Food Microbiology 85 (2020) 103282

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Biopreservation approaches to reduce Listeria monocytogenes in fresh T

vegetables
Bárbara Ramos, Teresa R.S. Brandão, Paula Teixeira, Cristina L.M. Silva*
Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327,
4169-005 Porto, Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: Two biopreservation approaches for fresh lettuce, rocket salad, parsley and spinach were studied.
Biopreservation The potential of Pediococcus pentosaceus DT016, as a protective culture, to suppress Listeria monocytogenes in
Listeria monocytogenes vegetables during storage was evaluated. The pathogen numbers in the vegetables inoculated with P. pentosaceus
Fresh vegetables DT016 were significantly (p < 0.01) lower throughout the storage period and, at the last storage day, a
Protective culture
minimum difference of 1.4 log CFU/g was reported when compared with the vegetables without the protective
Pediocin
Pediococcus pentosaceus DT016
culture. Moreover, by using two levels of L. monocytogenes (about 6 and 4 log CFU/g), it was observed that the
antagonist effect of P. pentosaceus was higher for the lower pathogen numbers.
The second approach evaluated a pediocin DT016 solution to inactivate and control L. monocytogenes pro-
liferation. The pathogen load was studied after washing with: water, chlorine and the pediocin solution and
along storage at 4 °C. Comparing the various washing solutions, the vegetables washed with pediocin presented
significantly (p < 0.01) lower pathogen numbers throughout storage, by a minimum of 3.2 and 2.7 log CFU/g,
than in vegetables washed with water and chlorine, respectively.
The proposed methodologies are promising alternatives to maintain the safety of fresh vegetables during
extended storage at refrigeration temperature.

1. Introduction Consumers' concern about chemical residues in food products has

Listeria monocytogenes is considered ubiquitous in the agricultural
environment and its occurrence in vegetables may be as high as 25 %
(Cordano and Jacquet, 2009; Crepet et al., 2007; Samara and
Koutsoumanis, 2009; Sant'Ana et al., 2012). In fact, raw vegetables
have been identified as a vehicle of transmission of foodborne out-
breaks and play an important role in listeriosis epidemiology (Luber
et al., 2011; Mir et al., 2018). This is of special concern because this
kind of food is eaten raw and relies only on cold storage to maintain its
safety, moreover Listeria has the ability to survive and multiply at re-
frigeration temperatures (Degli Esposti et al., 2018; McManamon et al.,
2019; Ramos et al., 2014).
Currently, commercial operations use wash treatments with anti-
microbials as the only step to reduce microbial populations on fresh
produce, being chlorine the most commonly used sanitizer. However,
numerous reports indicate that chlorine has limited antimicrobial effi-
cacy at the permitted levels, and it has been associated with the pro-
duction of potentially toxic substances (McManamon et al., 2019; São
José et al., 2014; Siroli et al., 2015a,b; Siroli et al., 2017).
led the food industry to seek novel and alternative technologies to
improve food quality and safety with the minimal use of chemicals
(Alegre et al., 2013). The use of generally recognized as safe (GRAS)
microorganisms, such as lactic acid bacteria (LAB) and/or their natural
metabolites, shows to be a promising alternative to maintain the food
safety and it is also perceived by the consumer as a natural food pre-
servation method (Engelhardt et al., 2015; Ramos et al., 2013).
Protective cultures of LAB have been developed over the last few
decades to increase the safety and shelf-life of fresh and minimally
processed vegetables (Siroli et al., 2015a,b; Siroli et al., 2015a,b; Trias
et al., 2008; Vescovo et al., 1996). The preservation abilities of LAB are
a result of several mechanisms of action, and are mainly related to the
production of antimicrobial compounds such as organic acids, hy-
drogen peroxide and bacteriocins (Allende et al., 2007).
Bacteriocins, ribosomally synthesized antimicrobial peptides, are
able to kill or inhibit the growth of other bacteria and are considered to
be safe natural biopreservatives (Barbosa et al., 2018; Martínez et al.,
2019). The use of these molecules in the washing of minimally pro-
cessed vegetables has been studied in recent years (Siroli et al., 2017).

*
Corresponding author.
E-mail address: clsilva@porto.ucp.pt (C.L.M. Silva).

https://doi.org/10.1016/j.fm.2019.103282
Received 15 January 2019; Received in revised form 21 July 2019; Accepted 26 July 2019
Available online 29 July 2019
0740-0020/ © 2019 Elsevier Ltd. All rights reserved.
B. Ramos, et al.
Moreover, several authors reported a reduction of 1.2 to 2.7 log units on
L. monocytogenes loads in vegetables washed with bacteriocin solutions
(Allende et al., 2007; Molinos et al., 2005; Randazzo et al., 2009; Siroli
et al., 2015a,b).
The effectiveness of bacteriocins is dependent on the chemical and
physical properties and on the storage conditions of foods (Aasen et al.,
2003; Molinos et al., 2005). In fact, food components can disturb bac-
teriocin's adsorption on to the vegetable's surface and even cause their
inactivation. In addition, bacteriocins can be affected by the natural
microbiota present on vegetables (Allende et al., 2007). So, the po-
tential use of each bacteriocin should be tested in the appropriate food
system.
The aim of this study was to evaluate two biopreservation ap-
proaches on the fate of L. monocytogenes in vegetables. As several re-
ports showed the incidence and prevalence of L. monocytogenes in let-
tuce, parsley, spinach and mixed salad vegetables (Zhu et al., 2017), the
methodologies were assessed in these vegetables and in rocket salad. A
bacteriocinogenic LAB, Pediococcus pentosaceus DT016, previously iso-
lated from lettuce, with antibacterial activity towards L. monocytogenes
and Listeria innocua (Ramos et al., 2016) was used as a protective cul-
ture or as a pediocin producer.
The first approach was to assess P. pentosaceus DT016 potential to
act as a protective culture towards L. monocytogenes in fresh vegetables.
The ability of the protective culture to grow and to exert its inhibitory
activity on the target pathogen in the different vegetables and at low
temperatures was studied. Moreover, two different L. monocytogenes
loads were evaluated to investigate if pathogen numbers could affect
the P. pentosaceus antagonist outcome.
The second biopreservation method explored the use of pediocin in
a washing step as a mean to reduce and control L. monocytogenes pro-
liferation in vegetables. Following vegetable's washing step, the im-
mediate reduction on the pathogen numbers was assessed along with
Listeria proliferation during the vegetable's storage. Additionally, results
were compared with the traditional methodology of chlorine and water
rinses.
2. Materials and methods
2.1. Vegetables
Whole wrapped iceberg lettuce (Lactuca sativa capitata) and packed
vegetables: rocket salad (Eruca sativa Mill.), spinach leaves (Spinacia
oleracea L.) and parsley (Petroselinum crispum Mill.) from Vitacress
Portugal S.A., were purchased from local retail establishments the day
before the experiment.
The external leaves and core of iceberg lettuce were discarded and
the rest was washed in tap water, drained, let too dry on sterile paper
and kept at 4 °C until use.
The other vegetables were removed from the bags and stored
overnight in air at 4 °C without any further discarding or processing
until the experiments.
2.2. Bacterial strains, media and culture conditions
The bacterial strains used in this work were: Pediococcus pentosaceus
DT016 and a three-strain cocktail of Listeria monocytogenes.
The LAB strain was grown for 24 h at 37 °C in de Man, Rogosa
Sharpe broth (MRS, Lab M, Lancashire, UK). P. pentosaceus DT016
second subculture was incubated in MRS broth (Lab M) at 37 °C for 24
h and washed twice by centrifugation (5000 rpm, 15 min, 4 °C) with
sterile distilled water. The final cell pellets were resuspended in dis-
tilled water so that the final cell numbers in the suspension were ap-
proximately 8 log CFU/mL.
The Listeria strains: 1334 serotype 1/2c, 1336 serotype 1/2b and
1092 serotype 4b (Escola Superior de Biotecnologia Culture Collection,
UCP) were grown independently for 24 h at 37 °C in Tryptic Soy Broth
2
Food Microbiology 85 (2020) 103282
(TSB, Lab M) with 0,6 % yeast extract -TSBYE (Lab M). 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 et al.,
2009). The cultures were washed as mentioned before with sterile
distilled water. The three pathogen cultures were mixed together in the
same proportion (1:1:1) and the cell pellets were resuspended in dis-
tilled water to obtain final bacterial levels of about 5 and 7 log CFU/mL.
2.3. Preparation of the washing solutions
A chlorine solution (NaOCl, 200 μg/mL) was prepared from a
commercial bleach disinfectant available for fresh fruits and vegetables
(1.15 mg/mL – AMUKINA, Farma-Lepori), and according to manufac-
turer's instructions.
Pediocin DT016, with an activity of 25,600 AU/mL against L.
monocytogenes strains 1334 and 1336, was obtained by the cultivation
of the producer strain P. pentosaceus DT016 in MRS broth (Lab M) for
18 h at 37 °C (Ramos et al., 2016). Then the culture was centrifuged
(5000 rpm, 15 min, 4 °C), the cell-free supernatant was filtered ster-
ilized (0.22 μm, Corning Incorporated, Germany) and the pH adjusted
to 6.5 with NaOH (1 N). The pediocin DT016 washing solution was
prepared by diluting 200 mL of the bacteriocinogenic supernatant in
1800 mL of sterile distilled water.
All solutions were prepared fresh before use.
2.4. Effect of Pediococcus pentosaceus DT016 on Listeria monocytogenes
populations inoculated in fresh vegetables
Pathogen concentrated suspensions were obtained as described
above. Antagonist, P. pentosaceus DT016, was tested at 7 log CFU/mL
and L. monocytogenes at 6 (L1) and 4 (L2) log CFU/mL.
The vegetables (1000 g) were immersed in suspensions (10 L)
containing the pathogen alone (L1 or L2), or the antagonist and the
pathogen (P + L1, P + L2), for 15 min and placed on sterile paper for
removing excess liquid at room temperature (20 °C). Vegetables in-
oculated exclusively with P. pentosaceus DT016 were also used to ana-
lyse the protective culture fate in the vegetables. To facilitate the at-
tachment of bacteria, samples were stored for 1 h at 4 °C and then
aseptically divided into 50 g sterile bags for storage over a 15-day
period at 4 °C.
Pathogen, LAB and mesophilic bacteria numbers were determined
immediately after inoculation and along storage period.
All experiments were made in three independent trials.
2.5. Effect of a pediocin DT016 washing solution on Listeria monocytogenes
populations inoculated in fresh vegetables
The vegetables (1000 g) were contaminated with the L. mono-
cytogenes cocktail (7 log CFU/mL) as described by Ramos et al. (2014).
In detail, the vegetables were dipped into a 10 L pathogen 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
facilitate the attachment of bacteria, samples were stored for 24 h at 4
°C before they were treated with the washing solutions.
Approximately 500 g + 500 g of each inoculated vegetable (lettuce,
rocket salad, spinach or parsley) were washed for 5 min, at room
temperature, in 2 L of the following solutions: (1) sterile distilled water
(H2O), (2) sodium hypochlorite 200 μg/mL solution (Chlorine), (3)
pediocin DT016 solution (Pediocin DT016).
After washing, the vegetables were placed on sterile absorbent
paper to allow removal of the excess liquid at room temperature (20
°C). The samples were aseptically divided into 50 g sterile bags for
storage over a 15-day period at 4 °C.
Listeria, lactic acid bacteria and mesophilic bacteria enumeration
was done before and immediately after the washing procedures and
B. Ramos, et al.
along the refrigerated storage.
Experiments using the water washings (sterile distilled water) were
performed as control.
All experiments were made in three independent trials.
2.6. Microbiological analyses
The inoculated vegetable samples with the pathogen and those
supplemented with LAB were analysed immediately after the treatment
and along the produce storage.
The control samples comprised: not inoculated vegetables, samples
inoculated only with the protective culture and samples inoculated only
with the target pathogen at two different loads.
The estimated protective culture load was further confirmed using
the control samples. The control samples, not inoculated with the target
pathogen, were checked for the presence of naturally occurring L.
monocytogenes.
The sampling period was determined by the overall vegetable's
perceivable fresh appearance. For lettuce samples the analyses were
performed along 7 days of storage (0, 3, 5 and 7 days); for rocket salad
the analyses were performed over 9 days of storage (0, 3, 5, 7 and 9
days), whereas for spinach and parsley the samples were evaluated
along 15 days of storage (0, 3, 5, 7, 9, 12 and 15 days).
In the trials involving the vegetables washings, the analyses were
performed before and immediately after treatment and along storage.
The sampling period for each vegetable was the same as for the ex-
periments mentioned above. The control samples, not inoculated with
the target pathogen, were checked for the presence of naturally oc-
curring L. monocytogenes.
Samples of 25 g of each vegetable were aseptically transferred to
225 mL of Buffered Peptone Water - BPW (Lab M) and homogenized in
a Stomacher (Lab-Blender 400, Seward Medical, London, UK) for 90 s.
Each sample and control sample were serially diluted and plated in
duplicate onto Tryptic Soy Agar-TSA (Lab M) to enumerate the meso-
philic bacteria (30 °C, 48 h), onto MRS agar (Lab M) to enumerate the
LAB population (37 °C, 48 h), and onto Palcam agar (Merck) plus se-
lective supplement (Merck) to estimate L. monocytogenes population
(37 °C, 48 h).
The non-Listeria populations were calculated as the difference be-
tween TSA counts and viable counts of L. monocytogenes. For the sam-
ples with protective culture the mesophilic populations were calculated
as the difference between TSA counts and viable counts in MRS. Mean
values of bacterial counts (CFU/g), from duplicate plate samples were
converted to log numbers for each combination.
The bacteriocinogenic activity in the vegetable samples was also
analysed. Samples (10 g each) of fresh vegetables were homogenized
with 0.02 N HCl (1:2 w/v) and centrifuged at 5000 rpm for 15 min.
The supernatants were filtered sterilized (0.22 μm, Corning
Incorporated, Germany) and the inhibitory activity was assayed by the
agar-spot test. Briefly, the L. monocytogenes strains (mentioned in sec-
tion 2.2.) were grown overnight at 37 °C in TSBYE (Lab M) and evenly
spread with a sterile cotton swab in TSAYE (Lab M). Plates were al-
lowed to set and 20 μL drops of vegetables samples were spotted on the
lawns of pathogens. Plates were incubated at 30 °C until confluent
growth was observed and then examined for zones of inhibition around
the inoculation spots.
2.7. Colour properties
The colour values (L*, a* and b*) of fresh and minimally processed
vegetables were assessed using a Minolta CR-400 colorimeter (Konica-
Minolta, Osaka, Japan), calibrated with a white standard tile. Lightness
value L* indicates how dark/light the sample is (ranges from black at 0
to white at 100). The chromaticity coordinate a* measures red when
positive and green when negative, and the chromaticity coordinate b*
measures yellow when positive and blue when negative.
3
Food Microbiology 85 (2020) 103282
Analysis was carried out on 10 pieces of each vegetable randomly
chosen from each bag at 3, 5, 7, 9, 12 and 15 days of storage at 4 °C.
The measures limit for lettuce and rocket salad was at the 7th and 9th
day of storage, respectively.
2.8. pH measurement
Five-gram sample of vegetable tissue was blended with an ultra-
turrax homogenizer (Ika digital T25, IKA®-Werke GmbH & Co. KG,
Staufen, Germany) for 5 min in 20 mL of ultra-pure water. The pH of
the slurry was measured at room temperature using a research pH-
meter (GLP 22, Crison Instruments, Barcelona, Spain).
2.9. Data analysis
A one-way ANOVA was used to assess the influence of the protective
culture on the L. monocytogenes, mesophilic and LAB load and on colour
properties along storage. The influence of the washing solutions on
Listeria, mesophilic and LAB loads and on colour coordinates was also
evaluated. Multiple comparisons on mean values of bacteria enumera-
tions and colour coordinates were evaluated by Tukey's post-hoc test
using SPSS statistics 22 (IBM, New York, USA). The level of significance
for all tests was 0.01.
3. Results and discussion
3.1. Effect of Pediococcus pentosaceusDT016 on Listeria monocytogenes
populations inoculated in fresh vegetables
The effect of in situ P. pentosaceus DT016 on the behaviour of L.
monocytogenes in fresh vegetables was determined during the storage
period at 4 °C (Fig. 1).
P. pentosaceus DT016 showed excellent adaptability to vegetables
stringent conditions, as it was able to survive and grow in all the ve-
getables at the selected temperature. When inoculated alone, the LAB
counts were around 7 log CFU/g in all the vegetables during the storage
period (data not shown). In the presence of the pathogen, P. pentosaceus
maintained levels above 6.2 log CFU/g along the storage period in all
the vegetables studied (Fig. 1).
In the analysis with the higher Listeria load (L1), L. monocytogenes
grew throughout the shelf-life period of all the vegetables, reaching +
high numbers (Fig. 1). In the presence of P. pentosaceus DT016, the
pathogen counts were reduced in all the vegetables. In the experiment
with the lower inoculum of L. monocytogenes (L2), a similar outcome
was achieved, although the Listeria reduction being higher (p > 0.01).
In detail, at the end of vegetables storage the pathogen final numbers
were 4.9, 4.9, 4.8 and 5.2 log CFU/g in lettuce, rocket salad, parsley
and spinach, respectively (Fig. 1).
The presence of bacteriocinogenic activity, by the direct application
of filtered vegetables samples to L. monocytogenes, was detected in all
the vegetables from the 3rd day of storage (700 AU/mL). The high
antimicrobial activity from the 3rd day is mainly due to bacteriocin
production during storage and cannot be attributed to a reduction in pH
or production of lactic acid, as the pH values (data not shown) re-
mained similar to the vegetables in the absence of the protective culture
(p > 0.01).
P. pentosaceus DT016 was able to produce the antimicrobial peptide
in all the vegetables, though the different conditions such as the natural
microbiota, the physical and chemical environment. This is an im-
portant feature as several microorganisms showed high biopreservation
potential in food models and that was not confirmed in real food ma-
trices or their action was affected by the food matrix (Allende et al.,
2007; Bennik et al., 1999; Collazo et al., 2019; McManamon et al.,
2019; Palmai and Buchanan, 2002).
The Listeria reductions accomplished in all the vegetables by the
protective culture ranged from 1.4 to 1.9 and 1.5 to 2.2 log units, for
B. Ramos, et al.
Fig. 1. Survival of Listeria monocytogenes in fresh vegetables (L1 and L2) and in vegetables with Pediococcus pentosaceus DT016 as a protective culture (P + L1 and
P + L2), during storage at 4 °C. P. pentosaceus DT016 concentration in the vegetables, along storage, is represented by the line. The vegetables are A: Lettuce; B:
Rocket salad; C: Parsley; D: Spinach. Error bars show standard deviation (SD).
the higher (L1) and lower pathogen (L2) load, respectively. These re-
sults are higher or identical than those obtained with other micro-
organisms (Cai et al., 1997; Collazo et al., 2019; McManamon et al.,
2019; Oliveira et al., 2015; Palmai and Buchanan, 2002; Siroli et al.,
2015a,b; Vescovo et al., 1996).
The addition of P. pentosaceus did not reduce the mesophilic bacteria
present in the vegetables. In fact, depending on vegetable, the samples
with the protective culture (P + L1 and P + L2) presented similar
(p > 0.01) or higher (p < 0.01) mesophilic loads than the samples in-
oculated exclusively with the investigated pathogenic microorganism
(L1 and L2) (Fig. 2). Iceberg samples presented equal mesophilic
numbers although during storage there was a difference between the
samples without protective culture (L1 and L2) and the one with LAB
culture (P + L1). For rocket salad and spinach samples, the differences
observed were attenuated during storage, and at the end of storage
period no distinction on the mesophilic numbers was observed
(p > 0.01). The mesophilic bacteria numbers were higher in parsley
samples with the protective culture (P + L1) than in the other samples
(P + L2, L1 and L2) (p < 0.01), at the 15th day of storage. At the end of
storage, in all samples, the total aerobic mesophilic cell loads ranged
between 6.0 and 7.0 log CFU/g.
The microbiota of foods have been recognized as having inhibitory
activity against some foodborne pathogens (Alegre et al., 2013; Allende
et al., 2007), so the fact that P. pentosaceus did not negatively affect the
mesophilic numbers can be an advantage.
3.2. Effect of a pediocin DT016 washing solution on Listeria monocytogenes
populations inoculated in fresh vegetables
Lettuce samples artificially contaminated with L. monocytogenes still
retained approximately 6.0 and 5.4 log CFU of viable Listeria/g after
washing with water and the chlorine solution (sodium hypochlorite
200 μg/mL) (Fig. 3 A). In samples treated with pediocin DT016 the
level of viable Listeria after washing was 2.1 and 1.6 log CFU/g lower
than the vegetables treated with water and chlorine solution, respec-
tively (p < 0.01).
Listeria counts increased throughout storage in the lettuce samples
4
Food Microbiology 85 (2020) 103282
treated with water and chorine solution, reaching large numbers of 7.0
and 6.3 log CFU/g, respectively. In the lettuce samples treated with the
pediocin, the pathogen counts decreased along storage until the day 7,
when it was observed a slight increase (Fig. 3 A). At the end of storage,
the washing with the pediocin resulted in a pathogen load significantly
lower than the other washings (p < 0.01), with reductions 3.2 and 2.7
log CFU/g higher than the water and chlorine washings, respectively.
The rocket salad washing procedure was able to remove in some
degree the pathogen from the vegetables. Immediately after washing, it
was observed a reduction in Listeria counts of 0.2, 0.7 and 2.0 log CFU/g
for water, chorine and pediocin (p < 0.01). L. monocytogenes increased
along storage in rocket salad washed with water and chlorine, reaching
numbers of 7.5 and 6.9 log CFU/g, respectively (Fig. 3 B). The pediocin
washing inhibited the pathogen proliferation along storage until the
day 7 (Fig. 3 B). For all the washings, the maximum pathogen load was
achieved at the last day of refrigerated storage. Listeria counts were
lower in the samples treated with pediocin by 3.0 and 2.1 log CFU/g
than the ones treated with water and chlorine (p < 0.01), respectively.
L. monocytogenes was reduced in parsley by the washing step with
water, chlorine and pediocin solutions by 0.6, 1.4 log and 2.9 log CFU/
g, respectively (p < 0.01). As shown in Fig. 3 C, the pathogen was able
to survive and grow during storage in parsley washed with water and
chlorine solution, reaching levels of 6.0 and 5.2 log CFU/g, respec-
tively. For parsley treated with the pediocin, viable Listeria were always
below detection limits (2 log CFU/g) from the washing until the 9th day
of storage (Fig. 3 C). From the 9th day to the end of storage, low levels
of the pathogen were detected with a maximum load of 3.1 log CFU/g
achieved at the end of storage. Along storage period, Listeria counts
were significantly lower in the parsley washed with pediocin than with
the other solutions (p < 0.01).
The washing of contaminated spinach led to different L. mono-
cytogenes reductions (p < 0.01), in the order of 0.2, 0.8 and 2.2 log
CFU/g for water, chlorine and pediocin solutions, respectively. During
the refrigerated storage the pathogen counts increased in the spinach
washed with water and chlorine solution (Fig. 3 D). For the spinach
washed with pediocin, the pathogen numbers continuous to decrease
along storage until the 9th day. Listeria counts were always lower in the
B. Ramos, et al. Food Microbiology 85 (2020) 103282

Fig. 2. Lactic acid bacteria and mesophilic populations present in fresh vegetables inoculated with Listeria monocytogenes loads (L1 and L2) and mesophilic popu-
lations present in (P + L1 and P + L2), during storage at 4 °C. The vegetables are A: Lettuce; B: Rocket salad; C: Parsley; D: Spinach. Error bars show standard
deviation (SD).

spinach treated with pediocin (p < 0.01) and at the end of storage
there was a difference in the pathogen population of about 2.5 and 1.9
log CFU/g between the samples washed with the pediocin and samples
treated with water and chlorine solution, respectively.
L. monocytogenes counts reduced in all the vegetables treated with
pediocin DT016 immediately after washing, and continuously de-
creased during storage until the 7th day for lettuce and rocket salad and
until the 9th day for parsley and spinach.
The continuous decrease in the pathogen load observed during
storage of the vegetables washed with the pediocin solution, suggests
that the adsorbed bacteriocin was still able to inhibit Listeria during
storage. Actually, it was found that the pediocin washing solution
showed residual activity (200 AU/mL) until the 7th day of storage (data
not shown).
The pediocin was unable to completely eliminate L. monocytogenes
during prolonged storage, this could be due to the interaction with
particular food components that may influence the adsorption of the
bacteriocin into the vegetables surface and even cause their

Fig. 3. Listeria monocytogenes population present in fresh vegetables before washing (BW) and after washing with water, chlorine and pediocin DT016 solutions,
during storage at 4 °C. The vegetables are A: Lettuce; B: Rocket salad; C: Parsley; D: Spinach. Error bars show standard deviation (SD).

5
B. Ramos, et al.
inactivation. Furthermore, it is important to notice that the pathogenic
numbers examined in vegetables are considerably higher than what is
expected in a natural contamination.
Pathogen populations were always lower in vegetables after
washing with the bacteriocin solution than in vegetables washed with
water or the chorine solution (p < 0.01).
Depending on the vegetable samples, for long storage, pediocin
solution highlighted a reduction between 2.5 and 3.2 log UCF/g on L.
monocytogenes numbers, compared with the control samples (distilled
water) (p < 0.01). These results are higher than those reported for
other bacteriocins, when compared to the control (water): nisin A
(McManamon et al., 2019); nisin (400 IU/mL) (Oliveira et al., 2015);
nisin and a bacteriocin RUC9 extract (Randazzo et al., 2009); solutions
containing nisin z, coagulin and a cocktail of nisin and coagulin
(Allende et al., 2007), enterocin AS-48 purified solutions (Molinos
et al., 2005); pediocin (48 μg/mL) and nisin (50 μg/mL) (Bari et al.,
2005).
It should be stated that, in this study, the bacteriocin was not added
in the purified form. Rather a fermented medium was used, MRS broth.
MRS is not a “food grade” medium, and further studies are needed on
the efficacy of fermented media obtained from food-grade matrices,
presumably vegetable juices.
In this study, Listeria monocytogenes were in the stationary growth
phase, with higher stress resistance (Miller et al., 2009), and the pa-
thogen had time to adhere to the vegetables surfaces and to adapt (24
h at 4 °C). In fact, washing with distilled water had little effect on L.
monocytogenes numbers, indicating that the pathogen had adhere to the
vegetable's surfaces.
Compared to other studies, the inoculation method, using higher
loads, using the stationary phase cells and letting the pathogen to ad-
here to the vegetables surfaces 24 h before the application of treat-
ments did not affect the effectiveness of the biopreservation agents. In
addition, pediocin DT016 maintained its effectiveness on all the vege-
tables, although there were differences on the pathogen's reductions
between vegetable's samples.
The pediocin solution did not significantly affect survival and pro-
liferation of the non-Listeria populations present in all the vegetables. In
fact, their load were comparable with the water washing (p > 0.01),
see Fig. 4. The chlorine solution resulted in lower mesophilic and LAB
Fig. 4. Mesophilic and lactic acid bacteria populations present in fresh vegetables before washing (BW) and after washing with water, chlorine and pediocin DT016
solutions, during storage at 4 °C. The vegetables are A: Lettuce; B: Rocket salad; C: Parsley; D: Spinach. Error bars show standard deviation (SD).
6
Food Microbiology 85 (2020) 103282
loads than the other solutions (p < 0.01). Natural microbiota present
in fresh vegetables can play an important role in maintaining quality
and safety and their removal may encourage the emergence of L.
monocytogenes and other foodborne pathogens organisms (Allende
et al., 2007; Iglesias et al., 2018; Ramos et al., 2013). In this respect,
this can be a positive feature when compared with chlorine rinses.
3.3. Colour
3.3.1. Colour properties of fresh vegetables inoculated with Pediococcus
pentosaceus DT016
Colour is one of the first quality characteristics evaluated by con-
sumers. Changes in colour were quantified in lettuce, rocket salad,
parsley and spinach. The colour coordinates of fresh vegetables and
vegetables with the protective culture, along refrigerated storage, are
shown in Fig. 5.
No noticeable changes in the colorimeter analyses were observed
between the samples off lettuce, rocket salad and parsley (p > 0.01).
With prolonged storage, the spinach samples with the protective culture
presented a lower a* and a higher b* than the fresh spinach (p < 0.01).
There was an increase in green colour coupled with an increase in
yellow aspect of these samples.
3.3.2. Colour properties of fresh vegetables washed with various solutions
Changes in colour were quantified in lettuce, rocket salad, parsley
and spinach after washing with water, chorine and pediocin DT016
solutions along refrigerated storage. The colour coordinates for the
treated vegetables along refrigerated storage, are shown in Fig. 6.
Lettuce, rocket salad and spinach samples presented similar colour
properties after the washing step, independently of the washing solu-
tion (p > 0.01). Along storage the colour coordinates maintained si-
milar values however, at the last day of storage there were differences
between the lettuce samples washed with the pediocin and chlorine
solutions. Chlorine washing resulted in a loss of the colour green of the
lettuce when compared with pediocin washing (p < 0.01).
For parsley samples, washing with chlorine and the pediocin solu-
tions resulted in an increase of green colour when compared with the
water washing (p < 0.01). Also, the samples washed with chlorine
showed an increase in yellow colour when compared with the others
B. Ramos, et al. Food Microbiology 85 (2020) 103282

Fig. 5. Colour coordinates of fresh vegetables and vegetables with Pediococcus pentosaceus DT016, as a protective culture, along storage at 4 C. The vegetables are A:
Lettuce; B: Rocket salad; C: Parsley; D: Spinach. Error bars show standard deviation (SD).

Fig. 6. Colour coordinates of fresh vegetables washed with water, chlorine and pediocin DT016 solutions, along storage at 4 °C. The vegetables are A: Lettuce; B:
Rocket salad; C: Parsley; D: Spinach. Error bars show standard deviation (SD).

7
B. Ramos, et al.
(p < 0.01). Along storage these differences were reduced and, at the
end of storage, parsley washed with the various solution presented si-
milar colour properties (p > 0.01).
4. Conclusions
Listeria monocytogenes was able to survive and grow in all the fresh
vegetables during storage.
The addition of P. pentosaceus DT016 to the vegetables reduced and
inhibited the pathogen populations.
The fresh vegetables showed to be a suitable environment for the
protective culture survival, growth and bacteriocin production. A great
advantage of this culture is the ability shown to produce pediocin in all
the vegetables, though the different conditions such as the natural
microbiota, the physical and chemical environment. Furthermore, the
addition of P. pentosaceus did not influence the colour parameters of
lettuce, rocket salad and parsley. Only after prolonged storage, the
spinach samples with the protective culture showed different a* and b*
properties.
The washing procedure of the vegetables with pediocin DT016
significantly reduced the initial load of L. monocytogenes and inhibited
the pathogen proliferation during prolonged storage at 4 °C. The pa-
thogen was able to survive and grow in the vegetables washed with
water and chorine solution, during the storage at 4 °C.
The pediocin washing did not affect the colour properties of lettuce,
rocket salad and spinach. Although the parsley samples washed with
the pediocin presented differences in green colour compared with the
samples washed water, at the end of storage all the colour parameters
were similar between the washings.
Compared to other studies, the inoculation method and load as well
as the incubation of contaminated vegetables for 24 h before the ap-
plication of treatments did not affect the effectiveness of the biopre-
servation agents.
In conclusion, the biopreservation approaches tested in this study
are very promising methods to reduce and control L. monocytogenes
proliferation in fresh vegetables. Combined treatments with other in-
hibitory agents or sanitizers and the use of more highly concentrated
bacteriocin solutions should be also considered to completely eradicate
the pathogen.
Acknowledgements
Financial support for authors Ramos B. and Brandão T.R.S. was
provided by FCT and Fundo Social Europeu (FSE) through fellowships
SFRH/BD/42169/2007 and SFRH/BPD/41419/2007, respectively. We
would also like to thank the scientific collaboration under the FCT
project UID/Multi/50016/2019.
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