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International Journal of Food Microbiology 123 (2008) 50 – 60

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Bioprotection of Golden Delicious apples and Iceberg lettuce against

foodborne bacterial pathogens by lactic acid bacteria
Rosalia Trias a , Lluís Bañeras b , Esther Badosa a , Emilio Montesinos a,⁎
a
Institute of Food and Agricultural Technology—CeRTA, CIDSAV, University of Girona, Campus Montilivi 17071, Girona, Spain
b
Molecular Microbial Ecology Group, IEA, University of Girona, Campus Montilivi 17071, Girona, Spain
Received 19 September 2007; received in revised form 19 November 2007; accepted 27 November 2007

Abstract

Lactic acid bacteria were isolated from fresh vegetables and fruit and its ability to inhibit the growth of foodborne human pathogens
(Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus) was tested using the
agar spot assay. Eighteen isolates showed a strong antagonistic capacity and were further characterised and identified using 16S rDNA sequencing
and API 50CH. Most of them pertained to Leuconostoc spp. and Lactobacillus plantarum, and a few corresponded to Weissella spp. and
Lactococcus lactis. Growth and efficacy of control of foodborne pathogen test bacteria by selected strains were tested in wounded Golden
Delicious apples and Iceberg lettuce leaf cuts. The strains grew on the substrates and did not cause negative effects on the general aspect of tissues
of apple or lettuce. Treatment of apple wounds and lettuce cuts with the antagonistic strains reduced the cell count of S. typhimurium and E .coli
by 1 to 2 log cfu/wound or g, whereas the growth of L. monocytogenes was completely inhibited. Results support the potential use of lactic acid
bacteria as bioprotective agents against foodborne human pathogens in ready-to-eat fresh fruit and vegetable products.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Lactic acid bacteria; Antagonistic activity; Foodborne pathogens; Ready-to-eat vegetables; Fresh fruit

1. Introduction are therefore critical steps in ensuring the safety of ready-to-eat

Ready-to-eat fresh vegetable and fruit products demand has
continuously increased in the last decades reflecting the
consumer's interest for fresh and healthy foods with an easy
way of preparation. Minimally processed vegetables and sliced
fruit belong to the low-acid foods, and exhibit a characteristic
high humidity. These facts together with the high number of cut
surfaces, can provide ideal conditions for microbial growth,
including foodborne pathogens and spoilage (Ongeng et al.,
2006). These products have been incriminated in outbreaks of
foodborne diseases caused by human pathogens like Escheri-
chia coli O157:H7 (Ackers et al., 1998), Listeria monocyto-
genes (Beuchat, 1996), Salmonella spp. (Lin et al., 1996; Salleh
et al., 2003), Staphylococcus aureus and Pseudomonas
aeruginosa (Viswanathan and Kaur, 2001). Safe production
methods and proper disinfection/decontamination procedures
⁎ Corresponding author. Tel.: +34 972418427; fax: +34 972418399.
E-mail address: emonte@intea.udg.edu (E. Montesinos).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.11.065
fresh fruit and vegetables (Zhang and Farber, 1996). Growing
concerns on the effects on human health, the development of
resistant strains of pathogens and the lack of continued approval
of some of the most effective chemical products, have justified
the search for alternatives to ensure food quality. Biological
control fits in well with this new tendency, and several bacteria
and yeasts have been identified as bioprotective agents
(Vermeiren et al., 2004). Several antagonistic micro-organisms
have been also used to inhibit the growth of foodborne bacterial
pathogens. Strains of Pseudomonas syringae have been used to
prevent the growth of E. coli in apple wounds (Janisiewicz
et al., 1999). A bioprotective method has been reported to
prevent the growth of L. monocytogenes and S. enterica in
fresh-cut apples using strains of Candida sp., Gluconobacter
sp., Discosphaerina sp. and Metschnikowia sp., but non-target
effects such as browning of fruit were observed (Leverentz
et al., 2006). However, there is still a need for new bioprotective
micro-organisms that fulfill desired characteristics such as
biosafety and limitation of non-target effects.
 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60
Lactic Acid Bacteria (LAB) are considered as food grade
micro-organisms and generally recognized as safe (GRAS) by
the USA Food and Drug Administration (FDA). LAB have
historically been used to preserve meat and dairy products
(Stiles and Holzapfel, 1997) and to bioprotect fermented
vegetables (Ruiz-Barba et al., 1994). The success of LAB in
preventing the growth and activity of foodborne pathogens and
spoilage micro-organisms in a large variety of foods may be due
to the diversity of mechanisms that can be combined. Among
the mechanisms that have been proposed for this activity, the
production of organic acids, bacteriocins and other low
molecular mass compounds, such as hydrogen peroxide and
diacetyl, are the most commonly referred in the literature
(Batish et al., 1997; Alakomi et al., 2000; Cleveland et al.,
2001). Previous works indicate the presence of LAB species
with antagonistic activity in vegetables (Gómez et al., 2002).
However, to our knowledge, the application of LAB isolated
from fresh fruit and vegetables as bioprotective agents against
foodborne pathogens has not been previously described in
detail.
The present work was aimed to the isolation of lactic acid
bacteria from fresh fruit and vegetables and the evaluation of its
antagonistic activity towards pathogen bacteria. The potential
use of LAB as bioprotective agents in fresh fruit and vegetables
is proposed as an optional method to circumvent the limitations
found with other antagonists such as Candida and Glucono-
bacter species.
2. Materials and methods
2.1. Isolation of lactic acid bacteria
Seven hundred samples of fresh fruit and vegetables
including 13 types of fruits, 19 types of vegetables, packaged
ready-to-eat salad vegetables and 4 types of fresh-cut individual
ingredients were used for the isolation of lactic acid bacteria.
Pieces or slices of fresh material were cut and weighted to
approximately 40 g and homogenized in 100 ml of peptone
water. Suspensions were ten fold serially diluted and plated on
de Man, Rogosa and Sharpe agar (MRS) (Oxoid, Hampshire,
UK). Plates were incubated at 23 °C for at least 48 h. Finally,
only two colonies of different shapes or colours from each
sample were randomly selected and purified by repetitive
plating. Each isolate was tested for catalase production and
Gram staining with the KOH string test (Suslow et al., 1982).
The morphology of the cells was determined by phase contrast
optical microscopy, using an Olympus BX50 (Olympus Optical
Co, Hamburg, Germany). Gram positive, catalase negative
bacilli or cocci were selected as putative lactic acid bacteria
(Schillinger and Lüke, 1987). Additionally, 12 commercial
dairy products such as milk (2), yoghurt (5) and cheese (5) were
used as natural sources for the isolation using the same methods
described above. Fourteen strains of LAB isolated from meat
subproducts with proven antagonistic activity were kindly
provided by Dr. Parés from the Food Technology Department of
the University of Girona (Dàvila et al., 2006). Leuconostoc
mesenteroides subsp. mesenteroides ATCC 8293, Ln. fallax
51
ATCC 700006, Lactococcus lactis subsp. lactis ATCC 15577,
Lc. lactis subsp. lactis ATCC 11454, Lc. lactis subsp. lactis
NCFB 1403, Lactobacillus plantarum ATCC 14917, Lb.
collinoides ATCC 27612, Lb. buchnerii ATCC 4005, Lb. brevis
ATCC 8287, Lb. sakei subsp. sakei ATCC 31063, Lb. delbruekii
subsp. lactis NCFB 970, Pediococcus dextrinicus ATCC
33087, and P. parvulus ATCC 19371, originally isolated from
vegetables, were obtained from the Spanish Type Culture
Collection (CECT). The final collection of LAB studied was
composed of 523 strains, 496 isolated in the present study.
2.2. In vitro assay of antagonistic activity
Screening for antagonistic activity was done using the agar
spot test in Lactose-Bromcresol Purple agar (LBP) and
modified MRS agar (MRS.02). LBP contained (g l− 1) tryptone
(20), yeast extract (5), lactose (10), gelatin (2.5), agar (15),
NaCl (0.4), sodium acetate (1.5), bromcresol purple (0.02)
(Panreac, Barcelona, Spain). This media has been previously
described for the detection of antagonistic LAB isolated from
Iceberg lettuce (Gómez et al., 2002). MRS agar was modified
according to Schillinger and Lüke (1989), with a 90% reduction
in glucose concentration and the removal of Tween 80. The
modifications were done in order to enhance the production of
hydrogen peroxide and bacteriocins by antagonistic LAB.
The antagonistic activity was assayed against the foodborne
bacteria E. coli ATCC 11775, L. monocytogenes ATCC 15313,
P. aeruginosa (EPS 1500 from our laboratory), S. typhimurium
LT2 ATCC 15277 and S. aureus ATCC 9144.
LAB isolates were spotted on LBP or MRS.02 agar plates and
incubated 24 h at 23 °C. 0.5 ml of a cell suspension containing
107 cfu ml− 1 were mixed in 4.5 ml of LBP or MRS.02 soft agar
(0.7% agar) and overlayed on the plate containing previously
grown colonies of LAB (Vermeiren et al., 2004). Plates were
incubated at 23 °C for 24 or 48 h, depending on the bacteria
tested, and diameters of the inhibition areas were measured.
Inhibition diameters (mm) were normalized for each experi-
mental condition by dividing values for the maximum value
observed. The relative values obtained (no dimension) were
ordered into five levels of activity and corresponding to high
(1.00 to 0.90), moderate (0.89 to 0.70), medium (0.69 to 0.40),
low (0.39 to 0.20) and without significant activity (0.19 to 0.00).
Isolates showing the best antagonistic activity towards each
bacteria were selected for further analysis. In addition, isolates
displaying activity against at least 3 micro-organisms were
included in the selection and used for further experiments.
Normalized antagonistic activities of the selected LAB were
used for hierarchical cluster analysis based on the squared
Euclidean distance using average linkage as the cluster
algorithm (SPSS 14.0, SPSS Inc., Chicago, USA).
2.3. Phenotypic characterization of LAB isolates
Isolates were tested for gas production from glucose using
48 h old cultures inoculated in MRS liquid medium containing
Durham tubes. Carbohydrate fermentation patterns were
performed using API 50 CH strips following the instructions
52 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60

Fig. 1. Scatter plots of normalized antagonistic activity of lactic acid bacteria isolates in MRS.02 and LBP media. Origin of isolates is indicated as, ○ vegetables and
fruit, ▼dairy products, ▽ meat subproducts, and ■ culture collection (CECT). Several isolates showing antagonistic properties have been conveniently identified. The
results of 523 isolates and strains were represented, thought many symbols correspond to more than one strain.
 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60
Fig. 2. Dendrogram based on the hierarchical cluster analysis of spectrum of antagonism, based on the squared Euclidean distance using average linkage as the cluster
algorithm. Identification of the selected isolates based on 16S rDNA and API 50CH are indicated. GenBank DNA accession numbers of 16srRNA sequences are indicated.
provided by the manufacturer (BioMérieux, Lyon, France).
Optimal growth conditions were determined in MRS medium at
different salinities (2, 4.5, and 6% NaCl) and pH values (4.5,
5.5, 6.5 and 7.5) using an automatic growth curve analyzer
(Bioscreen, Thermo LabSystems, MA, USA). All determina-
tions were done at 25 °C. Maximum growth rates at all
conditions were calculated from triplicate runs of OD600 vs.
time curves. Shaking periodicity was set at 30 min to prevent
sedimentation of cells and cultures were incubated for a
minimum of 48 h until stationary phases were reached.
2.4. 16S rDNA sequencing
Isolates were identified by the sequence of the 16S rDNA,
including the V1–V3 region (Tannock et al., 1999). Nucleic
acids were extracted from colonies grown on MRS agar using
the Wizard DNA extraction kit (Promega, Madison, US)
following the instructions provided by the manufacturer. PCR
amplification was performed with primers 27f and 1492r (Lane,
1991) in a total volume of 25 µl containing, 1× PCR buffer,
0.2 mM deoxynucleoside triphosphate, 4 µM of each primer,
3 mM MgCl2, 2.5 U Taq DNA polymerase (Invitrogen Life
Technologies, CA, USA), and 10 to 100 ng of template. Positive
PCR products were purified with QIAEX Desalting and
concentrating DNA solutions Kit (QIAGEN GmbH, Hilden,
Germany), prior to sequencing. All fragments were sequenced
in both strands using BigDye Terminator v3.0 Ready Reaction
53
cycle kit (PE Applied Biosystems) in a ABI Prism™ 310
sequencer, using primers 27f, 787f, 787r and 1492r (Lane,
1991). The sequences obtained were analyzed and aligned using
BioEdit Sequence Alignment Editor®, and homology search
was done using the BLAST program at NCBI database
(Alstchul et al., 1990). Sequences have been deposited in the
GenBank (Accession No EU074828 to EU074832, EU074834
to EU074843, EU074845, EU074846 and EU074848).
2.5. Growth of LAB and pathogen test bacteria on fruit and
vegetables
The ability of six selected LAB strains and three foodborne
human pathogen test bacteria to grow on plant tissue models
was carried out on Iceberg lettuce leaves and Golden Delicious
apples. All LAB isolates were naturally resistant to streptomy-
cin (50 µg ml− 1), which was used as a tool for selective
quantification. Rifampicin resistant mutants of L. monocyto-
genes and S. typhimurium were used for these experiments and
were obtained on Luria Bertani (LB) and Violet Red Bile
Glucose (VRBG) (Oxoid, Hampshire, UK) agar amended with
rifampicin (50 µg ml− 1) (Sigma, Missouri, USA). Similarly, a
nalidixic acid resistant mutant of E. coli was selected in Violet
Red Bile Lactose (VRBL) (Oxoid, Hampshire, UK) agar plates
supplemented with nalidixic acid (50 µg ml− 1).
The four outer leaves of Iceberg lettuce were discarded, and
inner leaves were cut in pieces of approximately 1 cm2 and
54 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60

Table 1
Normalized antagonistic activity⁎ observed for each strain of the two different clusters of LAB in the hierarchical cluster analysis
Cluster Strain Micro-organism inhibited
Gram + bacteria Gram − bacteria
L. monocytogenes S. aureus E. coli P. aeruginosa S. typhimurium
A ATCC 11454 0.60/0.31 1.00/0.75 0.17/0.37 0.17/0.29 0.50/0.11
SE303 0.80/0.69 0.92/0.63 0.13/0.17 0.00/0.18 0.42/0.00
CM160 1.00/0.81 0.00/0.00 0.00/0.00 0.00/0.00 0.08/0.05
CM135 0.40/1.00 0.25/0.00 0.00/0.00 0.00/0.06 0.17/0.05
B CC121 0.00/0.00 0.00/0.19 0.07/0.00 0.17/0.00 0.00/1.00
CM464 0.00/0.00 0.00/0.19 1.00/0.17 0.00/0.29 0.08/0.26
PM141 0.00/0.00 0.67/0.37 0.03/0.07 0.20/0.00 0.25/0.00
TM128 0.10/0.06 0.83/0.00 0.00/0.07 0.17/0.06 0.08/0.00
BC48 0.05/0.00 0.00/0.00 0.00/0.00 0.59/0.00 0.00/0.11
TC41 0.20/0.00 0.08/0.06 0.10/0.40 0.83/0.82 0.16/0.00
ATCC 4005 0.00/0.00 0.00/0.19 0.10/0.50 0.37/0.76 0.83/0.03
PC216 0.00/0.00 0.25/0.63 0.00/0.37 0.13/0.53 0.25/0.47
TC97 0.00/0.00 0.08/0.75 0.13/0.47 0.27/0.76 0.25/0.11
TC69 0.00/0.00 0.00/0.69 0.13/0.43 0.82/0.25 0.25/0.32
TC54 0.00/0.13 0.17/0.63 0.13/0.43 0.70/0.76 0.25/0.16
TM319 0.00/0.00 0.00/0.63 0.00/1.00 0.00/0.71 0.33/0.53
AC318 0.00/0.00 0.00/0.00 0.23/1.00 0.00/0.82 0.25/0.47
CC317 0.00/0.00 0.08/0.13 0.00/1.00 0.00/0.71 0.25/0.26
CC315 0.00/0.00 0.00/0.25 0.17/0.57 0.00/0.41 0.42/0.53
PM249 0.00/0.00 0.42/0.50 0.20/0.60 0.00/0.35 0.50/0.63
⁎Normalized antagonistic values (range from 0 to 1) were obtained by dividing values of the diameter of halos of inhibition (mm) for the maximum value observed.
Values in both media are indicated as MRS.02/LBP.

placed into sterile plastic bags containing 10 g of material. Bags
were inoculated with 1 ml of a suspension of the test strain to a
final concentration of 104 cfu g− 1 lettuce and gently shaken for
30 times to ensure a homogenous distribution of the inoculum.
The inoculated leaf cuts were left in a microbiological safety
cabinet at room temperature for 1 h to remove excess water
(Koseki and Isobe, 2005). Bags were sealed to preserve high
humidity and stored at 25 °C and treated as individual samples.
Bacterial enumeration was done at 1, 24, 72 and 96 h after the
inoculation of LAB and pathogens. Samples were extracted
with 40 ml of peptone water in a Stomacher blender (IUL
instruments, Barcelona, Spain) for 90 s.
Golden Delicious apples were disinfected by immersion in a
0.1% hypochlorite water solution for 10 min, followed by 2
consecutive 10 min washes of distilled sterile water (Francés et
al., 2006). Apples were exposed to room temperature to remove
excess water, and 6 wounds per fruit were made with a sterile
cork borer (5 mm diameter and 5 mm depth) to inoculate 20 µl
of the appropriate bacterial suspension to a final concentration
of 103 cfu wound− 1. Inoculated fruits were left to absorb the
excess liquid and were stored in plastic boxes on fruit pack trays
sealed with plastic bags at high humidity conditions at 25 °C.
Samples for bacterial enumeration corresponding to apple
cylinders containing the entire wound were taken at 1, 24, 72
and 96 h after the inoculation of LAB and pathogens. Cylinders
were obtained using a sterile cork borer of 10 mm diameter.
Three wounds of every fruit were mixed and extracted in a
Stomacher bag with 20 ml of peptone water for 90 s.
In both cases, Golden Delicious apples and Iceberg lettuces,
the resulting material was left to sediment and aliquots of 1 ml
were serially diluted and plated on selective media with the
corresponding antibiotic at a concentration of 50 µg ml− 1. MRS
medium supplemented with streptomycin was used for LAB,
VRBL supplemented with nalidixic acid for E. coli, and VRBG
and LB with rifampicin for S. typhimurium and L. mono-
cytogenes. Plates were incubated at 23 °C for 48 h. Spoilage of
the product by the inoculated bacteria was inspected by visual
comparison with non-treated controls. Unwanted modifications
such as browning, pectinolitic activities and olfactory modifica-
tions by inoculated bacteria were qualitatively inspected. All
experiments were run in triplicate.
2.6. Efficacy of LAB for inhibition of foodborne pathogens on
fruit and vegetables
The efficacy trials were performed on wounded Golden
Delicious apples and Iceberg lettuce cut leaves and consisted of
the application of both LAB and the corresponding pathogen
test bacteria. Apple wounds were inoculated with 20 µl of
inoculum, and lettuce cuts with 1 ml, and preparation and
inoculation protocols were done as described above. Lactic acid
bacteria were applied first at a concentration of 108 cfu
wound− 1 in apple and 108 cfu g− 1 on lettuce and were left to
absorb excess water during 1 h at room temperature. Pathogen
test bacteria were applied afterwards at densities of 103 cfu per
wound or g. The latter densities were chosen because it is well
above of pathogen concentrations found in fresh fruit and
vegetables (Salleh et al., 2003; Nguz et al., 2005). The six LAB
strains used for this assay were the same as for the colonization
experiments. A biological control agent of fungal infections,
 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60 55

Table 2 Antagonistic ability was evaluated for all 523 isolates from
Identification, origin and phenotipical characteristics of the selected LAB strains fresh fruit and vegetables and selected strains from other
Strain 16S rDNA Isolation Gas Optimum growth sources. Maximum diameters of inhibition zones varied
homology source production conditions according to the type of culture medium and test bacteria, and
pH %NaCl ranged from 12 to 30 mm being the highest found for E. coli.
TC41 Lb. plantarum Tomatoes − 6.5 (4.5–7.5) 4 (0–6) The use of two types of media has been determinant for the
TC54 Lb. plantarum Tomatoes − 7.5 (4.5–7.5) 4 (0–6) effective selection of appropriate candidates with exploitable
TC69 Lb. plantarum Tomatoes − 7.5 (4.5–7.5) 4 (0–6) bioprotective activities, since for most strains results differed on
TC97 Lb. plantarum Tomatoes − 6.5 (4.5–7.5) 4 (0–6)
MRS.02 and LBP in most of the test bacteria used. However,
CC121 Lb. plantarum Cucumber − 7.5 (4.5–7.5) 4 (0–6)
TM128 W. cibaria Tomatoes + 7.5 (4.5–7.5) 2 (0–6) some strains exhibited high antagonistic activity in both media.
BC48 W. cibaria Chards + 6.5 (4.5–7.5) 4 (0–6) For example, strains SE303 and CM160, active against L.
CC317 Ln. mesenteroides Persimmon + 5.5 (4.5–7.5) 4 (0–6) monocytogenes, the strains TC41 and TC54, active against P.
CM135 Ln. mesenteroides Cherries + 6.5 (4.5–7.5) 4 (0–6) aeruginosa, or the strain SE303, which inhibited S. aureus
CM160 Ln. mesenteroides Cherries + 5.5 (4.5–7.5) 2 (0–6)
(Fig. 1). A moderate to high inhibition on P. aeruginosa in
PC216 Ln. mesenteroides Apple + 7.5 (4.5–7.5) 2 (0–6)
PM249 Ln. mesenteroides Peach + 7.5 (4.5–7.5) 4 (0–6) MRS.02 and LBP medium was observed in approximately 2%
CC315 Ln. mesenteroides Persimmon + 7.5 (4.5–7.5) 4 (0–6) of tested strains, being these bacteria the most frequently
AC318 Ln. mesenteroides Aubergine + 7.5 (5.5–7.5) 4 (0–6) inhibited. Contrarily, S. typhimurium and E. coli were less
TM319 Ln. citreum Tomatoes + 7.5 (4.5–7.5) 2 (0–6) frequently inhibited (1.3% and 0.8%, respectively). Gram
CM464 Ln. mesenteroides Pumpkin + 7.5 (4.5–7.5) 4 (0–6)
positive bacteria were also inhibited by a low number of strains.
PM141 Ln. mesenteroides Peach + 7.5 (4.5–7.5) 4 (0–6)
SE303 Lc. lactis Soybean − 7.5 (5.5–7.5) 4 (0–6) The isolates obtained from fresh fruit and vegetables denoted a
sprouts higher activity than strains obtained from meat subproducts,
Ranges for favorable growth conditions are given in parentheses. dairy products and the CECT towards almost all test bacteria,
except for S. typhimurium when MRS.02 medium was used
(Fig. 1). The highest inhibition of this bacterium was achieved
Pantoea agglomerans EPS 125, was included as a reference with CECT strains.
strain. Resistance to ampicillin (50 µg ml− 1) was used as a Based on the intensity of antagonistic activity 18 isolates
selection method for its quantification in LB agar (Bonaterra were selected for further study: CC121, TM128, PM141,
et al., 2003; Francés et al., 2006). Both lettuce cuts and CM135, CM160, PC216, CM464, TC41, BC48, TC54, TC69,
wounded apples were maintained at 25 °C. Samples for TC97, CC317, AC318, TM319, PM249, CC315, SE303. The
bacterial enumeration of LAB and pathogen test bacteria were strains Lc. lactis ATCC 11454 and Lb. buchnerii ATCC 4005
taken at 0 and 48 h after inoculation of the pathogen, using the were included in the selection because these showed high
same methods and media described above, resulting the inhibition against S. aureus and S. typhimurium, respectively.
detection level of 70 cfu wound− 1 in apples and 40 cfu g− 1 in The selected isolates were identified on the basis of the
lettuce. All experiments were run in triplicate and controls sequence of the 16S rDNA and the API 50 CH utilization
consisting in water-inoculated products were also included to pattern. Both techniques coincided in the identification of all the
check for cross-contamination of samples. strains except for CC121, which differed at the species level,
and BC48 and TM128, which have different results for each
2.7. Data analysis method at the genera level (Fig. 2). Results showed a high
frequency of Leuconostoc spp. isolates among the bacteria
Significance of the effect of treatments was tested by analysis showing the best antagonistic capacities over a wide range of
of variance (ANOVA). Means of three replications were micro-organisms. The normalized values of antagonistic
separated using the Fisher's protected least significant differ- activity were used to run a hierarchical cluster analysis (Table
ence test at P ≤ 0.05. The analysis was performed with the GLM 1, Fig. 2). Cluster A, included 2 Lactococcus and 2 Leuconos-
procedure of the PC-SAS software (SAS Institute, Cary, NC, toc strains, exhibiting mainly activity against Gram positive
USA). bacteria. Cluster B was composed of 4 Lactobacillus isolates, 7
Leuconostoc and 2 Weissella, which mainly inhibited Gram
3. Results negative bacteria and in a minor degree the Gram positive S.
aureus.
3.1. Isolation, antagonistic activity and characterization of Growth of lactic acid bacteria was also compared at different
lactic acid bacteria pH and salinity conditions. Most of the isolates grew at all the
conditions tested with little variations in the growth rate.
Many vegetable products and fruits of different origins Maximum growth rates were recorded at 2 to 4% NaCl and at a
(72%) resulted in isolation of confirmed LAB colonies on MRS pH of 6.5 to 7.5 for most of the isolates. Only two strains,
agar plates indicating a wide distribution of lactic acid bacteria CC317 and CM160 showed a maximum growth rate at pH 5.5
in this type of food. LAB occurred at densities ranging from 102 (Table 2).
to 106 cfu g− 1, being fresh-cut vegetables the products with the In relation to metabolic characterization, all Leuconostoc
highest population levels. and Weissella isolates were heterofermentative and produced
56 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60
Fig. 3. Growth of LAB and foodborne pathogen test bacteria on Golden apple wounds and Iceberg lettuce leaf cuts at 25 °C during 5 days. Values are means of three
replications and mean standard errors are represented by bars. ● TM128, ■ SE303, ▼ PM249, □ L. monocytogenes ATCC 15313, ▽ S. typhimurium LT2, and ○ E.
coli ATCC 11775.
gas from glucose. On the contrary, Lactobacilllus and Lacto-
coccus isolates were all homofermentative.
3.2. Growth of LAB on fruit and vegetable tissue models
Growth of LAB in Golden Delicious apple wounds and
Iceberg lettuce cuts was studied with all members of cluster A,
strains CM160, CM135, SE303, and Lc. lactis ATCC 11454,
and some representatives of cluster B, strains PM249, TM128
(Fig. 2). Strains were selected according to their antagonistic
activity against the bacteria L. monocytogenes, E. coli, and S.
typhimurium. Lactic acid bacteria developed lower final
population levels in apple wounds than in lettuce cuts, whereas
foodborne pathogen test bacteria developed similarly in both
plant products. Foodborne pathogen test bacteria exhibited an
increase of 103 to 105 cfu wound− 1 or cfu g− 1 upon 24 h from
inoculation (Fig. 3). E. coli was the bacteria with the highest
growth, reaching the highest population levels in both fruit and
lettuce models. Lactic acid bacteria from clusters A and B
exhibited lower growth in apple and lettuce than pathogen
bacteria, having a maximum increase of 102 cfu wound− 1 or cfu
g− 1. Strain TM128 did not grew in any model, but maintained
initial population levels during a period of 5 days, whereas all
Leuconostoc (CM160, CM135 and PM249) and Lactococcus
strains (SE303 and ATCC11454) showed increased population
levels and maintained viable cell densities for 5 days. None of
the bacteria promoted spoilage reactions such as browning,
pectinolitic activity or production of off-odours in either apple
wounds or lettuce leaves within the incubation period.
3.3. Efficacy of LAB for inhibition of foodborne pathogens on
fruit and vegetables
The six selected LAB strains from clusters A and B were
tested as bioprotective agents against E. coli, S. typhimurium
and L. monocytogenes in apple wounds and lettuce cuts. LAB
strains considerably inhibited L. monocytogenes and S.
typhimurium whereas the reference postharvest biocontrol
strain P. agglomerans EPS125 inhibited all three foodborne
test bacteria, although inhibited more intensively the Gram
negative bacteria. The only exception was the LAB strain
SE303, which did not reduce population levels of any of the
pathogens tested. The effect found after treatment with LAB
was a reduction of growth on the Gram negative bacteria and
bactericide with L. monocytogenes, as final bacterial population
was lower than the initial.
Significant differences in the level of inhibition were found
between apple fruit and lettuce (P = 0.0235) and among the
different antagonistic strains used (P b 0.0001). Population
levels of the Gram positive bacteria L. monocytogenes were
significantly reduced with all LAB treatments, except with the
strain SE303, both in apple wounds (P = 0.0496) and lettuce cuts
 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60 57

Fig. 4. Effect of treatments with LAB on population levels of foodborne pathogens on apple and lettuce after two days from inoculation at 25 °C. Values are means of
three replications and bars in the columns represent the mean standard error. N: Below detection level. NTC: non-treated control. Significative different groups are
conveniently identified ⁎: Different treatments based on Fisher's unprotected test.

(P b 0.0001) (Fig. 4). The population levels of S. typhimurium
had a significant reduction (P = 0.0014) about 1–2 log cfu in
lettuce cuts by all strains except SE303, and similarly a
significative reduction (P = 0.0001) in apple wounds was
observed in the treatments with all strains except ATCC
11454. The treatment of apple wounds with P. agglomerans
EPS125 reduced populations of S. typhimurium below the
detection level. Fisher's unprotected test performed in the assay
with E. coli indicated that strain P. agglomerans EPS125
reduced significantly the growth of this pathogen in apple
wounds (below detection level), but the effect was no
significant for LAB strains (P = 0.1957). Strains ATCC 11454,
CM160, TM128, CM135, PM249 and EPS125 reduced the
population level of E. coli in lettuce cuts (P = 0.0224).
4. Discussion
In the present work we have focused on lactic acid bacteria
indigenous to fresh fruits and vegetables as potential biopro-
tective bacteria. We have isolated lactic acid bacteria from most
of samples and were especially abundant in ready-to-eat
products, suggesting a relatively important contribution to the
microbiota found in these products. The genera Lactobacillus
and Leuconostoc and to a less extent Weissella, Enterococcus
58 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60
and Lactococcus spp. were found to be the most abundant. This
could be attributed to the processing of raw vegetables, which
may increase nutrient availability and have greater colonizing
surfaces due to frequent cutting and slicing of original products,
which could be exploited by different kinds of bacteria (Ongeng
et al., 2006). The maximum population levels of LAB found in
the present work achieve 106 cfu g− 1. These results are
comparable with the amount of lactic acid bacteria found
naturally in ready-to-eat salads in other studies (Carlin et al.,
1989, Marchetti et al., 1992).
Although a high diversity of LAB is widely spread in fresh
fruit and vegetables only a considerably low percentage of
bacteria revealed to have inhibitory abilities against foodborne
bacterial pathogens. As shown in the results section the
simultaneous use of LBP medium and MRS.02 improved the
chance of obtaining positive results. For example, the use of two
different media has allowed us to find strain TM128, which is
active against S. aureus in MRS.02, and the strain CC121,
which is the best antagonist of S. typhimurium in LBP.
Additionally, strains with enhanced antagonistic activity in
both media, i.e. CM135 and CM160 (against L. monocyto-
genes), or PM249 (against S. typhimurium and E. coli) exhibited
the greatest activities in an ex vivo assay.
The high number of Leuconostoc strains with a significant
antagonistic activity found in the present work was of particular
interest. Leuconostoc species have their natural ecological niche
in green vegetation and roots, but they are also found in
fermented and refrigerated food products (Hemme and
Foucaud-Scheunemann, 2004). The use of Leuconostoc spp.
strains as additives in food is somehow limited to the
predominance of heterofermentative species, which may
negatively influence the organoleptic properties of food due to
gas production (Björkroth et al., 1998). All Leuconostoc strains
selected in this study were heterofermentative. Nevertheless, no
modifications in the general aspect of the fruit and vegetable
were observed for any of them when inoculated on either apples
or lettuce leaves for a period of 5 days.
Lactic acid bacteria grew well on vegetable cut surfaces.
Final population levels achieved by LAB strains were
considerably higher in lettuce cuts than in apple wounds. This
could be related to the high number of cut surfaces present in the
lettuce samples, which allow a better nutrient availability and
colonization. Moreover, we consider the partial inactivation of
LAB cells in apple wounds, as initial cell counts show lower
population than inoculated. However, after this initial inactiva-
tion of cells in apple wounds, population levels are maintained
or fewly increased. The growth of LAB in both vegetable and
fruit models was considered important, as other authors have
determined that establishing the growth of selected putative
protective cultures in experiments that resemble the real
conditions of application is of major concern in the assessment
of biocontrol methods (Wilson and Wisniewski, 1994; Holzap-
fel et al., 1995). It should be noticed that final population
densities of LAB strains in lettuce cuts compare well with the
growth of test bacteria specially Salmonella and Listeria strains,
excepting strain TM128, which did not increase population
levels in any of the substrates used. The growth of E. coli
significantly surpassed all tested LAB strains, including Leu-
conostoc strains CM135, CM160 and PM249. The highest
growth capacity of E. coli could explain the absence of in vivo
inhibition of this strain by LAB. Moreover, E. coli was
completely inhibited by the non LAB reference strain, EPS125,
which had a comparable growth capacity.
Efficacy trials were performed with high densities of lactic acid
bacteria to stablish potential inhibition of foodborne pathogens.
Isolated LAB interfered with the growth of two foodborne
pathogen bacteria tested, S. typhimurium, and L. monocytogenes
in apple wounds and lettuce cuts, but showed little effect over E .
coli. The inhibition had a bactericidal effect against L.
monocytogenes but not against Gram negative bacteria. Contra-
rily, P. agglomerans EPS125 inhibited all foodborne pathogen
bacteria used. This specificity of LAB for Gram positive bacteria
may be related to bacteriocin production. Experiments are under
progress to determine the mechanisms of inhibition of the LAB
strains. Further studies with inhibitory strains will stablish the
optimal application of bioprotective strains.
Outbreaks due to contaminated fresh vegetable products and
fruit represent a notable proportion of the total number of food-
related illnesses reported (Lynch et al., 2006). Previous works
have proven the effectiveness of micro-organisms other than
LAB as bioprotective agents (Janisiewicz et al., 1999; Leverentz
et al., 2006), and moreover, a combination of inoculated lytic
bacteriophages with nisin has also proven useful for the
inhibition of L. monocytogenes in both apples and honeydew
melon slices (Leverentz et al., 2003). We propose the use of
lactic acid bacteria as bioprotective agents for several reasons.
First, they are among the most studied bacteriocin producing
bacteria, and as shown, are relatively common in fresh
vegetables, reaching to densities over 106 cfu g− 1. Second,
these bacteria have been described extensively as bioprotective
agents against the colonization of a large number of bacterial
species. The present results demonstrate their potential applica-
tion on vegetable products inhibiting L. monocytogenes and S.
typhimurium. Other examples of the applicability of LAB for
preservation of food products showed the inhibition of L.
monocytogenes in meat (Schillinger and Lüke, 1989) and in
cheese (Buyong et al., 1998), Bacillus sp. in milk (Rossland
et al., 2003), or Salmonella spp. (Hudault et al., 1997). And
third, the results indicate the absence of browning and
pectinolytic activities which could be an obstacle for further
application.
It is known that lactic acid bacteria are generally regarded as
safe (GRAS) and usually fit all recommendations, including a
lack of evidences for pathogenicity to humans, to be used in
food products (Stiles and Holzapfel, 1997). These character-
istics make LAB the ideal candidates for the development of
bioprotective agents, providing a good antagonistic activity
towards target organisms. However, it has to be taken into
account that the possible involvement of certain LAB strains
was described in cases of sepsis, endocarditis or bacteremia
(Daniel et al., 2006), and consequently, each strain must be
carefully studied before commercial use.
In conclusion, the use of LAB as bioprotective agents in fresh
fruits and vegetables provided positive and encouraging results,
 R. Trias et al. / International Journal of Food Microbiology 123 (2008) 50–60
reinforced by the safe nature of this bacterial group. As shown, a
primary screening of 523 LAB isolates resulted in the selection of
18 putative protective cultures, with different inhibitory ranges
towards pathogenic micro-organisms associated with fresh fruits
and vegetables, specially for Salmonella spp. and Listeria spp..
However, further research to confirm whether the selected strains
do have a shelf-life prolonging effect on fresh fruit and vegetables,
and if they combine well with common practices in the food
industry, need to be addressed.
Acknowledgements
R. Trias was the recipient of a research grant (BR02/05) from
the University of Girona. This work was supported by the
Spanish Ministry of Education and Science (Instituto Nacional
de Investigación y Tecnología Agraria y Alimentaria, INIA
Project CAL03-084), European Union (FEDER), and Catalo-
nian Government (Comissió Interdepartamental de Ciència i
Tecnologia de Catalunya, GRC-2001SGR00293).
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