e.coli in Fresh Vegetables

7232019 ecoli in Fresh Vegetables

httpslidepdfcomreaderfullecoli-in-fresh-vegetables 18

Growth potential of Escherichia coli O157H7 on fresh-cut fruits (melon andpineapple) and vegetables (carrot and escarole) stored under different conditions

Maribel Abadias a Isabel Alegre b Marcia Oliveira b Rosa Altisent a Inmaculada Vintildeas b

a IRTA XaRTA-Postharvest 191 Rovira Roure 25198 Lleida Catalonia Spainb University of Lleida XaRTA-Postharvest 191 Rovira Roure 25198 Lleida Catalonia Spain

a r t i c l e i n f o

Article history

Received 8 July 2011

Received in revised form

14 February 2012

Accepted 28 February 2012

Keywords

Minimally processed fruits and vegetables

Survival

Population dynamics

Foodborne pathogens

Modi1047297ed atmosphere packaging

Storage conditions

a b s t r a c t

Minimally processed fruits and vegetables are ready-to-eat and do not require further treatment at

home These foods are usually stored in a modi1047297ed atmosphere and should be maintained at refrigerated

conditions until consumption These fruits and vegetables can become contaminated by foodborne

pathogens such as Escherichia coli O157H7 Salmonella and Listeria monocytogenes and it has been

demonstrated that current industrial sanitising washing treatments do not guarantee the total elimi-

nation 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

modi1047297ed atmosphere) and temperature (5 or 25 C) on the population dynamics of a strain of E coli

O157H7 For vegetables the growth in two 1047297lms which created different O2 and CO2 concentrations and

air were compared At 25 C growth of E coli O157H7 was higher in fresh-cut carrots than in endive

reaching populations between 70e84 log cfu g1 and 52e63 log cfu g1 after 3 days of storage

respectively In fruits E coli O157H7 grew well in fresh-cut melon regardless of the atmospheric

conditions in the package reaching populations of 85 and 89 log cfu g1 after 1 day of storage in

modi1047297ed atmosphere packaging (MAP) or under air conditions respectively No growth was observed in

the fresh-cut pineapple At 5

C E coli O157H7 did not grow but survived throughout the studied periodin 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

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

O157H7 Salmonella and Listeria monocytogenes Unfortunately ithas been demonstrated that current industrial sanitising washing

treatments do not guarantee the total elimination of the pathogen

when present (Abadias Alegre Usall Torres amp Vintildeas 2010

Abadias Usall Oliveira Alegre amp Vintildeas 2008 Beuchat1996 Parish

et al 2003) Outbreaks of E coli O157H7 infection have been linked

to the consumption of fresh fruits and vegetables such as alfalfa

and radish sprouts different lettuce varieties carrots spinach

unpasteurised apple cider berries and melon (Beuchat 1996

De Roever1998 Ethelberg et al 2010 Friesema et al 2008 Sodha

et al 2010) In 1994 an outbreak of non-O157H7 E coli (O11H7) in

the US was linked to pineapple consumption (Sivapalasingam

Friedman Cohen amp 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 40 00 people infected in Europe and 46

peoplehaving died fromSTEC 45 of them inGermany (ECDC 2011)

German of 1047297cials initially suspected cucumbers from Spain as the

source of contamination but further tests showed that those

vegetables did not contain the E coli O104H4 strain Epidemio-

logical 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 O157H7 could

survive andor grow in a range of minimally processed fruits and

vegetables such as shredded lettuce (Abdul-Raouf Beuchat amp Corresponding author Tel thorn34 973 032 850x1502 fax thorn34 973 238 301

E-mail address isabelabadiasirtacat (M Abadias)

Contents lists available at SciVerse ScienceDirect

Food Control

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e f o o d c o n t

0956-7135$ e see front matter 2012 Elsevier Ltd All rights reserved

doi101016jfoodcont201202032

Food Control 27 (2012) 37e44



Ammar1993 Francis amp OrsquoBeirne 2001 Oliveira et al 2010) sliced

cucumber (Abdul-Raouf et al 1993) shredded carrot (Abdul-Raouf

et al 1993) dry coleslaw mix (Francis amp OrsquoBeirne 2001) soybean

sprouts (Francis amp OrsquoBeirne 2001) packaged fresh-cut salad (Luo

He amp McEvoy 2010) apples (Alegre Abadias Anguera Oliveira amp

Vintildeas 2010 Dingman 2000) honeydew melon (Leverentz et al

2001 2003) and peaches (Alegre Abadias Anguera amp Vintildeas

2010) at abuse temperatures

Modi1047297ed 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 amp Hotchkiss 2006) MAPsystems 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 aerobic1047298ora

However under certain conditions the growth of some anaerobicor microaerophilic psychrotrophic microorganisms such as

L monocytogenes and Clostridium spp might be allowed or even

stimulated Moreover extending the shelf-life of minimally pro-

cessed 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 O157H7 One leafy vegetable (escarole) grated

root (carrot) and two fruits with different pH values (pineapple and

melon) were used

2 Material and methods

21 Microorganism and preparation of inoculums

Strain NCTC 12900 of E coli O157H7 (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 O157H7 The strain was adapted to grow on Tryp-

tone Soy Agar (TSA Oxoid CM0131) supplemented with

100 mg mL 1 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 mL 1 streptomycin

(TSBS) and incubated at 150 rpm for 18e20 h at 37 1 C After-

wards the culture was centrifuged at 9820 g for 10 min and the

resultant pellets were resuspended in saline peptone (SP 85 g L 1

NaCl and 1 g L 1

peptone) Bacterial concentrations were estimatedby comparing the suspension transmittance at 420 nm with

a previously determined standard curve Suspensions of about 10 7

and 105 cfu mL 1 were prepared to inoculate vegetables and fruits

respectively The concentration applied was con1047297rmed by plating

01 mL of an appropriately diluted culture on TSAS

22 Fruits and vegetables

E coli growth was studied in different vegetal matrices pine-

apple ( Ananas sativus L Del Monte Gold) melon (Cucumis melo L

var lsquoPiel de saporsquo) 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

23 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 mL 1) for 2 min at 150 rpm Inoculated vege-

tables were dried in a laminar 1047298ow biosafety cabinet Samples

(w15 g) of fresh-cut carrot or endive were packaged under three

different atmospheric conditions air and two passive modi1047297ed

atmospheres created by means of using different 35 mm-oriented

polypropylene (OPP) plastic 1047297lms (Amcor Flexibles Ledbury

Herefordshire UK) FILM I (35PA60) had an O2 and CO2 perme-

ability of 3500 cm3 lm1 day1 atm1 at 23 C and water steam

permeability of 09 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 09 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 weremade 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 surface-

sterilised with 70 ethanol and cut into 1-cm slices For experi-

ments 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 mL 1 suspension of E coli and tubes were incubated at 5

and 25 C for different periods of time For experiments with fresh-

cut fruit packaged in a modi1047297ed atmosphere melon and pineapple

slices were sliced through the equator with a sterile knife and 10-

mm-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 amp Saftner 2000 Leverentz et al 2001) and thepieces were subsequently inoculated with 15 mL of the107 cfu mL 1

suspension of E coli Fruit pieces were placed in polypropylene

trays sealed in a thermo-sealing machine with microperforated

1047297lm (40000 cc m2 day1 FFP Packaging Solutions Ltd North-

ampton 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

24 Microbial count

Populations of E coli in inoculum suspensions and in dips were

enumerated by surface plating duplicate samples (01 mL) which

were serially diluted in SP on TSAS plates followed by incubation at37 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 determi-

nations 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 1047297lter bag with 9 mL of SP homogenised in a stomacher

blender for 2 min at high speed (Bagmixer 100 Minimix Inter-

science) ten-fold diluted in SP plated in duplicate on TSAS and

incubatedat 371 C for 24 2 h Colonieswere 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 poly-

ethylene bag and pummelled with a Stomacher at 150 rpm for

M Abadias et al Food Control 27 (2012) 37 e4438



2 min Wash 1047298uid was serially diluted surface plated in duplicate

on TSAS and incubatedat 371 Cfor24 2 h as described before

Randomly selected presumptive E coli O157H7 colonies were

con1047297rmed using an E coli O157 Latex Test Kit (Oxoid Ltd Cam-

bridge UK)

25 Gas analysis

On each sampling date CO2 and O2 concentrations within three

of each package type were analysed using a handheld gas analyser

(CheckPoint O2CO2 PBI Dansensor Denmark) Gas extraction was

performed with a hypodermic needle inserted through an adhesive

septum previously 1047297xed to the bag

26 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 refrac-

tometer (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 with01 M NaOH solution to an end point of 81 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 1047298esh was also

measured using a puncture electrode for semi-solid and liquid

samples (ref 5232 Crison Abrera Barcelona Spain)

27 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 91 for Windows Cary NC) was performed Means were

separated using Duncanrsquos Multiple range test at the 5 signi1047297cancelevel

3 Results

31 Growth and survival of E coli O157H7 in fresh-cut escarole at

different temperatures and packaging atmospheres

The initial concentration of E coli on fresh-cut escarole was

between 43 and 45 log cfu g1 (Fig 1A) E coli grew in fresh-cut

escarole at 25 C regardless of the packaging 1047297lm In escarole

packaged in FILM I E coli signi1047297cantly increased during the1047297rstday

of storage however no signi1047297cant 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 63 log cfu g1 There were no signi1047297cant differ-

ences between the growth rates in FILM I and FILM II However

growth under airconditions was signi1047297cantlylower except at day 2

The O2 concentration signi1047297cantly 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 1047297nal values between 112 and 157 for both 1047297lms (Fig 2B)

At 5 C the E coli population signi1047297cantly 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 decreased07 log units in 8 days For fresh-cut escarole stored in FILM I and

FILM II bags the populations decreased 06 and 09 log units after 8

days and 08 and 10 log units after 10 days of storage at 5 C

respectively The O2 concentration inside FILM I bags signi1047297cantly

decreased from 207 to 94 after day 8 and then remained

unchanged (Fig 2A) and progressively decreased in FILM II The O2

concentration in FILM II bags was signi1047297cantly lower than in FILM I

with a 1047297nal value of 37 The CO2 concentration gradually

increased at 5 C but never exceeded 10 (Fig 2B)

32 Growth and survival of E coli O157H7 in grated carrots at


The growth of E coli in grated carrots packaged under differentatmospheric conditions and stored at 25 C was higher than that

25ordmC 0 1 2 3 25ordmC 0 1 2 3

Air 43 a x 52 b y 53 b y 52 b y Air 45 a x 84 a y 85 a z 84 a zy

Film I 45 a x 57 a y 60 a y 60 a y Film I 44 a x 70 c y 79 b z 80 a z

Film II 45 a x 57 a y 55 ab y 63 a z Film II 45 a x 77 b z 80 b z 70 a y

5ordmC 0 2 6 8 10 5ordmC 0 2 6 8

Air 43 a x 40 b y 37 ab x 36 b x nd Air 45 a z 45 a z 41 a y 40 a y

Film I 44 a z 43 a y 40 a x 39 a x 37 a w Film I 44 a z 43 a z 41 a z 32 a y

Film II 45 a z 43 a z 36 b y 36 b y 35 a y Film II 45 a z 43 a z 40 a y 38 a y

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12

E c o l i l o g c f u g - 1

Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

A

20

30

40

50

60

70

80

90

0 2 4 6 8 10

E c o l i l o g C F U g - 1

Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

B

Fig 1 Populations of Escherichia coli O157H7 (log cfu g1) inoculated by dipping in a suspension containing 105 cfu mL 1 in fresh-cut endive (A) and grated carrot (B) which were

packaged in different 1047297lms 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 signi 1047297cant differences (P lt 005) of E coli O157H7

populations among the tested 1047297

lms and different letters within rows (w x y z) indicate signi1047297

cant differences among the storage times

M Abadias et al Food Control 27 (2012) 37 e44 39



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

between 44 and 45 log cfu g1 Maximum growth was observed in

samples stored under air with a 4-log increase observed in pop-

ulations after 1 day of storage However after 3 days of storage

there were not signi1047297cant differences between E coli populations

The O2 concentration fell to 35 and 10 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 207 and 286 in FILM I

and FILM II bags respectively The CO2 concentration attained

a maximum level (346) 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

(Fig1B)Decreases observed after 8 days of storage at 5 C were05

13 and 07 log units in air FILM I and FILM II respectively but there

were no signi1047297cant differences (P lt 005) The O2 concentration

inside FILM I bags (Fig 3A) decreased during the 1047297rst day of storage

at 5 Cand then no signi1047297cant differences were observed In FILM II

bags the O2 concentration progressively decreased reaching 02

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 signi1047297cantly lower at each tested time

33 Growth and survival of E coli O157H7 in fresh-cut pineapple

at different temperatures and packaging atmospheres

The pineapples used had a high acid content (618 g citric

acid L 1 of juice) a pH of 359 and a soluble solids content of

140 Brix which corresponded to a maturity index (SSTA) of 23

25ordmC 0 1 2 3 25ordmC 0 1 2 3

Film I 206 a z 35 a y 26 a y 53 a y Film I 01 a y 207 b z 260 b z 195 b z

Film II 206 a z 10 b y 15 a y 12 b y Film II 01 a x 286 a y 346 a z 287 a y

5ordmC 0 2 6 8 5ordmC 0 2 6 8


Film II 206 a z 86 b y 18 b x 02 b w Film II 01 a w 78 a x 159 a y 180 a z

0

3

6

9

12

15

18

21

0 2 4 6 8 10

O 2

C o n c e n t r a t i o n

Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2-Film II-5ordmC

A

0

10

20

30

40

0 2 4 6 8 10

C O

2 C o n c e n t r a t i o n

Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I-5ordmC

CO2-Film II-5ordmC

B

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 signi1047297cant differences (P lt 005) of

gas concentrations among the tested 1047297



25ordmC 0 1 2 3 25ordmC 0 1 2 3

Film I 207 a z 80 a y 33 a x 03 a w Film I 02 a z 95 b y 135 a x 157 a w

Film II 207 a z 46 b y 05 b x 01 a x Film II 02 a x 120 a zy 125 a z 112 b y

5ordmC 0 2 6 8 10 5ordmC 0 2 6 8 10

Film I 207 a z 162 a y 135 a yx 94 a x 107 a x Film I 02 a x 35 b y 49 b zy 67 a z 59 b z

Film II 207 a z 148 b y 79 b x 57 a w 37 b v Film II 02 a w 49 a x 78 a y 79 a zy 83 a z

0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

O

2 c o n c e n t r a t i o n

Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2- Film II-5ordmC

A

0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I- 5ordmC

CO2-Film II-5ordmC

B



gas concentrations among the tested 1047297lms and different letters within rows (w x y z) indicate signi1047297cant differences among the storage times




In the case of fruit samples only storage under air conditions

and one MAP condition (one 1047297lm) were compared The initial

population was in the range of 49 and 53 log cfu g1 E coli was

unable to grow on fresh-cut pineapple at both studied tempera-

tures (Fig 4A) At25 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 O157H7 survived during the whole

experiment with a signi1047297cant 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 1047297nal value of 503 at the end of

the experiment (3 days) The packages reached anaerobic condi-

tions after 2 days of storage at 25 C

34 Growth and survival of E coli O157H7 in fresh-cut melon at


The acidity of the melon expressed in g of citric acid was very

low (114 g L 1) the pH was 594 and the soluble solids content was

96 Brix The obtained maturity index was higher than that of thepineapple (155) 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 signi1047297cantly between 10 and 14 days of storage in MAP

The CO2 and O2 patterns were similar to those obtained with fresh-

cut pineapple (Fig 5B)No signi1047297cant changes were observedat 5 C

but CO2 levels rose to 113 252 and 394 after 1 2 and 3 days of

storage at 25 C respectivelyAt thesame time theO2 concentration

decreased by 119 46 and 22 during that same time period

4 Discussion

This study compared the growth of a strain of E coli O157H7(NCTC 12900) on different fresh-cut fruits and vegetables stored

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 O157H7 growth was predom-

inantly dependent on the type of vegetableand temperature and to

a lesser extent the atmospheric condition

At 25 C E coli O157H7 grew on fresh-cut escarole carrot and

melon but was unable to grow on fresh-cut pineapple At 5 Cit 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 O157H7 are generally believed to be 5 C and 8 C

respectively (Palumbo Call Schultz amp Williams 1995 Rajkowski amp

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 O157H7 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

O157H7 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 amp Brackett 1990) inhibit

the growth of several fungi and bacteria (Kurosaki amp Nishi 1983)and thus may also be inhibitory or toxic to E coli O157H7 Francis

and OrsquoBeirne (2001) did not observe the growth of E coli O157H7

12900 in a dry coleslaw mix (80 cabbage thorn 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 patho-

genic 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 bettersurvival and growth on cut surfaces (Gleeson amp OrsquoBeirne 2005 Seo

25ordmC 0 1 2 3 25ordmC 0 1 2 3

Air 51 a z 49 a z 43 a y 40 a y Air 48 y 89 z 90 z 93 z

MAP 53 a z 50 a z 42 a y 25 b x MAP 50 x 85 y 88 zy 87 z

5ordmC 0 2 6 8 5ordmC 0 2 6 8 10 14

Air 49 a z 48 a z 47 a z nd Air 48 z 50 z 49 z 44 z nd nd

MAP 53 a z 48 a zy 42 a y 30 x MAP 50 z 52 z 50 z 48 z 48 z 35 y

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9 12 15

Storage time days

Air_25ordmC

Air-5ordmC

MAP-25ordmC

MAP-5ordmC

B

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9

E

c o

l i O 1 5 7 H

7

l o g c

f u g - 1

Storage time days

Air-25 ordmC

Air 5 ordmC

MAP-25ordmC

MAP-5ordmC

A

Fig 4 Population of Escherichia coli O157H7 (log cfu g1) spot inoculated with 15 mL of a suspension containing 107 cfu mL 1 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 signi1047297cant differences (P lt 005) of E coli O157H7 populations among the tested 1047297 lms and different letters within rows (w x y z) indicate

signi1047297





amp Frank 1999) Escarole has less cut surface area compared to the

carrot and E coli O157H7 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 Lwhich belongs to the same family as escarole (Asteraceae) have

been reported to have antibacterial activities (Petrovic Stanojkovic

Comic amp Curcic 2004) The survival of E coli O157H7 in other leafy

vegetables (mainly lettuce) has been reviewed by Delaquis Bach

and Dinu (2007) Populations of viable E coli O157H7 cells signif-

icantly decrease on lsquoIcebergrsquo lettuce stored at 5 C and signi1047297cantly

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 lsquoRomainersquo lettuce stored at 25 C but no

growth was observed at 5 C The presence of competing micro-

organisms 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 andorproduce 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 back-

ground microbiota did not affect the growth of E coli O157H7 and

L monocytogenes on lsquoRomainersquo lettuce (Oliveira Vintildeas Anguera amp

Abadias 2012)

Pineapple was largely unsuitable for E coli growth even at 25 C

At 1047297rst and due to other authorsrsquo results we presumed that the lack

of growth could be due to the low pH of pineapple (328e406) 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 lsquoGranny Smithrsquo fresh-

cut apples (pH between 329 and 335 and a titratable acidity of

79e84 g malic acid L 1 of juice) which would be more restrictive

E coli O157H7 is known to have a high tolerance to acidity

(Benjamin amp Datta 1995 Conner amp Kotrola 1995) Other authors

(Deng Ryu amp Beuchat 1999) noted that both the pH and the acid

present are also of importance In TSB-acidi1047297ed medium the order

of sensitivity for E coli O157H7 at a given pH is acetic acid gt citric

acid gt 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 O157H7 did not grow at 25 C

in TSBYE acidi1047297ed topH 45 using citricacid but theygrew at pH45

when malic acid was used Strawn and Danyluk (2010) found that

E coli O157H7 did not grow and survived poorly on fresh-cut

pineapples (pH ca 36) 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 O157H7 growth (Basset amp McClure

2008) and also because of the presence of a high percentage of

unfermentable 1047297bres which may decrease the availability of nutrients for E coli O157H7 metabolism (Mutaku Erku amp Ashena1047297

2005) In conclusion the inability of E coli O157H7 to grow on

fresh-cut pineapple at 25 C may be a consequence of a combina-

tion of factors such as low pH acid type and its concentration

presence of unfermentable 1047297bres and gas composition

In our study we also observed that populations of E coli

O157H7 on fresh-cut pineapples decreased faster at 25 C

compared to 5 C Similarly Han amp Linton (2004) found that E coli

O157H7 populations inoculated into strawberry juice (pH 36)

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 amp 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 lowtemperatures by the production of cold-shock proteins This

property of E coli O157H7 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 amp Ashena1047297 2003) Strawn and

Danyluk (2010) noted that the slower rate of E coli O157H7

decline at lower temperatures is likely impacted by the overall

reduced metabolism of the organism at cooler temperatures

In contrast E coli O157H7 populations sharply increased at 25 C

on fresh-cutmelon (approximately4 log unitsin 24 h) andremained

almostconstant at 5 CIn generalthe pHof melonsis not acidicand

they contain high amounts of sugars that could be readily used by

bacteria and other microorganisms The growth of other foodbornepathogens 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 thatSalmonella declined slightly throughout 12days of storage at 5 C on

fresh-cut cantaloupe honeydew and watermelon but signi1047297cantly

increased at 10 and22 CThe survivalof this bacterium inotherfruit

commodities such as strawberries (Knudsen Yamamoto amp Harris

2001) and apples (Conway et al 2000 Dingman 2000 Fisher amp

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 modi1047297ed which was mainly

a result of the respiration of the packaged produce The concen-

trations of CO2 and O2 in the bags mainly varied with the packaged

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O

2 c o n c e n t r a t i o n ( )

Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2

c o n c e n t r a t i o n ( )

Storage time days

B

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 signi1047297cant differences (P lt 005) of gas concentrations among tested 1047297lms and different letters within rows (w x y z) indicate

signi1047297cant differences among the storage times




product butnot with the1047297lm usedDissolved CO2 has been found to

inhibit microbial growth (Devlieghere amp Debevere 2000 Hotchkiss

amp 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

amp 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 concentra-

tions were achieved at 25 C and there was no growth inhibition

Similarly Hao and Brackett (1993) concluded that the growth of

E coli O157H7 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 O157H7 on shredded

lettuce stored at 13 or 22 C (Diaz amp Hotchkiss 1996) Francis and

OrsquoBeirne (2001) also found that a gas atmosphere that was

passively generated inside packs of lettuce and swedes was not

inhibitory to E coli O157H7 12900 Delaquis et al (2007) also

suggested that gas composition has no direct effect on E coli

O157H7 growth

5 Conclusions

The studied strain of E coli O157H7 survived at 5 C throughoutthe studied period in the four studied commodities Except for

pineapple at 25 C growth was very rapid and reached populations

between 52 and 89 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

O157H7 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 speci1047297c for the

pathogen of concern be applied at all stages of production Even

under refrigeration conditions (5 C) E coli O157H7 could survive

and be present at consumption thus serving as a risk forconsumers

as no speci1047297c disinfection measures are taken for ready-to-eatproducts

Acknowledgements

The authors are grateful to the Spanish Government [Ministerio

de Ciencia y Tecnologiacutea Research Project AGL-2004-06027 and

INIA researcher contract CTE35972003 (BOE 23122003)] and the

FEDER for their 1047297nancial support

References

Abadias M Alegre I Usall J Torres R amp Vintildeas I (2010) Evaluation of differentalternative substances to chlorine for reducing foodborne pathogens in fresh-cut apple processing Postharvest Biology and Technology 59 289e297

Abadias M Usall J Oliveira M Alegre I amp Vintildeas I (2008) Ef 1047297cacy of neutralelectrolysed water (NEW) for reducing microbial contamination on minimally-processed vegetables International Journal of Food Microbiology 123 151e158

Abdul-Raouf U M Beuchat L R amp Ammar M S (1993) Survival and growth of E coli O157H7 on salad vegetables Applied and Environmental Microbiology 591999e2006

Alegre I Abadias M Anguera M Oliveira M amp Vintildeas I (2010) Factors affectinggrowth of foodborne pathogens on minimally processed apples Food Microbi-ology 27 70e76

Alegre I Abadias M Anguera M amp Vintildeas I (2010) Fate of Escherichia coliSalmonella and Listeria innocua on minimally-processed peaches underdifferent storage conditions Food Microbiology 27 862e868

Babic I Watada A E amp Buta J G (1997) Growth of Listeria monocytogenesrestricted by native microorganisms and other properties of fresh-cut spinach

Journal of Food Protection 60 912e917Basset J amp McClure P (2008) A risk assessment approach for fresh fruits Journal of

Applied Microbiology 104 925e943Benjamin M M amp Datta A R (1995) Acid tolerance of enterohemorrhagic

Escherichia coli Applied and Environmental Microbiology 61 1669e

1672

Beuchat L R (1996) Pathogenic microorganisms associated with fresh produce Journal of Food Protection 59 204e216

Beuchat L R amp Brackett R E (1990) Inhibitory effects of raw carrots on Listeriamonocytogenes Applied and Environmental Microbiology 56 1734e1742

CDC (Centers for Disease Control and Prevention) (2011) Investigation updateOutbreak of Shiga toxin-producing E coli O104 (STEC O104H4) infections associ-ated with travel to Germany Available at httpwwwcdcgovprintdourlfrac14httpwwwcdcgovecoli2011ecoliO104 Accessed 280611

Conner D E amp Kotrola J S (1995) Growth and survival of E coli O157H7 underacidic conditions Applied and Environmental Microbiology 61 382e385

Conway W S Leverentz B amp Saftner R A (2000) Survival and growth of Listeriamonocytogenes on fresh-cut apple slices and its interaction with Glomerellacingulata and Penicillium expansum Plant Disease 84 177e181

Cutter C N (2002) Microbial control by packaging a review Critical Reviews inFood Science and Nutrition 42 151e161

De Roever C (1998) Microbiological safety evaluations and recommendations onfresh produce Food Control 9 321e347

Delaquis P Bach S amp Dinu L D (2007) Behavior of Escherichia coli O157H7 inleafy vegetables Journal of Food Protection 70 1966e1974

Deng Y Ryu J-H amp Beuchat L R (1999) Tolerance of acid-adapted and non-adapted Escherichia coli O157H7 cells to reduced pH as affected by type of acidulant Journal of Applied Microbiology 86 203e210

Devlieghere F amp Debevere J (2000) In1047298uence of dissolved carbon dioxide on thegrowth of spoilage bacteria LWT e Food Science and Technology 33 531e537

Diaz C amp Hotchkiss J H (1996) Comparative growth of E coli O157H7 spoilageorganisms and shelf life of shredded iceberg lettuce stored under modi1047297edatmospheres Journal of the Science of Food and Agriculture 70 433e438

Dingman D W (2000) Growth of Escherichia coli O157H7 in bruised apple (Malusdomestica) tissue as in1047298uenced by cultivar date of harvest and source Appliedand Environmental Microbiology 66 1077

e1083

ECDC (2011) European Centre for Disease Prevention and Control Shiga toxin- producing E coli (STEC) Update on outbreak in the EU 27 July 2011 StockholmECDC Available at httpecdceuropaeuenactivitiessciadviceListsECDC20ReviewsECDC_DispFormaspxListfrac14512ff74f2D77d42D4ad82Db6d62Dbf0f23083f30ampIDfrac141166ampRootFolderfrac142Fen2Factivities2Fsciadvice2FLists2FECDC20Reviews Accessed 160112

EFSA (2011) European Food Safety Authority Tracing seeds in particular fenugreek(Trigonella foenum-graecum) seeds in relation to the Shiga toxin-producing E coli(STEC) O104H4 2011 outbreaks in Germany and France Parma EFSA Available athttpwwwefsaeuropaeuensupportingdoc176epdf Accessed 160112

Eribo Bamp Ashena1047297 M (2003) Behaviourof Escherichia coli O157H7 during storagein pressure-treated orange juice Journal of Food Protection 62 1038e1040

Ethelberg S Lisby M Boumlttiger B Schultz A C Villif A Jensen T et al (2010)Outbreaks of gastroenteritis linked to lettuce Denmark January 2010 EuroSurveillance 15(6) piifrac1419484 Available at httpwwweurosurveillanceorgimagesdynamicEEV15N06art19484pdf Accessed 280612

Fisher T L amp Golden D A (1998) Fate of Escherichia coli O157H7 in ground apples

used in cider production Journal of Food Protection 61 1372e

1374Francis G Aamp OrsquoBeirne D (2001) Effects of vegetable type package atmosphere andstoragetemperature on growthand survival of Escherichiacoli O157H7 and Listeriamonocytogenes Journal of Industrial Microbiology and Biotechnology 27 111e116

Frank C Faber M S Askar M Bernard H Fruth A Gilsdorf A et al (2011) Largeand ongoing outbreak of haemolytic uraemic syndrome Germany May 2011Euro Surveillance 16 (21) piifrac1419878 Available at httpwwweurosurveillanceorgViewArticleaspxArticleidfrac1419878 Accessed 220611

Friesema I Sigmundsdottir G van der Zwaluw K Heuvelink A Schimmer B de Jager C et al (2008) An international outbreak of Shiga toxin-producingEscherichia coli O157 infection due to lettuce SeptembereOctober 2007 EuroSurveillance 13(50) piifrac1419065 Available at httpwwweurosurveillanceorgViewArticleaspxArticleIdfrac1419065 Accessed 230611 (Online)

Gleeson E amp OrsquoBeirne D (2005) Effects of process severity on survival and growthof Escherichia coli and Listeria innocua on minimally processed vegetables FoodControl 16 677e685

Han Y amp Linton R H (2004) Fate of Escherichia coli O157H7 and Listeria mono-cytogenes in strawberry juice and acidi1047297ed media at different pH values andtemperatures Journal of Food Protection 67 2443e2449

Hao Y Y amp Brackett R E (1993) Growth of Escherichia coli O157H7 in modi1047297edatmosphere Journal of Food Protection 56 330e332

Hotchkiss J H amp Banco M J (1992) In1047298uence of new packaging technologies onthe growth of microorganisms in produce Journal of Food Protection 55815e820

Janisiewicz W J Conway W S Brown M W Sapers G M Fratamico P ampBuchanan R L (1999) Fate of Escherichia coli O157H7 on fresh-cut apple tissueand its potential for transmission by fruit 1047298ies Applied and EnvironmentalMicrobiology 65 1e5

Knudsen D M Yamamoto S A amp Harris L J (2001) Survival of Salmonella sppand Escherichia coli O157H7 on fresh and frozen strawberries Journal of FoodProtection 64 1483e1488

Kurosaki Famp Nishi A (1983) Isolation and antimicrobial activity of the phytoalexin6-methoxymellein from cultured carrot cells Phytochemistry 22 669e673

Leverentz B Conway W S Alavidze Z Janisiewicz W J Fuchs Y Camp M Jet al (2001) Examination of bacteriophage as biocontrol method for Salmonellaon fresh-cut fruit a model study Journal of Food Protection 64 1116e1121

Leverentz B Conway W S Camp M J Janisiewicz W J Abuladze T Yang Met al (2003) Biocontrol of Listeria monocytogenes on fresh-cut produce by




treatment with lytic bacteriophages and a bacteriocin Applied and Environ-mental Microbiology 69 4519e4526

Luo Y He Q amp McEvoy J L (2010) Effect of storage temperature an duration onthe behavior of Escherichia coli O157H7 on packaged fresh-cut salad containingRomaine and Iceberg lettuce Journal of Food Science 75 M390eM397

Marques P A H F Worcman-Barninka D Lannes S C S amp Landgraf M (2001)Acid tolerance and survival of Escherichia coli O157H7 inoculated in fruit pulpsstored under refrigeration Journal of Food Protection 64 1674e1678

Mutaku E Erku W amp Ashena1047297 M (2005) Growth and survival of Escherichia coliO157H7 in fresh tropical fruit juices at ambient and cold temperatures Inter-

national Journal of Food Science and Nutrition 56 133e

139Oliveira M Usall J Solsona C Alegre I Vintildeas I amp Abadias M (2010) Effects of

packaging type and storage temperature on the growth of foodborne pathogenson shredded lsquoRomainersquo lettuce Food Microbiology 27 375e380

Oliveira M Vintildeas I Anguera M amp Abadias M (2012) Fate of Listeria mono-cytogenes and Escherichia coli O157H7 in the presence of natural backgroundmicrobiota on conventional and organic lettuce Food Control 25 678e683

Palumbo S A Call J E Schultz F J amp Williams A C (1995) Minimum andmaximum temperatures for growth and verotoxin production by hemorrhagicstrains of Escherichia coli Journal of Food Protection 58 352e356

Parish M E Beuchat L R Suslow T V Harris L J Garrett E H Farber J N et al(2003) Methods to reduceeliminate pathogens from fresh and fresh-cutproduce Comprehensive Reviews in Food Science and Food Safety 2 161e173

Petrovic J Stanojkovic A Comic L J amp Curcic S (2004) Antibacterial activity of Cichorium intybus L Fitoterapia 75 737e739

Rajkowski D T amp Marmer B S (1995) Growth of Escherichia coli O157H7 at1047298uctuating incubation temperatures Journal of Food Protection 58 1307e1313

Seo K H amp Frank J F (1999) Attachment of Escherichia coli O157H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demon-strated by using confocal scanning laser microscopy Journal of Food Protection62 3e9

Sivapalasingam S Friedman C R Cohen L amp Tauxe R V (2004) Fresh producea growing cause of outbreaks of foodborne illness in the United States 1973

through 1997 Journal of Food Protection 67 2342e

2353Sodha S V Lynch M Wannemuehler K Leeper M Malavet M Schaffzin J et al

(2010) Multistate outbreak of Escherichia coli O157H7 infections associatedwith a national fast-food chain 2006 a study incorporating epidemiologicaland food source traceback results Epidemiology and Infection 30 1e8

Strawn L K amp Danyluk M D (2010) Fate of Escherichia coli O157H7 and Salmo-nella on fresh and frozen cut pineapples Journal of Food Protection 73 418e424

Ukuku D O amp Sapers G M (2007) Effect of time before storage and storagetemperature on survival of Salmonella inoculated on fresh-cut melons FoodMicrobiology 24 288e295

Werner B G amp Hotchkiss J H (2006) Modi1047297ed atmosphere packaging InG M Sapers J R Gorny amp A E Yousef (Eds) Microbiology of fruits and vege-tables (pp 437e460) Boca Raton FL CRC Press




Ammar1993 Francis amp OrsquoBeirne 2001 Oliveira et al 2010) sliced

cucumber (Abdul-Raouf et al 1993) shredded carrot (Abdul-Raouf

et al 1993) dry coleslaw mix (Francis amp OrsquoBeirne 2001) soybean

sprouts (Francis amp OrsquoBeirne 2001) packaged fresh-cut salad (Luo

He amp McEvoy 2010) apples (Alegre Abadias Anguera Oliveira amp

Vintildeas 2010 Dingman 2000) honeydew melon (Leverentz et al

2001 2003) and peaches (Alegre Abadias Anguera amp Vintildeas

2010) at abuse temperatures

Modi1047297ed 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 amp Hotchkiss 2006) MAPsystems 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 aerobic1047298ora

However under certain conditions the growth of some anaerobicor microaerophilic psychrotrophic microorganisms such as

L monocytogenes and Clostridium spp might be allowed or even

stimulated Moreover extending the shelf-life of minimally pro-

cessed 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 O157H7 One leafy vegetable (escarole) grated

root (carrot) and two fruits with different pH values (pineapple and

melon) were used

2 Material and methods

21 Microorganism and preparation of inoculums

Strain NCTC 12900 of E coli O157H7 (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 O157H7 The strain was adapted to grow on Tryp-

tone Soy Agar (TSA Oxoid CM0131) supplemented with

100 mg mL 1 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 mL 1 streptomycin

(TSBS) and incubated at 150 rpm for 18e20 h at 37 1 C After-

wards the culture was centrifuged at 9820 g for 10 min and the

resultant pellets were resuspended in saline peptone (SP 85 g L 1

NaCl and 1 g L 1

peptone) Bacterial concentrations were estimatedby comparing the suspension transmittance at 420 nm with

a previously determined standard curve Suspensions of about 10 7

and 105 cfu mL 1 were prepared to inoculate vegetables and fruits

respectively The concentration applied was con1047297rmed by plating

01 mL of an appropriately diluted culture on TSAS

22 Fruits and vegetables

E coli growth was studied in different vegetal matrices pine-

apple ( Ananas sativus L Del Monte Gold) melon (Cucumis melo L

var lsquoPiel de saporsquo) 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

23 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 mL 1) for 2 min at 150 rpm Inoculated vege-

tables were dried in a laminar 1047298ow biosafety cabinet Samples

(w15 g) of fresh-cut carrot or endive were packaged under three

different atmospheric conditions air and two passive modi1047297ed

atmospheres created by means of using different 35 mm-oriented

polypropylene (OPP) plastic 1047297lms (Amcor Flexibles Ledbury

Herefordshire UK) FILM I (35PA60) had an O2 and CO2 perme-

ability of 3500 cm3 lm1 day1 atm1 at 23 C and water steam

permeability of 09 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 09 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 weremade 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 surface-

sterilised with 70 ethanol and cut into 1-cm slices For experi-

ments 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 mL 1 suspension of E coli and tubes were incubated at 5

and 25 C for different periods of time For experiments with fresh-

cut fruit packaged in a modi1047297ed atmosphere melon and pineapple

slices were sliced through the equator with a sterile knife and 10-

mm-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 amp Saftner 2000 Leverentz et al 2001) and thepieces were subsequently inoculated with 15 mL of the107 cfu mL 1

suspension of E coli Fruit pieces were placed in polypropylene

trays sealed in a thermo-sealing machine with microperforated

1047297lm (40000 cc m2 day1 FFP Packaging Solutions Ltd North-

ampton 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

24 Microbial count

Populations of E coli in inoculum suspensions and in dips were

enumerated by surface plating duplicate samples (01 mL) which

were serially diluted in SP on TSAS plates followed by incubation at37 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 determi-

nations 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 1047297lter bag with 9 mL of SP homogenised in a stomacher

blender for 2 min at high speed (Bagmixer 100 Minimix Inter-

science) ten-fold diluted in SP plated in duplicate on TSAS and

incubatedat 371 C for 24 2 h Colonieswere 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 poly-

ethylene bag and pummelled with a Stomacher at 150 rpm for








bridge UK)

25 Gas analysis






26 Quality of fruit



















3 Results
































25ordmC 0 1 2 3 25ordmC 0 1 2 3




5ordmC 0 2 6 8 10 5ordmC 0 2 6 8




20

30

40

50

60

70

80

90

0 2 4 6 8 10 12


Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

A

20

30

40

50

60

70

80

90

0 2 4 6 8 10


Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

B






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8



0

3

6

9

12

15

18

21

0 2 4 6 8 10

O 2


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2-Film II-5ordmC

A

0

10

20

30

40

0 2 4 6 8 10

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I-5ordmC

CO2-Film II-5ordmC

B






25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 10 5ordmC 0 2 6 8 10



0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

O


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2- Film II-5ordmC

A

0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I- 5ordmC

CO2-Film II-5ordmC

B





































4 Discussion






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8 10 14



00

10

20

30

40

50

60

70

80

90

100

0 3 6 9 12 15

Storage time days

Air_25ordmC

Air-5ordmC

MAP-25ordmC

MAP-5ordmC

B

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9

E

c o

l i O 1 5 7 H

7

l o g c

f u g - 1

Storage time days

Air-25 ordmC

Air 5 ordmC

MAP-25ordmC

MAP-5ordmC

A





signi1047297



























Abadias 2012)























C These researchers











































00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O


Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2


Storage time days

B


























O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083

















































bridge UK)

25 Gas analysis






26 Quality of fruit



















3 Results
































25ordmC 0 1 2 3 25ordmC 0 1 2 3




5ordmC 0 2 6 8 10 5ordmC 0 2 6 8




20

30

40

50

60

70

80

90

0 2 4 6 8 10 12


Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

A

20

30

40

50

60

70

80

90

0 2 4 6 8 10


Storage time days

Film I-25ordmC

Film II-25ordmC

Air-25ordmC

Film I-5ordmC

Film II-5ordmC

Air-5ordmC

B






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8



0

3

6

9

12

15

18

21

0 2 4 6 8 10

O 2


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2-Film II-5ordmC

A

0

10

20

30

40

0 2 4 6 8 10

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I-5ordmC

CO2-Film II-5ordmC

B






25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 10 5ordmC 0 2 6 8 10



0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

O


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2- Film II-5ordmC

A

0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I- 5ordmC

CO2-Film II-5ordmC

B





































4 Discussion






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8 10 14



00

10

20

30

40

50

60

70

80

90

100

0 3 6 9 12 15

Storage time days

Air_25ordmC

Air-5ordmC

MAP-25ordmC

MAP-5ordmC

B

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9

E

c o

l i O 1 5 7 H

7

l o g c

f u g - 1

Storage time days

Air-25 ordmC

Air 5 ordmC

MAP-25ordmC

MAP-5ordmC

A





signi1047297



























Abadias 2012)























C These researchers











































00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O


Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2


Storage time days

B


























O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083









































































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8



0

3

6

9

12

15

18

21

0 2 4 6 8 10

O 2


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2-Film II-5ordmC

A

0

10

20

30

40

0 2 4 6 8 10

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I-5ordmC

CO2-Film II-5ordmC

B






25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 10 5ordmC 0 2 6 8 10



0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

O


Storage time days

O2-Film I-25ordmC

O2-Film II-25ordmC

O2-Film I-5ordmC

O2- Film II-5ordmC

A

0

3

6

9

12

15

18

21

0 2 4 6 8 10 12

C O


Storage time days

CO2-Film I-25ordmC

CO2-Film II-25ordmC

CO2-Film I- 5ordmC

CO2-Film II-5ordmC

B





































4 Discussion






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8 10 14



00

10

20

30

40

50

60

70

80

90

100

0 3 6 9 12 15

Storage time days

Air_25ordmC

Air-5ordmC

MAP-25ordmC

MAP-5ordmC

B

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9

E

c o

l i O 1 5 7 H

7

l o g c

f u g - 1

Storage time days

Air-25 ordmC

Air 5 ordmC

MAP-25ordmC

MAP-5ordmC

A





signi1047297



























Abadias 2012)























C These researchers











































00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O


Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2


Storage time days

B


























O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083











































































4 Discussion






































25ordmC 0 1 2 3 25ordmC 0 1 2 3



5ordmC 0 2 6 8 5ordmC 0 2 6 8 10 14



00

10

20

30

40

50

60

70

80

90

100

0 3 6 9 12 15

Storage time days

Air_25ordmC

Air-5ordmC

MAP-25ordmC

MAP-5ordmC

B

00

10

20

30

40

50

60

70

80

90

100

0 3 6 9

E

c o

l i O 1 5 7 H

7

l o g c

f u g - 1

Storage time days

Air-25 ordmC

Air 5 ordmC

MAP-25ordmC

MAP-5ordmC

A





signi1047297



























Abadias 2012)























C These researchers











































00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O


Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2


Storage time days

B


























O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083



































































Abadias 2012)























C These researchers











































00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O


Storage time days

A

00

100

200

300

400

500

600

0 3 6 9 12 15

O 2

C O 2


Storage time days

B


























O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083































































O157H7 growth

5 Conclusions















Acknowledgements





References










1672













e1083


































































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e.coli in Fresh Vegetables

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