Food Microbiology 54 (2016) 11e19

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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
b
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

a r t i c l e i n f o a b s t r a c t

Article history: The intrinsic characteristics of minimally processed fruit may favor the growth of pathogens and spoilage
Received 3 June 2015 microorganisms. In this context, the use of natural alternatives to traditional chemical sanitizer agents
Received in revised form may represent a useful tool to increase the shelf-life and the safety of minimally processed fruit. The aim
4 November 2015
of this study was to evaluate the application of the nisin-producing Lactococcus lactis CBM21 as a po-
Accepted 6 November 2015
tential biocontrol agent in sliced apples combined or not with hexanal, 2-(E)-hexenal and citral through
Available online 10 November 2015
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
Keywords:
Sliced apples
storage. Moreover, this strain significantly increased the safety of the products, inhibiting the growth of
Biocontrol Listeria monocytogenes, especially when used in combination with the proposed natural antimicrobials.
Natural antimicrobials No negative effects on color parameters were observed after 14 days of storage in presence of the natural
Nisin antimicrobials. Furthermore, the addition of the biocontrol agent was positively perceived by panelists
Safety and shelf-life for product flavor 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.

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

The demand for minimally processed fruits and vegetables has
incessantly increased in the last years reflecting 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).
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 efficient 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 justified the search for substitutes to guarantee the
food safety and quality. In this context, the use of biocontrol
agents fits 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 vegeta-
bles (Bennik et al., 1999; Leroy et al., 2003; Vescovo et al., 1996).
The use of biocontrol agents, such as Candida spp., Gluconobacter

http://dx.doi.org/10.1016/j.fm.2015.11.004
0740-0020/© 2015 Elsevier Ltd. All rights reserved.
12 L. Siroli et al. / Food Microbiology 54 (2016) 11e19
spp., Discosphaerina spp. and Metschnikowia spp., have been re-
ported to inhibit the growth of L. monocytogenes, E. coli and Sal-
monella 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 Aero-
monas 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 fulfill the desired char-
acteristics, 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 (Ruiz-
Barba et al., 1994; Stiles and Holzapfel, 1997). Moreover, the capa-
bility of LAB to produce bacteriocins and other antimicrobial mol-
ecules, 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
first 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. mono-
cytogenes, 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
both in model and food systems (De Azeredo et al., 2011; Lo
pez-
Ga
lvez et al., 2009; Siroli et al., 2014). In particular, it is well
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 efficacy has already been
experienced in fruit-based salads in syrup (Belletti et al., 2008),
fruit-based soft drinks (Belletti et al., 2007) and minimally pro-
cessed 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 modified 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 first 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 antimicro-
bials 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 prod-
ucts, 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 pur-
chased 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 re-
ported 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 pu-
rified using the Wizard Genomic Purification Kit (Promega corpo-
ration, 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 amplification of the nisin-
encoding gene. The PCR mixture (50 ml) was composed by 2 mM
MgCl2, 0.2 mM of each primer, 0.2 mM of deoxyribonucleotide tri-
phosphates (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 final
extension at 72
C for 10 min. PCR products were visualized on 1.5%
agarose gel. The PCR resulting amplicons were purified with the
QIAquick PCR Purification 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 Strain Source Inhibition of Lb. plantarum ATCC 14917T

L. lactis subsp. cremoris CBM2 Mozzarella cheese e

L. lactis subsp. lactis CBM17 Mozzarella cheese e
L. lactis subsp. lactis CBM18 Mozzarella cheese e
L. lactis subsp. lactis CBM21 Mozzarella cheese þ
L. lactis subsp. cremoris CBM26 Mozzarella cheese e
L. lactis subsp. lactis CBM28 Mozzarella cheese e
L. lactis subsp. cremoris CBM34 Mozzarella cheese e
L. lactis subsp. lactis CBM36 Mozzarella cheese e
L. lactis subsp. lactis CBM38 Mozzarella cheese e
L. lactis subsp. lactis CBM39 Mozzarella cheese e
L. lactis subsp. lactis CBM41 Mozzarella cheese e
L. lactis subsp. lactis CBM43 Mozzarella cheese e
L. lactis subsp. lactis CBM44 Mozzarella cheese þ
L. lactis subsp. lactis CBM45 Mozzarella cheese e
L. lactis subsp. lactis CBM58 Mozzarella cheese e
L. lactis subsp. lactis CBM72 Mozzarella cheese e
L. lactis subsp. lactis CBM77 Mozzarella cheese e
L. lactis subsp. lactis RAC2410 Ragusano cheese e
L. lactis subsp. lactis RAC2412 Ragusano cheese e
L. lactis subsp. lactis PBCF56 Pecorino siciliano cheese e
L. lactis subsp. lactis RAC248 Ragusano cheese e
L. lactis subsp. lactis RALC5 Raw cow milk e
L. lactis subsp. lactis L2813A Soft cheese þ
L. lactis subsp. lactis 6049 Milk e
L. lactis subsp. lactis var. diacetylactis LMG 7949T e e
L. lactis subsp. lactis LMG 6890T e e
L. lactis subsp. lactis DSM 20729 e þ
L. lactis subsp. lactis Lc 2,12 G II p Caciotta cheese e
L. lactis subsp. lactis Lc 2,12 G II g Caciotta cheese e
L. lactis subsp. lactis M10G19 Apple e

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

Table 2 conditions were: different temperatures (4, 8, 15 and 30
C),

Antagonistic activity of Lactococcus lactis CBM21 against several microor-
different levels of sodium chloride (2, 4 and 6% w/w), high con-
ganisms determined by agar spot test.
centrations of sucrose (20% w/w) and low pH values (3.5, 4.0 and
Target strain Inhibition 4.5). The screening was performed on M17 broth (Oxoid Ltd), the
Bacillus cereus SV90 þ inoculum of the tested strain was 5 log cfu/mL in flasks of 50 ml.
Escherichia coli 555 e The flasks were stored at expected temperatures, while in case of
þþ
Enterococcus faecalis 29212
modified M17 broth at 30
C. The bacterial growth was monitored
Lactobacillus brevis IOEB9809 þþ
Lactobacillus casei V4B4 þþþ
every 12 h by spectrophotometer measurements of the optic den-
Lactobacillus plantarum CIT 3 þþþ sity and by plate counting. Three repetitions for each condition
Lactobacillus plantarum V7B3 þþþ were considered.
Lactobacillus rhamnosus C1112 þþþ The evaluation of the ability of the strain CBM21 to antagonize
Lactobacillus sakei S8 þþþ
the spoilage and pathogenic microorganisms listed in Table 2 was
Lactococcus lactis S1 þþþ
Listeria monocytogenes OSP 4 þþ performed through the agar spot assay (Schillinger and Lucke,
Listeria monocytogenes SCOTT A þþþ 1989).
Saccharomyces cerevisiae spa e The capability of L. lactis CBM21 to survive in the presence of the
Salmonella enteridis E5 e
natural antimicrobials hexanal, 2-(E)-hexenal and citral was eval-
Staphylococcus aureus F1 þþþ
uated determining the minimum inhibitory concentration (MIC)
Legend: - No inhibition zone; þ small inhibition zone (0.5e1 mm); þþ and minimum bactericidal concentration (MBC), as reported by
medium inhibition zone (1e2 mm); þþþ large inhibition zone (>2 mm).
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
2.4. Bacteriocin quantification, phenotypic characterization and (Corning Incorporated, NY, USA). Fifty mL of the tested natural an-
evaluation of antagonistic activity of Lactococcus lactis CBM21 timicrobials, properly diluted in M17 broth and conveyed through
96% ethanol (VWR international, PROLABO, France) were added to
The bacteriocin quantification was performed in duplicate on each well in order to obtain the required concentration of each
24-h-old cultures of L. lactis CMB21. Soluble nisin Z activity was compound in the final volume (200 mL), and with a constant
determined by a critical dilution assay as described by Amiali et al. amount of ethanol (1% v/v in wells). Microtiter plates were incu-
(1998). The activity of nisin Z was calculated with the following bated at 37
C and checked after 24 h. The MBC were determined by
formula: (1000/125)/D where D represents the highest dilution spotting 10 mL of each well after 24 h, onto M17 agar plates.
that prevents the growth of Lactobacillus plantarum ATCC 14917T MIC was defined as the lowest concentration of the compound
after an incubation of 18 h. preventing visible growth of the inoculated cells after 24 h. The
The nisin Z-producing L. lactis CBM21 was tested for its capa- MBC was defined as the lowest concentration of the compound that
bility to grow in different environmental conditions. The selected 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 nat-
ural antimicrobials, on the shelf-life and safety of minimally pro-
cessed apples was tested. Minimally processed apples were
prepared by following the protocol reported in Fig. 1. As antimi-
crobials, 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 con-
centration of the natural antimicrobials was selected on the basis of
their sensory compatibility and their efficacy in minimally pro-
cessed 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 dip-
ping 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, re-
suspended 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 modified 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%) (Sigma-
eAldrich), 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 se-
lective 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 8°C; 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 μm ckness bags:

3 2
permeability to CO2 at 23 °C: 2720 cm m /day,
3 2
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
efficacy of the different dipping conditions on product safety.

Run Dipping conditions

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

2 Dipping with 2-(E)-hexenal/hexanala
3 Dipping with 2-(E)-hexenal/hexanala and inoculation with L. monocytogenesb and E. colib
4 Dipping with 2-(E)-hexenal/hexanala and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib
5 Dipping with 2-(E)-hexenal/hexanala and inoculation with L. lactis CBM21c
6 Dipping with 2-(E)-hexenal/citrala
7 Dipping with 2-(E)-hexenal/citrala and inoculation with L. monocytogenesb and E. colib
8 Dipping with 2-(E)-hexenal/citrala and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib
9 Dipping with 2-(E)-hexenal/citrala and inoculation with L. lactis CBM21c
10 Conventional dipping and inoculation with L. monocytogenesb and E. colib
11 Conventional dipping and inoculation with L. lactis CBM21c
12 Conventional dipping and inoculation with L. lactis CBM21c and L. monocytogenesb and E. colib
a
Each natural antimicrobial was used at a concentration of 125 mg/L.
b
The inoculation level of L. monocytogenes and E. coli ranged between 3 and 4 log cfu/mL.
c
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 treat-
ments 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 color-
spectrophotometer mod. Colorflex (Hunterlab, USA). Color was
measured using the CIELab scale and Illuminant D65. The instru-
ment 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 sam-
ple, 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 flavor, taste, browning, firm-
ness, 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 Sta-
tistica software (version 8.0; StatSoft., Tulsa, Oklahoma, USA) by
two way-ANOVA followed by Tukey honest significant 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
amplified 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 in-
cubation. Also, the increase of NaCl concentration to 6% led to a
significant 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 significant 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 micro-
organisms, 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 Concentra-
tion) 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 anti-
microbials. 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 significant 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
significant (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 significant 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 figure 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

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 significantly delayed
the growth of yeasts (p < 0.05), allowing a significant 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 significantly 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 Pseu-
domonas graminis CPA-7 packaged in modified 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 antimicro-
bial 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 specifically sensitive to
nisin Z, such as the deliberately inoculated E. coli and the naturally
occurring yeasts. No significant 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 significantly 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 significant worsening of the color parameters were
observed as the time of storage increased independently on the
dipping solution adopted (significance not shown in Table 4).
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 significant 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 anti-
microbials 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 differ-
ences in terms of L* values were observed among the samples
added with the biocontrol agent and the controls. Moreover, sam-
ples inoculated only with the biocontrol agent, with or without the
inoculation of pathogens, showed a significant (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 antimi-
crobials significantly 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 an-
timicrobials were significantly 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
with those of Ro € ßle et al. (2010) that showed an acceptable
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 modified 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 significant differences be-
tween the control samples and samples supplemented with the

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 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.

Control 76.33 ±1.99ab 3.70 ±0.67a 76.51 ±2.39ab 2.34 ±0.31ab 77.96 ±2.54a 1.17 ±0.25b 74.35 ±4.47ab 0.93 ±0.06bc
Control þ pathogens 77.05 ±0.43ab 3.61 ±1.04a 77.28 ±2.41ab 2.34 ±0.38ab 76.67 ±2.31a 1.89 ±0.54b 70.24 ±3.69ab 0.47 ±1.29b
L. lactis CBM21 77.99 ±0.89ab 3.58 ±0.32a 76.41 ±1.65ab 1.98 ±0.28ab 72.84 ±3.20a 0.12 ±0.63a 68.96 ±3.13a 2.01 ±1.13a
CBM21 þ pathogens 75.78 ±0.68a 4.14 ±0.48a 74.96 ±1.86ab 2.20 ±0.84ab 72.34 ±1.24a 0.32 ±0.30a 68.82 ±5.43a 1.08 ±1.49ab
CBM21 þ pathogens þ hexanal/ 77.19 ±0.46ab 3.83 ±1.08a 74.51 ±1.25ab 1.79 ±0.28a 76.51 ±0.50a 1.75 ±0.51b 74.47 ±3.98b 1.18 ±0.76c
2-(E)-hexenal
CBM21 þ hexanal/2-(E)-hexenal 76.85 ±0.14ab 3.20 ±0.52a 73.77 ±2.80a 1.74 ±0.39a 73.83 ±2.02a 1.35 ±0.42b 74.18 ±2.11b 1.24 ±0.47c
CBM21 þ pathogens þ citral/ 77.82 ±0.24ab 3.65 ±0.70a 77.36 ±1.42ab 2.26 ±0.43ab 73.59 ±2.64a 1.49 ±0.57b 72.51 ±2.61ab 1.21 ±0.92c
2-(E)-hexenal
CBM21 þ citral/2-(E)-hexenal 76.86 ±0.88ab 3.43 ±0.46a 78.37 ±0.20ab 2.98 ±0.34b 72.14 ±3.95a 1.29 ±0.49b 73.64 ±2.02ab 2.03 ±1.22c
hexanal/2-(E)-hexenal 78.34 ±1.02b 3.18 ±0.19a 76.34 ±2.12ab 2.61 ±0.48ab 75.88 ±1.18a 1.89 ±0.18b 74.87 ±1.20b 2.16 ±0.43c
hexanal/2-(E)-hexenal þ 78.24 ±0.87b 3.60 ±0.06a 78.89 ±0.78ab 2.15 ±0.24ab 75.39 ±1.24a 1.37 ±0.11b 75.26 ±2.48b 2.37 ±0.33c
pathogens
citral/2-(E)-hexenal 77.51 ±0.78ab 3.52 ±0.16a 79.40 ±2.14b 2.87 ±0.23b 77.98 ±1.17a 1.90 ±0.17b 74.13 ±1.38b 1.86 ±0.15c
citral/2-(E)-hexenal þ 75.97 ±0.85ab 4.05 ±0.37a 78.72 ±1.84ab 2.89 ±0.08b 76.40 ±1.34a 1.53 ±0.28b 74.69 ±2.51b 2.17 ±0.56c
pathogens

Means of the same column, followed by different letters are significantly different (p < 0.05).
18 L. Siroli et al. / Food Microbiology 54 (2016) 11e19

Odor
A 6.0
Overall quality 5.0 Browning
4.0
3.0
Flavor 2.0 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
B 6.0
Overall quality 5.0 Browning
4.0
3.0
Flavor 2.0 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 flavor 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 flavor. 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 flavor 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 flavor, 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 flavor 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 pro-
cessed 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.
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