Food Microbiology 54 (2016) 11e19

Contents lists available at ScienceDirect

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

Use of a nisin-producing Lactococcus lactis strain, combined with

natural antimicrobials, to improve the safety and shelf-life of
minimally processed sliced apples
Lorenzo Siroli a, Francesca Patrignani a, Diana I. Serrazanetti b, Lucia Vannini a, b,
Elisa Salvetti c, Sandra Torriani c, Fausto Gardini a, b, Rosalba Lanciotti a, b, *
a

Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Campus of Food Science, Piazza Goidanich 60, 47521 Cesena,
FC, Italy
Interdepartmental Center for Industrial Agri-food Research, University of Bologna, Piazza Goidanich 60, 47521 Cesena, FC, Italy
c
Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 June 2015
Received in revised form
4 November 2015
Accepted 6 November 2015
Available online 10 November 2015

The intrinsic characteristics of minimally processed fruit may favor the growth of pathogens and spoilage
microorganisms. In this context, the use of natural alternatives to traditional chemical sanitizer agents
may represent a useful tool to increase the shelf-life and the safety of minimally processed fruit. The aim
of this study was to evaluate the application of the nisin-producing Lactococcus lactis CBM21 as a potential biocontrol agent in sliced apples combined or not with hexanal, 2-(E)-hexenal and citral through
microbiological, colorimetric, textural and sensory assessments.
The use of L. lactis CBM21 limited the growth of yeasts on apples below 5 log cfu/g during 28 days of
storage. Moreover, this strain signicantly increased the safety of the products, inhibiting the growth of
Listeria monocytogenes, especially when used in combination with the proposed natural antimicrobials.
No negative effects on color parameters were observed after 14 days of storage in presence of the natural
antimicrobials. Furthermore, the addition of the biocontrol agent was positively perceived by panelists
for product avor and odor.
Even if further studies are necessary, these results suggest that the combination of the considered
hurdles can represent a new strategy to prolong shelf-life and ensure the safety and quality of sliced
apples.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Sliced apples
Biocontrol
Natural antimicrobials
Nisin
Safety and shelf-life

1. Introduction
The demand for minimally processed fruits and vegetables has
incessantly increased in the last years reecting the interest of
consumers for fresh and healthy products with an easy way of
preparation (Allende et al., 2006). The intrinsic characteristics of
ready-to-eat fruits, such as the low acidity and high humidity,
together with the high number of cut surfaces, may favor the
growth of spoilage microorganisms and pathogens (Beuchat,
2002). These products have been implicated in outbreaks of
foodborne infections caused by human pathogens, such as

* Corresponding author. Department of Agricultural and Food Sciences, Alma

Mater Studiorum, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy.
E-mail address: rosalba.lanciotti@unibo.it (R. Lanciotti).
http://dx.doi.org/10.1016/j.fm.2015.11.004
0740-0020/ 2015 Elsevier Ltd. All rights reserved.

Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp.,

Staphylococcus aureus and Pseudomonas aeruginosa (Alegre et al.,
2010; Beuchat, 2002; Lanciotti et al., 2004). Consequently, the
use of raw materials of good quality and efcient decontamination
procedures are critical steps to ensure the safety of ready-to-eat
fresh fruits and vegetables (Gil et al., 2009; Francis et al., 2012).
The resistance of some pathogenic strains toward traditional
sanitizers has justied the search for substitutes to guarantee the
food safety and quality. In this context, the use of biocontrol
agents ts well with this new trend, and various microorganisms
have been proposed as bioprotective agents (Russo et al., 2014;
Vermeiren et al., 2004), Numerous studies have shown the good
potential of several microorganisms to inhibit the growth of
foodborne pathogens in minimally processed fruits and vegetables (Bennik et al., 1999; Leroy et al., 2003; Vescovo et al., 1996).
The use of biocontrol agents, such as Candida spp., Gluconobacter

12

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

spp., Discosphaerina spp. and Metschnikowia spp., have been reported to inhibit the growth of L. monocytogenes, E. coli and Salmonella enterica in fresh-cut apples, but negative effects, such as
browning of fruits, have been observed (Leverentz et al., 2006).
Scolari and Vescovo (2004) and Torriani et al. (1997) showed the
potential of a strain of Lactobacillus casei to increase the safety of
minimally processed vegetables due to the inhibition of Aeromonas hydrophila, Staph. aureus, E. coli and L. monocytogenes.
However, literature data do not exhaustively explain the effects of
biocontrol agents on the spoilage microbiota and more generally
on the shelf-life of products. For this reason, there is still a need for
new bioprotective microorganisms that fulll the desired characteristics, such as biosafety and limitation of non-target effects
(Trias et al., 2008).
Lactic acid bacteria (LAB) are generally recognized as safe
(GRAS) by the USA Food and Drug Administration (FDA), and their
use to preserve (through fermentation) meat and dairy products
and to bioprotect fermented vegetables is well documented (RuizBarba et al., 1994; Stiles and Holzapfel, 1997). Moreover, the capability of LAB to produce bacteriocins and other antimicrobial molecules, and their general acceptability in foods, make them
interesting alternatives to chemicals in food preservation. Several
authors have reported the good potential of bacteriocins to inhibit
Gram-positive bacteria both in model and in real food systems,
such as cheese, meat and ready-to-eat vegetables (Jamuna et al.,
2005; Loessner et al., 2003; Molinos et al., 2005). Nisin was the
rst characterized bacteriocin and it is produced by Lactococcus
lactis and in European Union it is allowed in some application as
food preservative (FDA, 1988; Jones et al., 2005). In fact, nisin, and
in particular the natural variant Z, is commonly used in various food
products in order to increase their microbiological safety, due to its
high solubility and stability (De Arauz et al., 2009). The activity of
nisin against Gram-positive bacteria, and particularly L. monocytogenes, is well-known both under laboratory conditions and in
foodstuffs (Cleveland et al., 2001; Yang et al., 2012). Recent studies
have shown synergistic effect of nisin when used in combination
with other food additives, such as sodium lactate, citric acid, phytic
acid, potassium sorbate and H2O2 in fresh cut lettuce and minimally
processed fruits and vegetables (Bari et al., 2005; Ukuku et al.,
2005).
Regarding other food additives, several studies have also shown
the good potential of essential oils and their principal components
to increase the safety and shelf-life of minimally processed fruits

pezboth in model and food systems (De Azeredo et al., 2011; Lo

lvez et al., 2009; Siroli et al., 2014). In particular, it is well
Ga
known the wide spectrum of antimicrobial activity of citral, a
mixture of the 2 isomers geranial and neral, and component of
several citrus essential oils, as well as of hexanal and 2-(E)-hexenal,
which are components of the aroma of many fruits and vegetables
(Belda-Galbis et al., 2013; Kubo and Fujita, 2001; Wuryatmo et al.,
2003). In addition, their antimicrobial efcacy has already been
experienced in fruit-based salads in syrup (Belletti et al., 2008),
fruit-based soft drinks (Belletti et al., 2007) and minimally processed sliced apples (Corbo et al., 2000; Serrano et al., 2008; Siroli
et al., 2014). In particular, Siroli et al. (2014) showed that the
addition of 2-(E)-hexenal in combination with hexanal or citral, at a
concentration of 125 mg/L, in the dipping solution of minimally
processed sliced apples packaged in modied atmosphere had a
positive effect on the product, due to their antimicrobial activity
against naturally occurring spoilage species, allowing the extension
of shelf-life up to 35 days, without detrimental effects on safety and
quality parameters.
In this perspective, the rst purpose of the present research was
to identify and characterize nisin-producing L. lactis strains and
evaluate the potential application of a selected nisin-producing

strain of L. lactis in minimally processed apples, combined or not

with other hurdles, such as hexanal, 2-(E)-hexenal and citral, to
inhibit the microbial growth. To assess their effects on product
safety, challenge tests with L. monocytogenes and E. coli were also
performed.
2. Material and methods
2.1. Natural antimicrobials
The tested compounds hexanal, 2-(E)-hexenal and citral were
purchased from SigmaeAldrich (Milano, Italy). These antimicrobials were selected on the basis of their antimicrobial activity and
their organoleptic compatibility with sliced apples (Siroli et al.,
2014).
2.2. Microbial strains and growth conditions
The strains used in this study are listed in Tables 1 and 2.
Lactococcus lactis strains (Table 1), all isolated from dairy products, except one isolated from minimally processed apples,
belong to the collection of the Department of Biotechnology
of Verona University. The strains listed in Table 2 belong to
the Department of Agricultural and Food Sciences of Bologna
University.
Before each trial, the L. lactis strains were preliminarily grown in
M17 broth incubated aerobically at 30
C for 24 h; the other LAB
strains were grown in de Man Rogosa and Sharpe (MRS) broth at
37
C, 24 h; pathogenic strains in Brain Heart Infusion broth (BHI) at
37
C, 24 h. Finally, S. cerevisiae cells were grown in Sabouraud
Dextrose broth (SAB) at 37
C for 48 h. All these media were purchased from Oxoid Ltd (Basingstoke, UK) and utilized according to
manufacturer's instruction.
2.3. Screening for nisin-producing Lactococcus lactis strains
The 30 strains of L. lactis, (Table 1) were screened for their
capability to produce nisin. The L. lactis strains was pre-cultured as
reported above, then an agar spot test, following the method reported by Schillinger and Lucke (1989) was used to verify the
antimicrobial activity against L. plantarum ATCC 14917T, which was
shown to be sensitive to nisin (Rossi et al., 2008). The cultures
showing an inhibition zone larger than 2 mm around the spot were
considered in the subsequent steps.
Total genomic DNA was extracted from microbial cells and puried using the Wizard Genomic Purication Kit (Promega corporation, Madison, WI, USA), following the manufacturer's
recommendations.
The primers reported by De Vos et al. (1993) (forward: 50 -CGC
GAG CAT AAT AAA CGG CT-30 ; reverse: 50 -GGA TAG TAT CCA TGT
CTG AAC-30 ) were employed for the amplication of the nisinencoding gene. The PCR mixture (50 ml) was composed by 2 mM
MgCl2, 0.2 mM of each primer, 0.2 mM of deoxyribonucleotide triphosphates (dNTPs), 0.02 U/ml Taq polymerase, 1  PCR buffer and
approximately 20 mg of genomic DNA. Thermocycling conditions
were: preliminary denaturation at 94
C for 5 min; 30 cycles at
93
C for 2 min, 54
C for 1 min, 72
C for 1.5 min, then a nal
extension at 72
C for 10 min. PCR products were visualized on 1.5%
agarose gel. The PCR resulting amplicons were puried with the
QIAquick PCR Purication Kit (Qiagen, USA) and sequenced at BMR
Genomics sequencing centre (Padua, Italy). Sequences were then
compared with those available in GenBank database retrieved
through BLASTn searches and then aligned using the GeneDoc 2.7
software.

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

13

Table 1
List of Lactococcus lactis strains screened in this study, source of isolation and eventual inhibition of the nisin-sensitive indicator strain.
Species
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.

lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis

subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.
subsp.

cremoris
lactis
lactis
lactis
cremoris
lactis
cremoris
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis
lactis var. diacetylactis
lactis
lactis
lactis
lactis
lactis

Strain

Source

Inhibition of Lb. plantarum ATCC 14917T

CBM2
CBM17
CBM18
CBM21
CBM26
CBM28
CBM34
CBM36
CBM38
CBM39
CBM41
CBM43
CBM44
CBM45
CBM58
CBM72
CBM77
RAC2410
RAC2412
PBCF56
RAC248
RALC5
L2813A
6049
LMG 7949T
LMG 6890T
DSM 20729
Lc 2,12 G II p
Lc 2,12 G II g
M10G19

Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Mozzarella cheese
Ragusano cheese
Ragusano cheese
Pecorino siciliano cheese
Ragusano cheese
Raw cow milk
Soft cheese
Milk
e
e
e
Caciotta cheese
Caciotta cheese
Apple

e
e
e

e
e
e
e
e
e
e
e

e
e
e
e
e
e
e
e
e

e
e
e

e
e
e

Legend: : Inhibition observed; -: no inhibition observed.

Table 2
Antagonistic activity of Lactococcus lactis CBM21 against several microorganisms determined by agar spot test.
Target strain

Inhibition

Bacillus cereus SV90

Escherichia coli 555
Enterococcus faecalis 29212
Lactobacillus brevis IOEB9809
Lactobacillus casei V4B4
Lactobacillus plantarum CIT 3
Lactobacillus plantarum V7B3
Lactobacillus rhamnosus C1112
Lactobacillus sakei S8
Lactococcus lactis S1
Listeria monocytogenes OSP 4
Listeria monocytogenes SCOTT A
Saccharomyces cerevisiae spa
Salmonella enteridis E5
Staphylococcus aureus F1

e
e

Legend: - No inhibition zone; small inhibition zone (0.5e1 mm);

medium inhibition zone (1e2 mm); large inhibition zone (>2 mm).

2.4. Bacteriocin quantication, phenotypic characterization and

evaluation of antagonistic activity of Lactococcus lactis CBM21
The bacteriocin quantication was performed in duplicate on
24-h-old cultures of L. lactis CMB21. Soluble nisin Z activity was
determined by a critical dilution assay as described by Amiali et al.
(1998). The activity of nisin Z was calculated with the following
formula: (1000/125)/D where D represents the highest dilution
that prevents the growth of Lactobacillus plantarum ATCC 14917T
after an incubation of 18 h.
The nisin Z-producing L. lactis CBM21 was tested for its capability to grow in different environmental conditions. The selected

conditions were: different temperatures (4, 8, 15 and 30
C),

different levels of sodium chloride (2, 4 and 6% w/w), high concentrations of sucrose (20% w/w) and low pH values (3.5, 4.0 and
4.5). The screening was performed on M17 broth (Oxoid Ltd), the
inoculum of the tested strain was 5 log cfu/mL in asks of 50 ml.
The asks were stored at expected temperatures, while in case of
modied M17 broth at 30
C. The bacterial growth was monitored
every 12 h by spectrophotometer measurements of the optic density and by plate counting. Three repetitions for each condition
were considered.
The evaluation of the ability of the strain CBM21 to antagonize
the spoilage and pathogenic microorganisms listed in Table 2 was
performed through the agar spot assay (Schillinger and Lucke,
1989).
The capability of L. lactis CBM21 to survive in the presence of the
natural antimicrobials hexanal, 2-(E)-hexenal and citral was evaluated determining the minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC), as reported by
Siroli et al. (2014). For the determination of MIC values, 150 mL of
M17 broth inoculated at three different levels (2, 4 or 6 log cfu/mL)
of the L. lactis CBM21 were added to 200 mL microtiter wells
(Corning Incorporated, NY, USA). Fifty mL of the tested natural antimicrobials, properly diluted in M17 broth and conveyed through
96% ethanol (VWR international, PROLABO, France) were added to
each well in order to obtain the required concentration of each
compound in the nal volume (200 mL), and with a constant
amount of ethanol (1% v/v in wells). Microtiter plates were incubated at 37
C and checked after 24 h. The MBC were determined by
spotting 10 mL of each well after 24 h, onto M17 agar plates.
MIC was dened as the lowest concentration of the compound
preventing visible growth of the inoculated cells after 24 h. The
MBC was dened as the lowest concentration of the compound that
caused the death of the inoculated cells, corresponding to no

14

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

growth after 24 h of incubation at 37
C of a 10 mL spot plated onto

M17 agar.
2.5. Preparation of minimally processed apples added of
Lactococcus lactis CBM21 alone or in combination with natural
antimicrobials
The effect of L. lactis CBM21, alone or in combination with natural antimicrobials, on the shelf-life and safety of minimally processed apples was tested. Minimally processed apples were
prepared by following the protocol reported in Fig. 1. As antimicrobials, the mixtures citral/2-(E)-hexenal and hexanal/2-(E)hexenal were employed using 125 mg/L for each compound. The
natural antimicrobials were added in the dipping solution (in the
presence of ascorbic acid (0.5%) and citric acid (1%) and conveyed
through 1% ethanol. Samples treated in the process of dipping with
only ascorbic acid and citric acid were used as controls. The concentration of the natural antimicrobials was selected on the basis of
their sensory compatibility and their efcacy in minimally processed sliced apples showed in previous studies (Belletti et al.,
2008; Lanciotti et al., 2004; Siroli et al., 2014). Challenge tests
with L. monocytogenes Scott A and E. coli 555 were performed in
order to evaluate the effects of the added biocontrol agent and
natural antimicrobials on the safety of the products, the pathogens
were inoculated together and simultaneously in the different dipping solutions at levels ranging between 3 and 4 log cfu/mL. The
supplementation of the biocontrol agent (7e8 log cfu/mL) and/or
pathogens (3e4 log cfu/mL) occurred in the dipping solution. The
pre-cultivated biocontrol agent was inoculated in M17 broth (Oxoid

Ltd) and grown for 24 h at 30
C, then, for each condition, 500 mL of

microbial culture for 5 L of washing solution were centrifuged at
8000 rpm for 15 min, washed one time in physiological solution, resuspended in 50 mL of physiological solution and added to dipping
solution. The pre-cultivated pathogens were inoculated and grown
in Brain Heart Infusion (BHI, Oxoid Ltd) for 24 h, properly diluted in
physiological solution and then inoculated in the dipping solution
in order to obtain a load of 3e4 log cfu/mL. All the conditions
employed are reported in Table 3. As reported in Fig. 1, the dipping
phase had a duration of 2 min, with a temperature of the washing
water of 13
C and manual agitation. After the dipping, apples were
dried with paper, packaged in active modied atmosphere with 7%
O2 and 0% CO2 and stored at 6
C up to 28 days. For each condition
50 bags containing 50 g of sliced apples were prepared.
2.6. Microbiological analyses
During storage, the LAB and yeasts cell loads were detected by
plate counting on MRS agar (Oxoid Ltd) supplemented with
cycloheximide (0.05%) (SigmaeAldrich) and Sabouraud Dextrose
agar (SAB, Oxoid Ltd.) with chloramphenicol (0.02%) (SigmaeAldrich), respectively. After homogenization, 10 g samples
were serially diluted in physiological solution (0.9% (w/v) NaCl).
MRS plates were incubated at 37
C for 48 h, while SAB plates
were incubated at 30
C for 48 h. The inoculated pathogens
L. monocytogenes and Escherichia coli were detected by plate
counting on Listeria Selective Agar Base (LSO, Oxoid Ltd.) with selective listeria supplement (Oxoid Ltd.) and Violet Red Bile Agar
(VRBA, Oxoid Ltd.) with 4-methylumbelliferyl-b-D-glucuronide

Apple Golden Delicious

purchased at a local retailer

Washing
2 min with running water;
manual agita on 8C; apples : water 1:10 w/v

Drying with paper

Peeling and manual slicing

3

into 1,5 cm cubes

Dipping for 2 min with ascorbic acid (0.5%) and citric acid (1%);
eventual addi on of natural an microbials and or biocontrol agent and or pathogenic species;
manual agita on, 13 C;
apple : water, 1:5 (w/v);
drying with paper

Packing in a 59 m ckness bags:

3

permeability to CO2 at 23 C: 2720 cm m /day,

3

permeability to O2 at 23 C 986 cm m /day ,

modified atmosphere (7% O2 0% CO2 )
(apples : headspace, 1:1)
Storage, 6 C
Fig. 1. Working protocol used to prepare sliced apples; the addition of lactic acid bacteria and/or natural antimicrobials was performed during the dipping phase; samples dipped
with only citric and ascorbic acid represented the controls.

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

15

Table 3
Experimental conditions used in the dipping phase of sliced Golden delicious apples. In addition to the conventional dipping (run 1), the inoculation
of the biocontrol agent Lactococcus lactis CBM21 and the supplementation of the natural antimicrobials 2-(E)-hexenal, hexanal and citral were
considered. In the same conditions, Listeria monocytogenes and Escherichia coli were inoculated together and simultaneously in order to evaluate the
efcacy of the different dipping conditions on product safety.
Run

Dipping conditions

1
2
3
4
5
6
7
8
9
10
11
12

Conventional
Dipping with
Dipping with
Dipping with
Dipping with
Dipping with
Dipping with
Dipping with
Dipping with
Conventional
Conventional
Conventional

a
b
c

dipping (control): 0.5% ascorbic acid; 1.0% citric acid

2-(E)-hexenal/hexanala
2-(E)-hexenal/hexanala and inoculation with L. monocytogenesb and E. colib
2-(E)-hexenal/hexanala and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib
2-(E)-hexenal/hexanala and inoculation with L. lactis CBM21c
2-(E)-hexenal/citrala
2-(E)-hexenal/citrala and inoculation with L. monocytogenesb and E. colib
2-(E)-hexenal/citrala and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib
2-(E)-hexenal/citrala and inoculation with L. lactis CBM21c
dipping and inoculation with L. monocytogenesb and E. colib
dipping and inoculation with L. lactis CBM21c
dipping and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib

Each natural antimicrobial was used at a concentration of 125 mg/L.

The inoculation level of L. monocytogenes and E. coli ranged between 3 and 4 log cfu/mL.
The inoculation level of L. lactis CBM21 ranged between 7 and 8 log cfu/mL.

(Oxoid Ltd.), respectively. Plates were incubated at 37
C for 24 h.

Microbiological analyses were performed immediately after treatments and after 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26 and 28 days.
2.7. Color analyses and panel test
Surface color of sliced apples was measured using a colorspectrophotometer mod. Colorex (Hunterlab, USA). Color was
measured using the CIELab scale and Illuminant D65. The instrument was calibrated with a white tile (L*98.03, a* e 0.23, b* 2,05).
Results were expressed as L* (luminosity), a* (red-green index) and
b* (yellow-blue index).
At each storage time, 20 readings were obtained for each sample, measuring three slices for each package.
A panel test was performed for sliced apples after 1 and 4 days of
storage. Thirty untrained consumers composed the panel, and the
quality parameters evaluated were avor, taste, browning, rmness, crispiness, sweetness, bitterness, acidity, juiciness and overall
quality. Consumers evaluated four different conditions: control
apples, apples added with citral/2-(E)-hexanal, apples added with
hexanal/2-(E)-hexanal, and apples added with the biocontrol agent.
2.8. Statistical analysis
Microbiological as well as color data were analyzed using Statistica software (version 8.0; StatSoft., Tulsa, Oklahoma, USA) by
two way-ANOVA followed by Tukey honest signicant difference
(HSD) test at p < 0.05 level in order to monitor changes over time as
well as differences between treatments.
3. Results and discussion
3.1. Selection of a nisin Z-producing Lactococcus lactis strain
Among the L. lactis strains taken into consideration, four strains,
i.e. CBM21, CBM44, L2813A and DSM 20729 (positive control), were
able to inhibit the indicator strain L. plantarum ATCC 14917T, as
reported in Table 1. The strains CBM21 and DSM 20729 were shown
to harbor the nisin-encoding gene according to PCR analyses. The
amplied PCR product of L. lactis CBM21 was subsequently
sequenced and showed a 100% similarity value with nisin Z.
A soluble nisin Z activity of 512 AU/mL was observed after 24 h,
which is the same activity detected for other bacteriocin-producing

L. lactis strains in acidic conditions (Cheigh et al., 2002) similar to

those of ready-to-eat fruits as apples.
Since the nisin Z variant is characterized by a better solubility in
food systems compared to the other nisin variants (De Arauz et al.,
2009), the strain CBM21 was selected as potential biocontrol agent
on food products.
3.2. Growth characteristics and evaluation of the antimicrobial
activity of Lactococcus lactis CBM21
The strain CMB21 was analyzed for growth characteristics to
assess its potential use in minimally processed apples. The results
showed that this strain was able to grow within 24 h at 30
C and
15
C, with 2% and 4% NaCl, and with 20% sucrose. In contrast, at
4
C and 8
C the stationary phase was reached after 7 days of incubation. Also, the increase of NaCl concentration to 6% led to a
signicant decrease of the growth kinetics of the strain, which
however, reached the stationary phase of growth after 5 days of
incubation at 30
C. The pH values of 4.5 and 4.0 allowed the strain
to reach levels higher than 108 cfu/mL in 2 and 5 days, respectively.
A pH value of 3.5 did not allow the growth of the bacterium, at least
after 15 days of incubation at 30
C. However, in the latter condition
no signicant decrease in the viability of the strain CBM21 was
observed. Considering the pH value of the Golden delicious apples
selected (4.1), the strain showed a potential maintenance of the
viability in the conditions employed in these experimental
conditions.
The antagonistic activity of L. lactis CBM21 was evaluated
against nisin-sensitive Gram-positive and Gram-negative microorganisms, as well as a strain of S. cerevisiae. The results are shown
in Table 2. In particular, L. lactis CBM21 showed a high antagonistic
activity (diameter of inhibition higher than 3 mm) against
L. monocytogenes Scott A, S. aureus F1, L. casei V4B4, L. rhamnosus
C111, L. sakei S8, L. lactis S1 and the two L. plantarum V4B4 and V7B3
strains considered. In contrast, L. lactis CBM21 did not show
antagonistic activity against the Gram-negative bacteria nor the
yeast considered: it is well known the activity of nisin is quite
ineffective against Gram-negative bacteria and yeasts unless the
outer membrane is compromised or damaged (Helander and
Sandholm, 2000).
Considering that sliced apples had pH values of 4.1 0.2, and the
storage conditions of minimally processed fruits rarely maintained
the cold chain at 4
C, the selected strain show good growth

16

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

characteristics for the application in a real system.

In this perspective, the MIC (Minimum Inhibitory Concentration) and MCB (Minimum Bactericidal Concentration) values of
citral, hexanal and 2-(E)-hexenal were determined to evaluate the
combined effect of the selected biocontrol agent and natural antimicrobials. Lactococcus lactis CBM 21 was extremely resistant
against citral and hexanal (MIC and MCB higher than 700 mg/L)
when inoculated at a level of 104 cfu/mL; MIC values were always
higher than 200 mg/L towards 2-(E)-hexenal. Generally, literature
data indicate that the shelf-life signicant increases using less than
50 mg/L of 2-(E)-hexenal in products with minimally processed
fruits (Lanciotti et al., 2003, 2004) and citral and hexanal have a
good antimicrobial potential associated with a good sensory
compatibility at concentration less than 200 mg/L (Belletti et al.,
2008).
3.3. Effects of Lactococcus lactis CBM21, in combination with
natural antimicrobials, on the microbiological quality of minimally
processed apples
The biocontrol agent was able to survive also in the presence of
the natural antimicrobials; in fact, in each condition adopted,
L. lactis CBM21 maintained a level of about 7 log cfu/g during the
storage (28 days at 6
C). In the control samples, LAB never
exceeded 3 log cfu/g.

As shown in Fig. 2A, the biocontrol agent limited the growth of

L. monocytogenes when used alone, but especially when used in
combination with the proposed natural antimicrobials.
The higher effectiveness against L. monocytogenes was observed
when the strain CBM21 was used in combination with hexanal/2(E)-hexenal. In fact, in these conditions, L. monocytogenes showed
signicant (p < 0.05) viability losses compared to the other samples
supplemented with the biocontrol agent at the end of storage
period. By contrast, L. monocytogenes was able to grow, although
very slowly, in the controls and in the samples added with hexanal/
2-(E)-hexenal and citral/2-(E)-hexenal in the absence of the
biocontrol agent. The major differences of Listeria monocytogenes
mean cell loads were observed at the end of storage time (28 days).
In fact, the cell loads detected were 1.7, 5.5, 5.0 and 4.7 log cfu/g for
samples dipped in hexanal/2-(E)-hexenal in combination with
CBM21, controls, citral/2-(E)-hexenal and hexanal/2-(E)-hexenal,
respectively (Fig. 2A). The effectiveness of the L. lactis CBM21 to
prevent the growth of L. monocytogenes can be attributed to nisin
production as the antilisterial activity of nisin is well documented
(Cosentino et al., 2012; Yang et al., 2012).
As for E. coli, the low storage temperature did not allow its
proliferation, independently on the conditions employed in the
dipping processes. However, the use of natural antimicrobials
slightly increased the kinetics of death of the target microorganism
but without signicant differences (p > 0.05) (Fig. 2B).

Fig. 2. A, B e Growth during storage at 6
C of Listeria monocytogenes (A) and Escherichia coli (B) cell loads in relation to the washing conditions employed. The gure shows the
conditions in which L. monocytogenes and E. coli were inoculated in the dipping solution in the presence of the compounds reported in the legend; samples dipped only with citric
and ascorbic acid without any other antimicrobial compound and inoculated with pathogens were used as controls.

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

17

L. lactis CBM21 caused a slight worsening of the colorimetric

measurement indexes (Table 4). In fact, samples inoculated with
the biocontrol agent, with or without the inoculation of pathogens,
were characterized by a signicant decrease of L* (luminosity) after
23 days of storage compared to the samples added with the mixture
hexanal/2-(E)-hexenal (including samples where the natural antimicrobials were added in combination with the strain CBM21) and
the samples added with the mixture citral/2-(E)-hexenal but
without the presence of the strain CBM21. By contrast, no differences in terms of L* values were observed among the samples
added with the biocontrol agent and the controls. Moreover, samples inoculated only with the biocontrol agent, with or without the
inoculation of pathogens, showed a signicant (p < 0.05) increase
of a* (red index) after 16 days compared to all the other samples.
The combination of the biocontrol agent with the natural antimicrobials signicantly reduce the negative effects of the strain
CBM21 on the color parameters L* and a*. In fact, after 23 days the
retention of L* and a* indexes in samples added with the strain
CBM21 in combination with both the two mixtures of natural antimicrobials were signicantly higher than in those dipped with the
strain CBM21 alone and in the presence of pathogens. However, the
negative effects of the strain CBM21 used alone were detected after
16 days of storage, which represent a longer storage period than the
normal shelf-life of these products. These results are in agreement
le et al. (2010) that showed an acceptable
with those of Ro
retention of the color indexes in sliced apples inoculated with the
probiotic strain L. rhamnosus GG up to 10 days of storage at 2e4
C.
The presence of natural antimicrobials mitigated the worsening of
the color indexes. On the other hand, previous literature data
showed that hexanal and 2-(E)-hexenal can positively affect the
color retention of sliced apples packaged in modied atmosphere
mainly due to the inhibition of polyphenol oxidase (Corbo et al.,
2000; Siroli et al., 2014). Contrarily, a cytotoxic effect of citral on
fruit tissues were observed by Belletti et al. (2008) in fruit-based
salads. However, these authors used higher concentrations of
citral and supplemented the natural antimicrobial directly in the
product syrup instead in the dipping solution according to the
present study.
Panel test performed after 1 and 4 days of storage at 6
C
(Fig. 3A, B), showed that the addition of the biocontrol agent had a
positive effect on the overall acceptability after 1 day of storage. In
addition, the panelists evidenced no signicant differences between the control samples and samples supplemented with the

Yeasts were able to overcome the level of 6 log cfu/g (which is

considered the spoilage threshold for this type of product) only in
the control samples subjected to the conventional dipping and in
the control samples which were previously inoculated with the
considered pathogens after 23 days of storage. The addition of the
biocontrol agent and/or natural antimicrobials signicantly delayed
the growth of yeasts (p < 0.05), allowing a signicant increase of
the product shelf-life. In fact, the yeast cell loads in all the samples
supplemented with L. lactis CBM21 and/or natural antimicrobials
did not reach 6 log cfu/g also after 28 days of refrigerated storage
while samples dipped only with citric and ascorbic acid overcame
that level after 23 days of storage (data not shown).
These results showed that the selected nisin-Z producer L. lactis
CBM21 signicantly increased the safety and shelf-life of sliced
apples. In particular, it was able to inhibit both the pathogenic
bacteria inoculated as well as yeasts, which represent the main
spoilage agents for this type of product. The effects obtained against
L. monocytogenes were absolutely relevant and comparable to those
observed for other biocontrol agents used on fresh-cut apples.
Alegre et al. (2013) observed a reduction of 2.5 log cfu/g of
L. monocytogenes in Golden Delicious apples inoculated with Pseudomonas graminis CPA-7 packaged in modied atmosphere and
stored for 7 days at 10
C. Also Abadias et al. (2009) obtained similar
results using Candida sake CPA-1 as a biocontrol agent. In this case
the inhibition was attributed to the competition for space and for
nutrients. However, it is possible that the selected strain was able to
produce other molecules with antimicrobial activity; in fact it is
well known that LAB can produce a broad spectrum of antimicrobial substances (volatile ketoacids, furanones, diacetyl, lactic acid,
hydrogen peroxide) (Schillinger et al., 1996). The selected strain, in
particular when combined with the natural antimicrobials
employed, inhibited microorganisms not specically sensitive to
nisin Z, such as the deliberately inoculated E. coli and the naturally
occurring yeasts. No signicant differences were observed in the
death kinetics of the inoculated strain of E. coli in relation to the
dipping solution adopted. In fact, the E. coli strain used was not able
to grow at the conditions adopted, and the presence of the
biocontrol agent did not signicantly accelerate its viability loss.
The changes overtime of L* and a* in relation to the dipping
solutions adopted and the time of storage are reported in Table 4. As
expected a signicant worsening of the color parameters were
observed as the time of storage increased independently on the
dipping solution adopted (signicance not shown in Table 4).

Table 4
Evolution of color parameters L* and a* of sliced apples immediately after treatment and after 9, 16 and 23 days of storage at 6
C.
0 days

Control
Control pathogens
L. lactis CBM21
CBM21 pathogens
CBM21 pathogens hexanal/
2-(E)-hexenal
CBM21 hexanal/2-(E)-hexenal
CBM21 pathogens citral/
2-(E)-hexenal
CBM21 citral/2-(E)-hexenal
hexanal/2-(E)-hexenal
hexanal/2-(E)-hexenal
pathogens
citral/2-(E)-hexenal
citral/2-(E)-hexenal
pathogens

9 days

16 days

23 days

L*

a*

L*

a*

L*

a*

L*

a*

Means S.D.

Means S.D.

Means S.D.

Means S.D.

Means S.D.

Means S.D.

Means S.D.

Means S.D.

3.70
3.61
3.58
4.14
3.83

76.51
77.28
76.41
74.96
74.51

2.34
2.34
1.98
2.20
1.79

77.96
76.67
72.84
72.34
76.51

1.17
1.89
0.12
0.32
1.75

74.35
70.24
68.96
68.82
74.47

76.33
77.05
77.99
75.78
77.19

1.99ab
0.43ab
0.89ab
0.68a
0.46ab

0.67a
1.04a
0.32a
0.48a
1.08a

2.39ab
2.41ab
1.65ab
1.86ab
1.25ab

76.85
77.82

0.14ab 3.20 0.52a 73.77

0.24ab 3.65 0.70a 77.36

2.80a 1.74 0.39a 73.83

1.42ab 2.26 0.43ab 73.59

2.02a 1.35 0.42b 74.18

2.64a 1.49 0.57b 72.51

2.11b 1.24 0.47c

2.61ab 1.21 0.92c

76.86
78.34
78.24

0.88ab 3.43 0.46a 78.37

1.02b 3.18 0.19a 76.34
0.87b 3.60 0.06a 78.89

0.20ab 2.98 0.34b 72.14

2.12ab 2.61 0.48ab 75.88
0.78ab 2.15 0.24ab 75.39

3.95a 1.29 0.49b 73.64

1.18a 1.89 0.18b 74.87
1.24a 1.37 0.11b 75.26

2.02ab 2.03 1.22c

1.20b 2.16 0.43c
2.48b 2.37 0.33c

77.51
75.97

0.78ab 3.52 0.16a 79.40

0.85ab 4.05 0.37a 78.72

2.14b 2.87 0.23b

1.84ab 2.89 0.08b

1.17a 1.90 0.17b 74.13

1.34a 1.53 0.28b 74.69

1.38b
2.51b

Means of the same column, followed by different letters are signicantly different (p < 0.05).

0.31ab
0.38ab
0.28ab
0.84ab
0.28a

77.98
76.40

2.54a
2.31a
3.20a
1.24a
0.50a

0.25b
0.54b
0.63a
0.30a
0.51b

4.47ab 0.93 0.06bc

3.69ab 0.47 1.29b
3.13a
2.01 1.13a
5.43a
1.08 1.49ab
3.98b 1.18 0.76c

1.86 0.15c
2.17 0.56c

18

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

Odor
6.0

A
Overall quality

Browning

5.0

4.0
3.0

2.0

Flavor

Firmness

1.0

0.0
Bi erness

Crispiness

Acidity

Juiciness

Swee ng
Control

L. lac s CBM21

citral/2-(E)-hexenal

hexanal/2-(E)-hexenal

Odor
6.0

B
Overall quality

5.0

Browning

4.0
3.0
2.0

Flavor

Firmness

1.0
0.0
Bi erness

Crispiness

Acidity

Juiciness
Swee ng

Control

L. lac s CBM21

citral/2-(E)-hexenal

hexanal/2-(E)-hexenal

Fig. 3. A, B e Sensory data of sliced apples, in relation to the washing conditions used, after 1 day (A) and 4 days (B) of storage at 6
C.

biocontrol agent in relation to all the other quality parameters

considered. After four days of storage, panelists perceived the
browning of the samples added with L. lactis CBM21 similar to the
control. The addition of citral/2-(E)-hexenal was perceived as
negative for the avor and the odor of the sliced apples by the
assessors, both after 1 and 4 days, mainly due to the presence of
citral that is characterized by a lemongrass avor. Belletti et al.
(2008) previously evidenced the negative effect of citral on the
apples odor in fruit salad in syrup. Contrarily, the addition of citral/
2-(E)-hexenal dramatically reduced the browning of the product
after 4 days of storage. According to the panelists, the addition of
hexanal/2-(E)-hexenal was more compatible with apple avor and
all the other quality parameters. Moreover, samples added with this
mixture of natural antimicrobials were perceived quite similar to
the controls after 1 days of storage, while, after 4 days of storage,
their quality parameters resulted better, in particular the avor, the
odor and the overall quality with respect to all the other samples.
4. Conclusions
Since literature data concerning minimally processed fruits are

mostly focused on the effects of biocontrol agents on pathogenic

microorganisms, without any reference to the shelf-life of the
product, these results can be useful to better understand the effects
of biocontrol agents on spoilage microorganisms. The use of L. lactis
CBM21 limited the growth of yeasts on apples below 5 log cfu/g
during the storage considered. The addition of the strain CBM21
affected the quality parameters of apples such as color after 16 days
of storage at 6
C. However, the recognized average shelf-life of this
category of products is generally less than this period (about 10
days). Moreover, the negative effects due to the inclusion of the
biocontrol agent after 16 days of storage was balanced by the
presence of the natural antimicrobials, particularly hexanal. The
panelists were not able to recognize the presence of the biocontrol
agent on sliced apples after 1 and 4 days of storage. Moreover, the
addition of the biocontrol agent was perceived as positive for
product avor and odor.
Therefore, the selected biocontrol agent, and in particular its
combination with natural antimicrobials, may represent a good
strategy to increase the safety and the shelf-life of minimally processed fruits. Furthermore, since important health properties have
been attributed to LAB, their use in this kind of products could also

L. Siroli et al. / Food Microbiology 54 (2016) 11e19

contribute to increase the healthy properties of these products

(Russo et al., 2014). However, the introduction of the biocontrol
agent can be further optimized, focusing on the level and mode of
inoculation and to limit the negative effects observed on the color
parameters.
Acknowledgment
This experimental research was supported by the national
project AGER-STAY FRESH 2010 2370.
References
Abadias, M., Usall, J., Alegre, I., Torres, R., Vinas, I., 2009. Fate of Escherichia coli in
apple and reduction of its growth using the postharvest biocontrol agent
Candida sake CPA-1. J. Sci. Food Agric. 89, 1526e1533.
 as, I., 2010. Factors affecting
Alegre, I., Abadias, M., Anguera, M., Oliveira, M., Vin
growth of foodborne pathogens on minimally processed apples. Food Microbiol.
27, 70e76.
Alegre, I., Vinas, I., Usall, J., Anguera, M., Altisent, R., Abadias, M., 2013. Antagonistic
effect of Pseudomonas graminis CPA-7 against foodborne pathogens in fresh-cut
apples under simulated commercial conditions. Food Microbiol. 33, 139e148.

s-Barber

Allende, A., Toma
an, F.A., Gil, M.I., 2006. Minimal processing for healthy
traditional foods. Trends Food Sci. Tech. 17, 513e519.
Amiali, M.N., Lacroix, C., Simard, R.E., 1998. High nisin Z production by Lactococcus
lactis UL719 in whey permeate with aeration. World J. Microbiol. Biotechnol. 14,
887e894.
Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K., Kawamoto, S., 2005.
Combined efcacy of nisin and pediocin with sodium lactate, citric acid, phytic
acid, and potassium sorbate and EDTA in reducing the Listeria monocytogenes
population of inoculated fresh-cut produce. J. Food Prot. 68, 1381e1387.

rez, M.C., Leufve

n, A., Martnez, A., Rodrigo, D., 2013.
Belda-Galbis, C.M., Pina-Pe
Impact assessment of carvacrol and citral effect on Escherichia coli K12 and
Listeria innocua growth. Food Control. 33, 536e544.
Belletti, N., Sado Kamdem, S., Patrignani, F., Lanciotti, R., Covelli, A., Gardini, F., 2007.
Antimicrobial activity of aroma compounds against Saccharomyces cerevisiae
and improvement of microbiological stability of soft drinks as assessed by logistic regression. Appl. Environ. Microbiol. 73, 5580e5586.
Belletti, N., Lanciotti, R., Patrignani, F., Gardini, F., 2008. Antimicrobial efcacy of
citron essential oil on spoilage and pathogenic microorganisms in fruit-based
salads. J. Food Sci. 73, 331e338.
Bennik, M.H.J., van Overbeek, W., Smid, E.J., Gorris, L.G.M., 1999. Biopreservation in
modified atmosphere stored mungbean sprouts: the use of vegetableassociated bacteriocinogenic lactic acid bacteria to control the growth of Listeria monocytogenes. Lett. Appl. Microbiol. 28, 226e232.
Beuchat, L.R., 2002. Ecological factors inuencing survival and growth of human
pathogens on raw fruits and vegetables. Microbes Infect. 4, 413e423.
Cheigh, C.I., Choi, H.J., Park, H., Kim, S.B., Kook, M.C., Kim, T.S., Hwang, J.K., Pyun, Y.R.,
2002. Inuence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi. J. Biotechnol.
95, 225e235.
Cleveland, J., Montville, T.J., Nes, I.F., Chikindas, M.L., 2001. Bacteriocins: safe, natural
antimicrobials for food preservation. Int. J. Food Microbiol. 71, 1e20.
Corbo, M.R., Lanciotti, R., Gardini, F., Sinigaglia, M., Guerzoni, M.E., 2000. Effects of
hexanal, (E)-2-hexenal, and storage temperature on shelf life of fresh sliced
apples. J. Agr. Food Chem. 48, 2401e2408.
Cosentino, S., Fadda, M.E., Deplano, M., Melis, R., Pomata, R., Pisano, M.B., 2012.
Antilisterial activity of nisin-like bacteriocin-producing Lactococcus lactis subsp.
lactis isolated from traditional Sardinian dairy products. J. Biomed. Biotechnol.
2012, 376428.
De Arauz, L.J., Jozala, A.F., Mazzola, P.G., Vessoni-Penna, T.C., 2009. Nisin biotechnological production and application: a review. Trends Food Sci. Tech. 20,
146e154.
De Azeredo, G.A., Stamford, T.L.M., Nunes, P.C., Neto, N.J.G., de Oliveira, M.E.C., de
Souza, E.L., 2011. Combined application of essential oils from Origanum vulgare
L. and Rosmarinus ofcinalis L. to inhibit bacteria and autochthonous microora
associated with minimally processed vegetables. Food Res. Int. 44, 1541e1548.
De Vos, W.M., Mulders, J.W.M., Siezen, R.J., Hugenholtz, J., Kuipers, O., 1993. Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl.
Environ. Microbiol. 59, 213e218.
FDA, 1988. Nisin preparation: afrmation of GRAS status as a direct human food
ingredient. Fed. Regist. 54, 11247e11251.
Francis, G.A., Gallone, A., Nychas, G.J., Sofos, J.N., Colelli, G., Amodio, M.L., Spano, G.,
2012. Factors affecting quality and safety of fresh-cut produce. Crit. Rev. Food
Sci. Nutr. 52, 595e610.

pez-G

Gil, M.I., Selma, M.V., Lo
alvez, F., Allende, A., 2009. Fresh-cut product sanitation and wash water disinfection: problems and solutions. Int. J. Food
Microbiol. 134, 37e45.
Helander, I.M., Sandholm, T.M., 2000. Permeability barrier of the gram-negative
bacterial outer membrane with special reference to nisin. Int. J. Food

19

Microbiol. 60, 153e161.

Jamuna, M., Babusha, S.T., Jeevaratnam, K., 2005. Inhibitory efficacy of nisin and
bacteriocins from Lactobacillus isolates against food spoilage and pathogenic
organisms in model and food systems. Food Microbiol. 22, 449e454.
Jones, E., Salin, V., Williams, G.W., 2005. Nisin and the Market of Commercial
Bacteriocins. TAMRC Consumer and Product Research Report No. CP-01-05.
Kubo, I., Fujita, K., 2001. Naturally occurring anti-Salmonella agents. J. Agr. Food
Chem. 49, 5750e5754.
Lanciotti, R., Belletti, N., Patrignani, F., Gianotti, A., Gardini, F., Guerzoni, M.E., 2003.
Application of hexanal, (E)-2-hexenal, and hexyl acetate to improve the safety
of fresh-sliced apples. J. Agr. Food Chem. 51, 2958e2963.
Lanciotti, R., Gianotti, A., Patrignani, F., Belletti, N., Guerzoni, M.E., Gardini, F., 2004.
Use of natural aroma compounds to improve shelf-life and safety of minimally
processed fruits. Trends Food Sci. Tech. 15, 201e208.
Leroy, F., Foulquie-Moreno, M.R., De Vuyst, L., 2003. Enterococcus faecium RZS C5, an
interesting bacteriocin producer to be used as a co-culture in food fermentation. Int. J. Food Microbiol. 88, 235e240.
Leverentz, B., Conway, W.S., Janisiewicz, W., Abadias, M., Kurtzman, C.P.,
Camp, M.J., 2006. Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar poona on fresh-cut apples with
naturally occurring bacterial and yeast antagonists. Appl. Environ. Microbiol.
72, 1135e1140.
Loessner, M., Guenther, S., Steffan, S., Scherer, S., 2003. A pediocin-producing
Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies
cheese surface microbial ripening consortium. Appl. Environ. Microbiol. 69,
1854e1857.

pez-Ga

lvez, F., Allende, A., Selma, M.V., Gil, M.I., 2009. Prevention of Escherichia
Lo
coli cross-contamination by different commercial sanitizers during washing of
fresh-cut lettuce. Int. J. Food Microbiol. 133, 167e171.

pez, R., Maqueda, M.,
Molinos, A.C., Abriouel, H., Omar, B.N., Valvidia, E., Lucas-Lo
Martinez-Canamero, M., Galvez, A., 2005. Effect of immersion solutions containing enterocin AS-48 on Listeria monocytogenes in vegetable foods. Appl.
Environ. Microbiol. 71, 7781e7787.
Rossi, F., Capodaglio, A., Dellaglio, F., 2008. Genetic modication of Lactobacillus
plantarum by heterologous gene integration in a not functional region of the
chromosome. Appl. Microbiol. Biotechnol. 80, 79e86.
le, C., Auty, M.A.E., Brunton, N., Gormley, L.T., Butler, F., 2010. Evaluation of
Ro
fresh-cut apples slices enriched with probiotic bacteria. Innov. Food Sci. Emerg.
Tech. 11, 203e209.
Ruiz-Barba, J.L., Cathcart, D.P., Warner, P.J., Jimenez-Diaz, R., 1994. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture in Spanishstyle green olive fermentations. Appl. Environ. Microbiol. 60, 2059e2064.
Russo, P., de Chiara, M.L.V., Vernile, A., Amodio, M.L., Arena, M.P., Capozzi, V.,
Spano, G., 2014. Fresh-cut pineapple as a new carrier to drive probiotic lactic
acid bacteria. Biomed. Res. Int. 2014, 1e9.
Schillinger, U., Lucke, F.K., 1989. Antibacterial activity of Lactobacillus sake isolated
from meat. Appl. Environ. Microbiol. 55, 1901e1906.
Schillinger, U., Geisen, R., Holzapfel, W.H., 1996. Potential of antagonistic microorganisms and bacteriocins for the biological preservation of foods. Trends Food
Sci. Tech. 71, 58e64.
Scolari, G., Vescovo, M., 2004. Microbial antagonism of Lactobacillus casei added to
fresh vegetables. Ital. J. Food Sci. 16, 465.

n, F., Valverde, J.M., Zapata, P.J., Castillo, S.,
Serrano, M., Martnez-Romero, D., Guille
Valero, D., 2008. The addition of essential oils to MAP as a tool to maintain the
overall quality of fruits. Trends Food Sci. Tech. 19, 464e471.
Siroli, L., Patrignani, F., Serrazanetti, D.I., Tabanelli, G., Montanari, C., Tappi, S.,
Rocculi, P., Gardini, F., Lanciotti, R., 2014. Efcacy of natural antimicrobials to
prolong the shelf-life of minimally processed apples packaged in modify atmosphere. Food Control. 46, 1e9.
Stiles, M., Holzapfel, W., 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36, 1e29.
Torriani, S., Orsi, C., Vescovo, M., 1997. Potential of Lactobacillus casei, culture
permeate, and lactic acid to control microorganisms in ready-to-use vegetables.
J. Food Prot. 12, 1564e1567.
Trias, R., Baneras, L., Badosa, E., Montesinos, E., 2008. Bioprotection of Golden Delicious apples and iceberg lettuce against foodborne bacterial pathogens by
lactic acid bacteria. Int. J. Food Microbiol. 123, 50e60.
Ukuku, D.O., Bari, M.L., Kawamoto, S., Isshiki, K., 2005. Use of hydrogen peroxide in
combination with nisin, sodium lactate and citric acid for reducing transfer of
bacterial pathogens from whole melon surfaces to fresh-cut pieces. Int. J. Food
Microbiol. 104, 225e233.
Vermeiren, L., Devlieghere, F., Debevere, J., 2004. Evaluation of meat born lactic acid
bacteria as protective cultures for the biopreservation of cooked meat products.
Int. J. Food Microbiol. 96, 149e164.
Vescovo, M., Torriani, S., Orsi, C., Macchiarolo, F., Scolari, G., 1996. Application of
antimicrobial-producing lactic acid bacteria to control pathogens in ready-touse vegetables. J. Appl. Microbiol. 81, 113e119.
Wuryatmo, E., Klieber, A., Scott, E.S., 2003. Inhibition of citrus postharvest pathogens by vapor of citral and related compounds in culture. J. Agr. Food Chem. 51,
2637e2640.
Yang, E., Fan, L., Jiang, J., Doucette, C., Fillmore, S., 2012. Antimicrobial activity of
bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts.
AMB Express 2, 48.