. Results
3.1. Growth and survival of E. coli O157:H7 in fresh-cut escarole at
different temperatures and packaging atmospheres
The initial concentration of E. coli on fresh-cut escarole was
between 4.3 and 4.5 log cfu g1 (Fig. 1A). E. coli grew in fresh-cut
escarole at 25
C regardless of the packaging lm. In escarole
packaged in FILM I, E. coli signicantly increased during the rst day
of storage; however, no signicant increase was observed at later
time points. In contrast, when the fresh-cut escarole was packaged
in FILM II, maximum growth was achieved after three days of
storage, reaching 6.3 log cfu g1. There were no signicant differences between the growth rates in FILM I and FILM II. However,
growth under air conditions was signicantly lower except at day 2.
The O2 concentration signicantly decreased (Fig. 2A) and reached
anaerobic conditions after 2 and 3 days of storage for FILM II and
FILM I, respectively. CO2 levels increased during the experiment,
with nal values between 11.2 and 15.7% for both lms (Fig. 2B).
At 5
C, the E. coli population signicantly declined in fresh-cut
escarole during storage (Fig. 1A). Nevertheless, it survived
throughout the storage period under all packaging conditions.
When the samples were stored in air, the population decreased
0.7 log units in 8 days. For fresh-cut escarole stored in FILM I and
FILM II bags, the populations decreased 0.6 and 0.9 log units after 8
days and 0.8 and 1.0 log units after 10 days of storage at 5
C,
respectively. The O2 concentration inside FILM I bags signicantly
decreased from 20.7% to 9.4% after day 8 and, then, remained
unchanged (Fig. 2A) and progressively decreased in FILM II. The O2
concentration in FILM II bags was signicantly lower than in FILM I,
with a nal value of 3.7%. The CO2 concentration gradually
increased at 5
C but never exceeded 10% (Fig. 2B).

2.5. Gas analysis

On each sampling date, CO2 and O2 concentrations within three
of each package type were analysed using a handheld gas analyser
(CheckPoint O2/CO2, PBI Dansensor, Denmark). Gas extraction was
performed with a hypodermic needle inserted through an adhesive
septum previously xed to the bag.
2.6. Quality of fruit
Soluble solids and the titratable acidity of each batch of melon
and pineapple used for the experiments were determined. Soluble
solids concentration (SS) was determined by measuring the
refractive index of the juice of a piece of fruit in a digital refractometer (model PR-100, ATAGO, Tokyo, Japan), and the data were
expressed in
Brix. Titratable acidity (TA) was measured as follows:
10 mL of pulp juice was diluted with 10 mL of H2O and titrated with
0.1 M NaOH solution to an end point of 8.1 using phenolphthalein
as an indicator. The results were expressed in grams of citric acid
per litre of melon or pineapple juice. The pH of the esh was also
measured using a puncture electrode for semi-solid and liquid
samples (ref. 5232, Crison, Abrera, Barcelona, Spain).
2.7. Statistical analysis
All experiments were performed in triplicate and repeated
twice. Therefore, the reported data represent the means of six
values. Population data were transformed to log, and the General
Linear Model procedure (GLM) of SAS software (SAS Institute,
version 9.1 for Windows, Cary, NC) was performed. Means were
separated using Duncans Multiple range test at the 5% signicance
level.

9.0
8.0

3.2. Growth and survival of E. coli O157:H7 in grated carrots at

different temperatures and packaging atmospheres
The growth of E. coli in grated carrots packaged under different
atmospheric conditions and stored at 25
C was higher than that

9.0

Film I-25C

39

Film II-25C

Film I-25C
Film II-25C

8.0

Air-25C

Air-25C

Film I-5C

7.0

Film II-5C

E. coli, log CFU g-1

E. coli, log cfu g-1

Film I-5C
Air-5C

6.0
5.0

4.0

Film II-5C

7.0

Air-5C

6.0
5.0
4.0

3.0

3.0

2.0

2.0
0

4
6
Storage time, days

10

12

10

Storage time, days

25C
Air
Film I
Film II

0
4.3 a x
4.5 a x
4.5 a x

1
5.2 b y
5.7 a y
5.7 a y

2
5.3 b y
6.0 a y
5.5 ab y

3
5.2 b y
6.0 a y
6.3 a z

5C
Air
Film I
Film II

0
4.3 a x
4.4 a z
4.5 a z

2
4.0 b y
4.3 a y
4.3 a z

6
3.7 ab x
4.0 a x
3.6 b y

8
3.6 b x
3.9 a x
3.6 b y

10
nd
3.7 a w
3.5 a y

25C
Air
Film I
Film II

0
4.5 a x
4.4 a x
4.5 a x

1
8.4 a y
7.0 c y
7.7 b z

2
8.5 a z
7.9 b z
8.0 b z

3
8.4 a zy
8.0 a z
7.0 a y

5C
Air
Film I
Film II

0
4.5 a z
4.4 a z
4.5 a z

2
4.5 a z
4.3 a z
4.3 a z

6
4.1 a y
4.1 a z
4.0 a y

8
4.0 a y
3.2 a y
3.8 a y

Fig. 1. Populations of Escherichia coli O157:H7 (log cfu g1) inoculated by dipping in a suspension containing 105 cfu mL1 in fresh-cut endive (A) and grated carrot (B), which were
packaged in different lms and stored at 25
C or 5
C. The data represent the mean of six determinations. Bars represent standard deviation of the mean. When vertical bars are not
visible, they are smaller than the symbol size. For each storage temperature, different letters (a, b, c) within columns indicate signicant differences (P < 0.05) of E. coli O157:H7
populations among the tested lms, and different letters within rows (w, x, y, z) indicate signicant differences among the storage times.

40

M. Abadias et al. / Food Control 27 (2012) 37e44

O2 concentration, %

18

21

O2-Film I-25C
O2-Film II-25C

CO2 concentration, %

21

O2-Film I-5C
O2- Film II-5C

15
12
9
6
3

18

CO2-Film I-25C
CO2-Film II-25C

CO2-Film I- 5C
CO2-Film II-5C

15
12
9
6
3

0
0

10

12

4
6
8
Storage time, days

Storage time, days

25C
Film I
Film II

0
20.7 a z
20.7 a z

1
8.0 a y
4.6 b y

2
3.3 a x
0.5 b x

3
0.3 a w
0.1 a x

5C
Film I
Film II

0
20.7 a z
20.7 a z

2
16.2 a y
14.8 b y

6
13.5 a yx
7.9 b x

8
9.4 a x
5.7 a w

10
10.7 a x
3.7 b v

10

12

25C
Film I
Film II

0
0.2 a z
0.2 a x

1
9.5 b y
12.0 a zy

2
13.5 a x
12.5 a z

3
15.7 a w
11.2 b y

5C
Film I
Film II

0
0.2 a x
0.2 a w

2
3.5 b y
4.9 a x

6
4.9 b zy
7.8 a y

8
6.7 a z
7.9 a zy

10
5.9 b z
8.3 a z

Fig. 2. O2 (A) and CO2 (B) concentrations (%) inside fresh-cut endive packaged in FILM I or FILM II and stored at 25
C or 5
C. Bars represent standard deviation of the mean. When
vertical bars are not visible, they are smaller than the symbol size. For each storage temperature, different letters (a, b, c) within columns indicate signicant differences (P < 0.05) of
gas concentrations among the tested lms, and different letters within rows (w, x, y, z) indicate signicant differences among the storage times.

were no signicant differences (P < 0.05). The O2 concentration

inside FILM I bags (Fig. 3A) decreased during the rst day of storage
at 5
C, and then no signicant differences were observed. In FILM II
bags, the O2 concentration progressively decreased, reaching 0.2%
at the end of the experiment. The concentration of CO2 (Fig. 3B)
increased in FILM II bags throughout the experiment and reached
18% by the end of the experiment. The concentrations in FILM I bags
were signicantly lower at each tested time.

observed for fresh-cut endive (Fig. 1B). Initial populations were

between 4.4 and 4.5 log cfu g1. Maximum growth was observed in
samples stored under air, with a 4-log increase observed in populations after 1 day of storage. However, after 3 days of storage,
there were not signicant differences between E. coli populations.
The O2 concentration fell to 3.5 and 1.0% in FILM I and FILM II bags,
respectively, after 1 day of storage at 25
C (Fig. 3A). The CO2
concentration inside the MAP bags rose greatly after 1 day of
storage at 25
C (Fig. 3B), reaching levels of 20.7 and 28.6% in FILM I
and FILM II bags, respectively. The CO2 concentration attained
a maximum level (34.6%) after 2 days of storage in FILM II bags.
Similar to the results achieved with fresh-cut escarole, the E. coli
populations decreased in fresh-grated carrots stored at 5
C
(Fig. 1B). Decreases observed after 8 days of storage at 5
C were 0.5,
1.3 and 0.7 log units in air, FILM I and FILM II, respectively, but there
21

The pineapples used had a high acid content (6.18 g citric

acid L1 of juice), a pH of 3.59 and a soluble solids content of
14.0
Brix, which corresponded to a maturity index (SS/TA) of 2.3.
40
CO2 Concentration, %

18
O2 Concentration,%

3.3. Growth and survival of E. coli O157:H7 in fresh-cut pineapple

at different temperatures and packaging atmospheres

15
12
9

O2-Film I-25C
O2-Film II-25C

O2-Film I-5C
O2-Film II-5C

CO2-Film I-25C
CO2-Film II-25C
CO2-Film I-5C

30

CO2-Film II-5C

20

10

0
0

10

Storage time, days

25C

Film I
Film II

5C

Film I
Film II

0
20.6 a z
20.6 a z

1
3.5 a y
1.0 b y

2
2.6 a y
1.5 a y

3
5.3 a y
1.2 b y

25C

0
20.6 a z
20.6 a z

2
12.0 a y
8.6 b y

6
9.6 a y
1.8 b x

8
12.2 a y
0.2 b w

5C

Film I
Film II

Film I
Film II

4
6
Storage time, days

10

0
0.1 a y
0.1 a x

1
20.7 b z
28.6 a y

2
3
26.0 b z 19.5 b z
34.6 a z 28.7 a y

0
0.1 a y
0.1 a w

2
5.6 b z
7.8 a x

6
8.0 b z
15.9 a y

8
5.2 b z
18.0 a z

Fig. 3. O2 (A) and CO2 (B) concentrations (%) inside fresh-cut endive packaged in FILM I or FILM II and stored at 25
C or 5
C. Bars represent standard deviation of the mean. When
vertical bars are not visible, they are smaller than the symbol size. For each storage temperature, different letters (a, b, c) within columns indicate signicant differences (P < 0.05) of
gas concentrations among the tested lms, and different letters within rows (w, x, y, z) indicate signicant differences among the storage times.

M. Abadias et al. / Food Control 27 (2012) 37e44

In the case of fruit samples, only storage under air conditions

and one MAP condition (one lm) were compared. The initial
population was in the range of 4.9 and 5.3 log cfu g1. E. coli was
unable to grow on fresh-cut pineapple at both studied temperatures (Fig. 4A). At 25
C, the population remained constant after day
2, which was when it started to decrease at both atmospheric
conditions. This decrease was faster in fresh-cut pineapple stored in
MAP than in air. At 5
C, E. coli O157:H7 survived during the whole
experiment, with a signicant population decrease after 8 days of
storage in MAP. In fresh-cut pineapple stored at 5
C in MAP, CO2
and O2 concentrations remained almost constant, with values close
to those of atmospheric conditions (Fig. 5A). By contrast, at 25
C,
the CO2 concentration increased rapidly, with values higher than
38% after 2 days of storage and a nal value of 50.3% at the end of
the experiment (3 days). The packages reached anaerobic conditions after 2 days of storage at 25
C.
3.4. Growth and survival of E. coli O157:H7 in fresh-cut melon at
different temperatures and packaging atmospheres
The acidity of the melon, expressed in g of citric acid, was very
low (1.14 g L1), the pH was 5.94 and the soluble solids content was
9.6
Brix. The obtained maturity index was higher than that of the
pineapple (15.5). Under these conditions, E. coli grew very well at
25
C regardless of the packaging atmosphere, with an increase of
4-log units after one day of storage (Fig. 4B). No growth was
observed at 5
C, but cells survived throughout the storage and
decreased signicantly between 10 and 14 days of storage in MAP.
The CO2 and O2 patterns were similar to those obtained with freshcut pineapple (Fig. 5B). No signicant changes were observed at 5
C,
but CO2 levels rose to 11.3, 25.2 and 39.4% after 1, 2 and 3 days of
storage at 25
C, respectively. At the same time, the O2 concentration
decreased by 11.9, 4.6 and 2.2% during that same time period.
4. Discussion
This study compared the growth of a strain of E. coli O157:H7
(NCTC 12900) on different fresh-cut fruits and vegetables stored

E. coli O157:H7, log cfu g -1

10.0

under different atmosphere and temperature conditions. A leafy

vegetable (escarole), a root (carrot) and two types of fruit with
different pHs and acidities (melon and pineapple) were selected.
Our results have shown that E. coli O157:H7 growth was predominantly dependent on the type of vegetable and temperature and, to
a lesser extent, the atmospheric condition.
At 25
C, E. coli O157:H7 grew on fresh-cut escarole, carrot and
melon but was unable to grow on fresh-cut pineapple. At 5
C, it did
not grow but survived throughout the studied period of time in all
tested commodities. The lower limits of growth for generic E. coli
and E. coli O157:H7 are generally believed to be 5
C and 8
C,
respectively (Palumbo, Call, Schultz, & Williams, 1995; Rajkowski &
Marmer, 1995). With regards to vegetables stored at 25
C, growth
was higher on fresh-cut carrots than on escarole. Abdul-Raouf et al.
(1993) reported that populations of E. coli O157:H7 on shredded
carrots decreased similarly but survived during a 14-day incubation
period at 5
C and that an atmospheric gas composition did not
affect this behaviour. These same authors also reported that E. coli
O157:H7 grew in large inoculum samples of shredded carrots
stored at 12 and 21
C. However, a known carrot phytoalexin,
6-methoxymellein, has been demonstrated to display antibacterial
effects towards Listeria species (Beuchat & Brackett, 1990), inhibit
the growth of several fungi and bacteria (Kurosaki & Nishi, 1983),
and, thus, may also be inhibitory or toxic to E. coli O157:H7. Francis
and OBeirne (2001) did not observe the growth of E. coli O157:H7
12900 in a dry coleslaw mix (80% cabbage 20% carrot) stored at
4
C, and they presumed that the inclusion of carrots in the mixture
may have also affected its survival. However, we have not observed
this antibacterial effect on this same strain.
Growth differences on the different matrices could be due to
different factors: the lower amount of cut surface, nutrient
composition and availability, pH, water activity, the presence of
antimicrobial substances, such as polyphenols, and background
microbiota, among others. It has been shown that certain pathogenic bacteria attach better to the cut or injured surfaces of fruits
and vegetables, and it is likely that the attachment and possible
penetration of pathogens into wounded tissue results in a better
survival and growth on cut surfaces (Gleeson & OBeirne, 2005; Seo

10.0

9.0

Air-25 C

9.0

Air 5 C

41

Air_25C

Air-5C

8.0

MAP-25C

8.0

MAP-25C

7.0

MAP-5C

7.0

MAP-5C

6.0

6.0

5.0

5.0

4.0

4.0

3.0

3.0

2.0

2.0

1.0

1.0
0.0

0.0
0

Storage time, days

25C

Air
MAP
5C

Air
MAP

12

15

Storage time, days

0
5.1 a z
5.3 a z

1
4.9 a z
5.0 a z

2
4.3 a y
4.2 a y

3
4.0 a y
2.5 b x

0
4.9 a z
5.3 a z

2
4.8 a z
4.8 a zy

6
4.7 a z
4.2 a y

8
nd
3.0 x

25C

Air
MAP
5C

Air
MAP

0
4.8 y
5.0 x

1
8.9 z
8.5 y

2
9.0 z
8.8 zy

3
9.3 z
8.7 z

0
4.8 z
5.0 z

2
5.0 z
5.2 z

6
4.9 z
5.0 z

8
4.4 z
4.8 z

10
nd
4.8 z

14
nd
3.5 y

Fig. 4. Population of Escherichia coli O157:H7 (log cfu g1) spot inoculated with 15 mL of a suspension containing 107 cfu mL1 in fresh-cut pineapple (A) and melon (B), packaged in
MAP (diamonds) or air (triangles) and stored at 25
C (open symbols) or 5
C (full symbols). Data represent the mean of six determinations (three replications and two experiment
repetitions). Bars represent standard deviation of the mean. When vertical bars are not visible, they are smaller than the symbol size. For each storage temperature, different letters
(a, b, c) within columns indicate signicant differences (P < 0.05) of E. coli O157:H7 populations among the tested lms, and different letters within rows (w, x, y, z) indicate
signicant differences among the storage times.

42

M. Abadias et al. / Food Control 27 (2012) 37e44

60.0

50.0

B
50.0

O2 / CO2 concentration (%)

O2 / CO2 concentration (%)

60.0

40.0
30.0
20.0
10.0

0.0

40.0

30.0
20.0

10.0
0.0

12

15

Storage time, days

12

15

Storage time, days

Fig. 5. O2 and CO2 concentrations (%) inside fresh-cut pineapple (A) and fresh-cut melon (B), which were packaged in MAP and stored at 25
C or 5
C. The data represent the mean
of six determinations. Bars represent standard deviation of the mean. When vertical bars are not visible, they are smaller than the symbol size. For each storage temperature,
different letters (a, b, c) within columns indicate signicant differences (P < 0.05) of gas concentrations among tested lms, and different letters within rows (w, x, y, z) indicate
signicant differences among the storage times.

& Frank, 1999). Escarole has less cut surface area compared to the
carrot, and E. coli O157:H7 cells most likely could not properly
attach to the surface of the escarole. Moreover, some aqueous
extracts from roots and the aerial parts of Cichorium intybus L.,
which belongs to the same family as escarole (Asteraceae), have
been reported to have antibacterial activities (Petrovic, Stanojkovic,
Comic, & Curcic, 2004). The survival of E. coli O157:H7 in other leafy
vegetables (mainly lettuce) has been reviewed by Delaquis, Bach,
and Dinu (2007). Populations of viable E. coli O157:H7 cells significantly decrease on Iceberg lettuce stored at 5
C and signicantly
increased during storage at 12 or 21
C (Abdul-Raouf et al., 1993).
Recently, Oliveira et al. (2010) also observed the growth of this
same strain on fresh-cut Romaine lettuce stored at 25
C, but no
growth was observed at 5
C. The presence of competing microorganisms on the surfaces of fresh produce has also been reported
to contribute to pathogen reduction. The typical microbiota present
on fresh vegetables is composed of many species and might
compete with pathogens for physical space and nutrients and/or
produce antagonistic compounds that negatively affect the viability
of pathogens (Parish et al., 2003). Babic, Watada, and Buta (1997)
reported that background microbiota on spinach inhibits the
growth of L. monocytogenes. However, different levels of background microbiota did not affect the growth of E. coli O157:H7 and
L. monocytogenes on Romaine lettuce (Oliveira, Vias, Anguera, &
Abadias, 2012).
Pineapple was largely unsuitable for E. coli growth even at 25
C.
At rst and due to other authors results, we presumed that the lack
of growth could be due to the low pH of pineapple (3.28e4.06), as
the concentrations of O2 and CO2 were initially similar to those
achieved in fresh-cut melon, which did not inhibit growth.
However, Alegre, Abadias, Anguera, Oliveira et al. (2010) found that
this same strain was able to grow at 25
C on Granny Smith freshcut apples (pH between 3.29 and 3.35 and a titratable acidity of
7.9e8.4 g malic acid L1 of juice), which would be more restrictive.
E. coli O157:H7 is known to have a high tolerance to acidity
(Benjamin & Datta, 1995; Conner & Kotrola, 1995). Other authors
(Deng, Ryu & Beuchat, 1999) noted that both the pH and the acid
present are also of importance. In TSB-acidied medium, the order
of sensitivity for E. coli O157:H7 at a given pH is acetic acid > citric
acid > malic acid, and the major acids in pineapple and apple are
citric and malic, respectively. Similarly, Conner and Kotrola (1995)
found that three isolates of E. coli O157:H7 did not grow at 25
C
in TSBYE acidied to pH 4.5 using citric acid, but they grew at pH 4.5
when malic acid was used. Strawn and Danyluk (2010) found that
E. coli O157:H7 did not grow and survived poorly on fresh-cut
pineapples (pH ca. 3.6) held at 23, 12 and 4
C. These researchers

stated that this result could be because this pH is below the

minimum pH limit for E. coli O157:H7 growth (Basset & McClure,
2008) and also because of the presence of a high percentage of
unfermentable bres, which may decrease the availability of
nutrients for E. coli O157:H7 metabolism (Mutaku, Erku, & Ashena,
2005). In conclusion, the inability of E. coli O157:H7 to grow on
fresh-cut pineapple at 25
C may be a consequence of a combination of factors such as low pH, acid type and its concentration,
presence of unfermentable bres and gas composition.
In our study, we also observed that populations of E. coli
O157:H7 on fresh-cut pineapples decreased faster at 25
C
compared to 5
C. Similarly, Han & Linton (2004) found that E. coli
O157:H7 populations inoculated into strawberry juice (pH 3.6)
decreased rapidly at 37
C but remained constant at 5
C. Because
these bacteria are more easily injured at a higher temperature
under more acidic conditions, Han & Linton (2004)linkconclu ded
that even the mechanisms for bacterial inactivation at 37
C are not
well understood and that bacteria may be protected at low
temperatures by the production of cold-shock proteins. This
property of E. coli O157:H7 to acquire an increased survival rate at
lower pHs and lower temperatures has also been observed in other
plant products, such as pineapple juice (Mutaku et al., 2005), fruit
pulps (Marques, Worcman-Barninka, Lannes, Landgraf, 2001) and
canned tomato products (Eribo & Ashena, 2003). Strawn and
Danyluk (2010) noted that the slower rate of E. coli O157:H7
decline at lower temperatures is likely impacted by the overall
reduced metabolism of the organism at cooler temperatures.
In contrast, E. coli O157:H7 populations sharply increased at 25
C
on fresh-cut melon (approximately 4 log units in 24 h) and remained
almost constant at 5
C. In general, the pH of melons is not acidic, and
they contain high amounts of sugars that could be readily used by
bacteria and other microorganisms. The growth of other foodborne
pathogens such as Salmonella and L. monocytogenes on fresh-cut
honeydew melon stored at 10
C has been reported (Leverentz
et al., 2001, 2003). Ukuku and Sapers (2007) also found that
Salmonella declined slightly throughout 12 days of storage at 5
C on
fresh-cut cantaloupe, honeydew and watermelon but signicantly
increased at 10 and 22
C. The survival of this bacterium in other fruit
commodities such as strawberries (Knudsen, Yamamoto, & Harris,
2001) and apples (Conway et al., 2000; Dingman, 2000; Fisher &
Golden, 1998; Janisiewicz et al., 1999) has been investigated.
Fresh-cut fruits and vegetables were sealed in packages and
initially enclosed with air. During the storage process, the gas
atmosphere within the packages was modied, which was mainly
a result of the respiration of the packaged produce. The concentrations of CO2 and O2 in the bags mainly varied with the packaged

M. Abadias et al. / Food Control 27 (2012) 37e44

product but not with the lm used. Dissolved CO2 has been found to
inhibit microbial growth (Devlieghere & Debevere, 2000; Hotchkiss
& Banco, 1992), affecting the lag phase, maximum growth rate and/
or maximum population densities reached, and levels in excess of
5% in MAP systems have been found to be bacteriostatic (Hotchkiss
& Banco, 1992). Moderate levels of CO2 of 20e60% have been found
to inhibit the growth of Pseudomonas spp. and Moraxella spp.
(Cutter, 2002). However, in our study, even high CO2 concentrations were achieved at 25
C, and there was no growth inhibition.
Similarly, Hao and Brackett (1993) concluded that the growth of
E. coli O157:H7 was not inhibited by gas mixtures containing up to
10% CO2 at 5 or 10
C. Other workers reported that 30% CO2 had no
inhibitory effect on the growth of E. coli O157:H7 on shredded
lettuce stored at 13 or 22
C (Diaz & Hotchkiss, 1996). Francis and
OBeirne (2001) also found that a gas atmosphere that was
passively generated inside packs of lettuce and swedes was not
inhibitory to E. coli O157:H7 12900. Delaquis et al. (2007) also
suggested that gas composition has no direct effect on E. coli
O157:H7 growth.
5. Conclusions
The studied strain of E. coli O157:H7 survived at 5
C throughout
the studied period in the four studied commodities. Except for
pineapple, at 25
C, growth was very rapid and reached populations
between 5.2 and 8.9 log cfu g1 after 24 h. This work emphasises
the importance of strict temperature control from processing to
consumption: transportation, distribution, storage and handling in
supermarkets and by consumers. An abusive storage temperature
could result in an undesirable and hazardous rise of an E. coli
O157:H7 population. It is essential that the contamination of
produce be minimised through the use of good agricultural and
strict hygiene practices and that HACCP programs specic for the
pathogen of concern be applied at all stages of production. Even
under refrigeration conditions (5
C), E. coli O157:H7 could survive
and be present at consumption, thus serving as a risk for consumers
as no specic disinfection measures are taken for ready-to-eat
products.
Acknowledgements
The authors are grateful to the Spanish Government [Ministerio
de Ciencia y Tecnologa, Research Project AGL-2004-06027 and
INIA researcher contract CTE/3597/2003 (BOE 23/12/2003)] and the
FEDER for their nancial support.
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