Balsamic vinegar from Modena: An easy and effective approach to reduce Listeria monocytogenes from lettuce

Abstract

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (6–7 log CFU/mL) of Listeria monocytogenes on Iceberg lettuce, the antilisterial properties of balsamic vinegar from Modena, white wine vinegar and acetic acid solutions were investigated.
Different proportions of the vinegars, acetic acid (58.7 g/L), and deionized water were evaluated to determine the role of those solutions at the stage of washing Iceberg lettuce to remove L. monocytogenes. The maximum observed log reduction of L. monocytogenes was 2.15 ± 0.04 for balsamic vinegar (50% (v/v)), 1.18 ± 0.06 for white wine vinegar ((50% (v/v)) and 1.13 ± 0.06 for acetic acid ((50% (v/v)). Washing with water only reduces 0.05 ± 0.04 log CFU/mL of L. monocytogenes numbers.
Listeria reductions observed for balsamic vinegar are similar or higher than those of chlorine-based sanitizers evaluated in other studies with lettuce. In the case of balsamic vinegar solutions, Listeria inhibition followed a linear reduction according to the model: Log (N/N0) = −4.09 × balsamic vinegar proportion % (v/v) − 0.13; R² = 0.95. Balsamic vinegar washings may be a promising method for reducing other foodborne pathogens present in produce or other foods, at home and retail environments.

Full text

Balsamic vinegar from Modena: An easy and effective approach to
reduce Listeria monocytogenes from lettuce
B. Ramos, T.R.S. Brandão, P. Teixeira, C.L.M. Silva
*
CBQF eCentro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Centro Regional do Porto da Universidade Católica Portuguesa, Rua Dr.
António Bernardino Almeida, 4200-072 Porto, Portugal
article info
Article history:
Received 5 August 2013
Received in revised form
19 January 2014
Accepted 25 January 2014
Keywords:
Listeria monocytogenes
Iceberg lettuce
Balsamic vinegar from Modena
Acetic acid
Antilisterial activity
abstract
The microbiological safety of fresh produce is a significant concern of consumers and industry. After
applying at an inoculated level (6e7 log CFU/mL) of Listeria monocytogenes on Iceberg lettuce, the
antilisterial properties of balsamic vinegar from Modena, white wine vinegar and acetic acid solutions
were investigated.
Different proportions of the vinegars, acetic acid (58.7 g/L), and deionized water were evaluated to
determine the role of those solutions at the stage of washing Iceberg lettuce to remove L. monocytogenes.
The maximum observed log reduction of L. monocytogenes was 2.15 0.04 for balsamic vinegar (50% (v/
v)), 1.18 0.06 for white wine vinegar ((50% (v/v)) and 1.13 0.06 for acetic acid ((50% (v/v)). Washing
with water only reduces 0.05 0.04 log CFU/mL of L. monocytogenes numbers.
Listeria reductions observed for balsamic vinegar are similar or higher than those of chlorine-based
sanitizers evaluated in other studies with lettuce. In the case of balsamic vinegar solutions, Listeria in-
hibition followed a linear reduction according to the model: Log (N/N
0
)¼4.09 balsamic vinegar
proportion % (v/v) 0.13; R
2
¼0.95. Balsamic vinegar washings may be a promising method for reducing
other foodborne pathogens present in produce or other foods, at home and retail environments.
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
The incidence of foodborne infections caused by bacterial
pathogens continues to be a problem in industrialized nations and
developing countries (Chang & Fang, 2007; Ramos, Miller, Brandão,
Teixeira, & Silva, 2013). Bacteria most frequently linked to food
outbreaks are Salmonella spp., Escherichia coli,Listeria mono-
cytogenes and Shigella spp. (Rico, Martin-Diana, Barat, & Barry-
Ryan, 2007; Senorans, Ibanez, & Cifuentes, 2003; Warriner, 2005).
Gastrointestinal disease caused by L. monocytogenes is rare
compared to other agents of foodborne illness, but invasive liste-
riosis can be very severe and has a high fatality rate (Little et al.,
2007; Miller, Ramos, et al., 2009; Nastou et al., 2012). This path-
ogen is considered ubiquitous in nature environment and produce
is likely to be contaminated (Sant’Ana, Igarashi, Landgraf, Destro, &
Franco, 2012).
Several studies have reported that the occurrence of
L. monocytogenes in ready-to-eat vegetables in several parts of the
world may be as high as 25% (Cordano & Jacquet, 2009; Crepet,
Albert, Dervin, & Carlin, 2007; Sant’Ana et al., 2012). This is of
special concern because this kind of food is likely to be consumed
raw, relying only on cold storage to maintain their safety, but Lis-
teria has the ability to survive and multiply at refrigeration tem-
peratures (Luber et al., 2011; Miller et al., 2011).
Vegetable consumption has grown over the last two decades,
especially lettuce that on average is eaten by a third of the popu-
lation once a week (Doménech, Botella, Ferrús, & Escriche, 2013).
Raw vegetables have been identified as a vehicle of transmission of
foodborne outbreaks and play an important role in listeriosis
epidemiology. Improper temperature control, poor cleanliness and
inappropriate refrigerator management have been identified as
critical factors in foodborne listeriosis (Luber et al., 2011; Ramos
et al., 2013; Sant’Ana et al., 2012).
Proper food handling at home can maintain the hazard at a safe
level and even reduce it. Thus, it is important to develop strategies
to control L. monocytogenes in the home environment (Doménech
et al., 2013; Shen, Geornaras, Kendall, & Sofos, 2009).
Retail environments also play a role in the contamination of
foods and/or amplification of L. monocytogenes, however linking a
specific retail environment to an outbreak of infection is difficult to
prove (Varma et al., 2007). Interventions directed at home and
retail environments may be an important way to reduce sporadic
*Corresponding author. Tel.: þ351 22 5580058; fax: þ351 22 5090351.
E-mail address: clsilva@porto.ucp.pt (C.L.M. Silva).
Contents lists available at ScienceDirect
Food Control
journal homepage: www.elsevier.com/locate/foodcont
0956-7135/$ esee front matter Ó2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2014.01.029
Food Control 42 (2014) 38e42

disease, which represents the greatest burden of L. monocytogenes
infection (Varma et al., 2007).
In the home or restaurant, fewer options are available for
effective washing of vegetables than in a modern processing plant.
There are various chemical compounds available in these envi-
ronments that can be useful for sanitizing fresh produce, particu-
larly vinegar which contains acetic acid (Shen et al., 2009; Yang,
Kendall, Medeiros, & Sofos, 2009).
Vinegar and vinegar-based solutions are commonly used as
dressings for salads and appetizers and have been studied with
favourable results for their efficacy in removing pathogens from fresh
fruits andvegetables (Chang & Fang, 2007; Sengun& Karapinar, 2004;
Shen et al., 2009; Vijayakumar & Wolf-Hall, 2002). In addition, con-
sumers are increasingly avoiding consumption of foods treated with
preservatives of chemical origin and so vinegar solutions can be an
appealing natural alternative (Sengun & Karapinar, 2004).
The purpose of this study was to determine the antimicrobial
activity of vinegar solutions on L. monocytogenes inoculated onto
lettuce. With this aim in mind, inactivation of this microorganism
with different washing solutions was determined.
2. Materials and methods
2.1. Preparation of L. monocytogenes inoculum
2.1.1. Cultures
A three-strain composite of L. monocytogenes was used in this
study. These strains were 1334 serotype 1/2c, 1336 serotype 1/2b
and 1092 serotype 4b (Escola Superior de Biotecnologia, UCP).
L. monocytogenes strains were grown independently for 24 h at
37

C in 50 mL Tryptic Soy Broth (Lab M, Lancashire, UK) with 0.6%
yeast extract - TSBYE (Lab M).
2.1.2. Preparation of cultures
The second subculture of each strain was incubated at 37

C for
24 h to yield stationary phase cultures. This cell growth phase was
chosen due to its higher stress resistance than exponential phase
cells (Miller, Gil, Brandão, Teixeira, & Silva, 2009).
The three cultures were mixed together in the same proportion,
and washed twice by centrifuging (5000 rpm, 5 min, 4

C) with
sterile distilled water. The cell pellets were resuspended in distilled
water so that final cell numbers in the suspension were approxi-
mately 6e7 log CFU/mL.
2.2. Procedure for inoculating lettuce
Iceberg lettuce (Lactuca sativa capitata) was purchased from a
local supermarket, outer layers were removed, and the leaves were
portioned by hand. All lettuces were kept at 4e5

C between the
time of purchase and initiation of experiments, and were then used
immediately.
Lettuce was dipped into L. monocytogenes culture suspension for
15 min and placed on sterile paper for removing excess liquid at
room temperature (20

C) and transferred to sterile bags. To facil-
itate the attachment of bacteria, samples were stored for 24 h at 4e
5

C before they were treated with the solutions.
2.3. Preparation of treatment solutions
Preliminary studies, using the agar diffusion method, were
made to establish the antilisterial activity of vinegars commercially
available: traditional balsamic vinegar from Modena (details
further in text; referred to only as balsamic vinegar), rice, fruit,
white and red wine and cider vinegars. Balsamic vinegar showed
the best antilisterial activity and for that reason was chosen for
future analyses (data not shown). Wine vinegar (later referred to
only as white vinegar) was also selected due to its common use and
presence in households.
Vinegars and acetic acid solutions, for the dipping treatment,
were prepared immediately before use. The control solution was
done with sterile distilled water alone.
Vinegar solutions were made by dilution in distilled water, to
achieve the following vinegar proportions: 15, 20, 37 and 50% (v/v).
Acetic acid concentration of vinegars, determined according to
NP 3264:1989, was 58.7 and 61.5 g acetic acid/L, for balsamic and
wine vinegar, respectively.
A solution with a concentration of 58.7 g acetic acid/L was
prepared from glacial acetic acid (Panreac, Barcelona, Spain) and
diluted in distilled water in the same proportions as the previous
vinegar solutions, resulting in the following acetic acid weight
percentages: 0.9, 1.2, 2.2 and 2.9.
The pH value of the solutions was measured using a pH meter
(GLP 22, Crison Instruments, Spain) and the mean values are pre-
sented in Table 1.
2.4. Washing treatments
Inoculated lettuce, 50 g approximately, was added to 1 L of the
treatment solutions (at 20

C), sufficient to cover all pieces, and left
for 15 min at room temperature. After the samples were removed,
they were placed on sterile absorbent paper to allow removal of the
excess liquid.
All experiments were made in triplicate.
2.5. Enumeration of L. monocytogenes
Listeria enumeration was done before and after the washing
procedures. A 25 g sample of lettuce was aseptically transferred to
225 mL of buffered peptone water (Lab M, Lancashire, UK) in a
stomacher bag and homogenized in a Stomacher (Lab-Blender 400,
Seward Medical, London, UK) for 90s. Each sample was serially
diluted and plated in duplicate onto Palcam agar (Merck, Darm-
stadt, Germany) plus selective supplement (Merck, Darmstadt,
Germany). Typical colonies were counted after incubation at 37

C
for 48 h to determine the survival of L. monocytogenes.
Mean values of bacterial counts (CFU/g), from duplicate plate
samples were converted to log numbers for each combination.
2.6. Data analysis
In terms of microbial loads, the treatment effects were assessed
by calculating the reduction of microbial content in relation to fresh
untreated samples, expressed in terms of log-cycles (i.e. log (N/N
0
),
Table 1
pH values for the different tested solutions.
Compound Proportion % (v/v) pH
Balsamic vinegar from Modena 15 3.38
20 3.35
37 3.29
50 3.26
White vinegar 15 3.05
20 3.00
37 2.93
50 2.89
CH
3
COOH 58.7 g/L 15 2.92
20 2.88
37 2.73
50 2.65
B. Ramos et al. / Food Control 42 (2014) 38e42 39

where N
0
is the sample initial microbial load and Nis the microbial
load after treatment). Microsoft
Ò
Excel 2010 (Microsoft Corpora-
tion, Washington, USA) was used for all calculations analysis.
A two-way ANOVA was used to assess the influence of treatment
solutions and corresponding concentrations on Listeria inactiva-
tion. Multiple comparisons on mean values of Listeria enumerations
were evaluated by Tukey’s post-hoc test using SPSS statistics 20
(IBM, New York, USA). The level of significance for all tests was 0.05.
3. Results and discussion
Viable L. monocytogenes reductions obtained after washing were
relative to populations on inoculated lettuce (positive control). The
inoculation level used in the experiment was higher than natural
contamination to allow valid observation of bacterial reductions
after washing with different solutions.
This study revealed that the usual method, for home and retail
environments, of water dipping lettuce with water is not effective
in removing Listeria from lettuce. Water dipping only decreased
0.05 0.04 log of L. monocytogenes inoculated on lettuce.
As Fig. 1 shows, all the treatment solutions significantly decrease
L. monocytogenes population (p<0.05) comparing with water
dipping.
Balsamic vinegar solutions resulted in the greatest reductions in
viable number of L. monocytogenes, with the exception of 15% (v/v)
proportion solutions, where white vinegar solutions showed
greater reductions. A higher quantity of balsamic vinegar (20% v/v)
was required to exceed the maximum effect against
L. monocytogenes of acetic acid and white vinegar solutions (1.13e
1.18 log CFU/g reduction). Maximum reduction was achieved by
dipping lettuce in 50% (v/v) of balsamic vinegar
(2.15 0.04 log CFU/g). In such conditions, the colour and perceived
texture of the lettuce were retained.
The various proportions of balsamic vinegar washings resulted
in different bacteria reductions, and the increment of vinegar so-
lution % was followed with increasing reductions of bacterial
numbers (p<0.05). In fact, Listeria destruction followed a linear
reduction according to the model: Log (N/N
0
)¼4.09 balsamic
vinegar proportion % (v/v) 0.13; R
2
¼0.95. Data and model fitare
shown in Fig. 2.
Populations of L. monocytogenes were reduced
(0.86 0.02 log CFU/g) significantly (p<0.05) when the samples
were dipped in 15% (v/v) of acetic acid solution. However,
increasing the proportion of acetic acid from 20 to 50% (v/v) did not
result in any further decrease (1.13 log CFU/g; p>0.05). For
increased proportions of white vinegar, different reductions were
obtained (p<0.05) with the exception of 37% (v/v).
Overall acetic acid and white vinegar solutions showed similar
efficiency on removing L. monocytogenes from lettuce (p>0.05).
According to the present results, dipping lettuce in 1.0e2.9% g/L
acetic acid resulted in 1.08e1.13 log CFU/g reduction. Akbas and
Olmez (2007b) reported that populations of L. monocytogenes on
Iceberg lettuce were reduced (0.9 log CFU/g). In a study by Samara
and Koutsoumanis (2009), dipping lettuce in 0.5% and 1.0% acetic
acid reduced L. monocytogenes by less than 1 log CFU/cm
2
. Our data
is in general agreement with most other studies reporting that
acetic acid concentrations of up to w1.0% are unlikely to reduce
L. monocytogenes populations by more than about 1 log CFU/g
(Nastou et al., 2012; Samara & Koutsoumanis, 2009; Zhang & Farber,
1996). Other studies with organic acids (0.25 g/100 g citric acid plus
0.50 g/100 g ascorbic acid) reported similar Listeria reductions to
those obtained with white wine vinegar and acetic acid solutions
(Ölmez & Temur, 2010).
Efficacy of balsamic vinegar at 50% (v/v) to decontaminate
L. monocytogenes from lettuce surfaces was similar or higher to
those of chlorine-based sanitizers evaluated in other studies (Akbas
& Olmez, 2007a, 2007b; Behrsing, Winkler, Franz, & Premier, 2000;
Doménech et al., 2013; Kilonzo-Nthenge, Chen, & Godwin, 2006;
Park et al., 2011; Zhang & Farber, 1996). Ölmez and Temur (2010)
reported similar Listeria reductions, 2.3 and 2.2 log CFU/g with
chlorine (100 mg/L) and ozone (2 mg/L) respectively. Park et al.
(2011) reported higher antilisterial activity linked to malic, citric
and lactic acid, however in that study they only allowed bacterial
attachment for 3 h. This increase in the efficacy of sanitizing
treatments may be explained by the initiation of biofilm formation
whereas there is an increase in the development of cell aggregates
after 24 h of incubation (Ells & Truelstrup Hansen, 2006; Koseki,
Yoshida, Isobe, & Itoh, 2001). The strength of attachment is a
main factor affecting the efficacy of sanitizing treatments (Ölmez &
Temur, 2010).
The higher antimicrobial activity of balsamic vinegar solutions
was not only due to hydrogen ion effect, since the pH of the bal-
samic vinegar solutions ranged from 3.26 to 3.38 while the white
vinegar and the acetic acid solutions had pH values of 2.89e3.05
and 2.65e2.92, respectively. Also the acid present in all the solu-
tions was mostly present in undissociated form (pH <pKa
(CH
3
COOH)).
The stronger bactericidal effect of balsamic vinegar may be also
related to the presence of compounds with antimicrobial proper-
ties resulting from the fermentation of grape juice and from grape
juice itself. It is known that grapes contain a number of phenolic
compounds that exhibit antilisterial activity, particularly polymeric
phenolic compounds: resveratrol, vanillic acid, caffeic acid, gallic
acid and flavonoids (rutin and quercetin) (Baydar, Özkan, & Sa
gdiç,
Fig. 1. Antilisterial activity of the different washing solutions and proportions, shown in terms of Log (N/N
0
). For a given washing proportion, values with different letters differ
significantly (p<0.05).
B. Ramos et al. / Food Control 42 (2014) 38e4240

2004; Oliveira et al., 2013; Rhodes, Mitchell, Wilson, & Melton,
2006; Rodríguez Vaquero, Alberto, & Manca de Nadra, 2007). In
fact Plessi, Bertelli, and Miglietta (2006) found three of these
antilisterial compounds, vanillic acid, gallic acid and caffeic acid in
traditional balsamic vinegar from Modena.
4. Conclusions
All tested solutions showed higher bactericidal effects against
the L. monocytogenes strains than water, although the balsamic
vinegar activity was clearly higher.
Balsamic vinegar showed similar and even better effectiveness
than chlorine-based sanitizers on removing L. monocytogenes from
lettuce surface, even though the time of storage of inoculated let-
tuce allowed the formation of biofilms. The presence of phenolic
compounds naturally presented in grape and grape juices may be
responsible for its high antilisterial activity.
Balsamic vinegar washing seems to be a promising method to
reduce L. monocytogenes present in produce at home and retail
environments. Good results at home or retail environment may be
achieved simply adding 50 mL of vinegar to 250 mL of water and
dipping the vegetables for 15 min (approximately 1 log reduction
can be attained).
Balsamic vinegar may be a promising effective solution to
inhibit other food pathogens present on produce surface or other
foods. There is a lack of studies with these vinegars and it is an
important resource for households and food establishments due to
is availability and organic nature.
Acknowledgements
This work was supported by National Funds from FCT eFun-
dação para a Ciência e a Tecnologia through project PEst-OE/EQB/
LA0016/2011.
Financial support for authors Ramos B., Miller F.A. and Brandão
T.R.S. was provided by FCT and Fundo Social Europeu (FSE) through
fellowships SFRH/BD/42169/2007, SFRH/BPD/65041/2009 and
SFRH/BPD/41419/2007, respectively.
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