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Food Microbiology 47 (2015) 74 e 84 Contents lists available at ScienceDirect Food Microbiology journal homepage:

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Food Microbiology

journal homepage: www.elsevier.c om/locate/fm

journal homepage: www.elsevier.c om/locate/fm Lactic acid bacteria and natural antimicrobials to improve

Lactic acid bacteria and natural antimicrobials to improve the safety and shelf-life of minimally processed sliced apples and lamb's lettuce

Lorenzo Siroli a , Francesca Patrignani a , Diana I. Serrazanetti b , Giulia Tabanelli b , Chiara Montanari b , 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, Italy b Interdepartmental Center for Industrial Agri-food Research, University of Bologna, Piazza Goidanich 60, 47521 Cesena (FC), Italy

of Bologna, Piazza Goidanich 60, 47521 Cesena (FC), Italy article info Article history: Received 14 July

article info

Article history:

Received 14 July 2014 Received in revised form 22 October 2014 Accepted 20 November 2014 Available online 28 November 2014

Keywords:

Minimally processed products Biocontrol agents Natural antimicrobials Lactic acid bacteria Safety and shelf-life

abstract

Outbreaks of food-borne disease associated with the consumption of fresh and minimally processed fruits and vegetables have increased dramatically over the last few years. Traditional chemical sanitizers are unable to completely eradicate or kill the microorganisms on fresh produce. These conditions have stimulated research to alternative methods for increasing food safety. The use of protective cultures, particularly lactic acid bacteria (LAB), has been proposed for minimally processed products. However, the application of bioprotective cultures has been limited at the industrial level. From this perspective, the main aims of this study were to select LAB from minimally processed fruits and vegetables to be used as biocontrol agents and then to evaluate the effects of the selected strains, alone or in combination with natural antimicrobials (2-(E)-hexenal/hexanal, 2-(E)-hexenal/citral for apples and thyme for lamb's let- tuce), on the shelf-life and safety characteristics of minimally processed apples and lamb's lettuce. The results indicated that applying the Lactobacillus plantarum strains CIT3 and V7B3 to apples and lettuce, respectively, increased both the safety and shelf-life. Moreover, combining the selected strains with natural antimicrobials produced a further increase in the shelf-life of these products without detrimental effects on the organoleptic qualities.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Fruits and vegetables are strongly recommended in the human diet due to their vitamins, antioxidants, mineral and dietary ber content. They are generally consumed fresh, minimally processed, pasteurized or cooked by boiling in water or microwaving. Although treating food with heat increases the product safety and shelf-life, it also decreases the nutritional properties and sensorial features of the raw materials, whereas fresh produce and mini- mally processed products are characterized by a short shelf-life due to rapid microbial spoilage ( Di Cagno et al., 2008 ). In addi- tion, outbreaks of food-borne disease associated with the con- sumption of fresh and minimally processed fruits and vegetables,

* Corresponding author. Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy. Tel.: þ 39 0547 338132. E-mail address: rosalba.lanciotti@unibo.it (R. Lanciotti).

0740-0020/ © 2014 Elsevier Ltd. All rights reserved.

primarily due to Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes , have increased dramatically since the 1970s ( Abadias et al., 2011 ). In fact, numerous studies have re- ported the presence of pathogenic species, including L. mono- cytogenes , Salmonella spp., Yersinia enterocolitica , Aeromonas hydrophila and Staphylococcus aureus ( Alegre et al., 2010 ), on fresh produce and related minimally processed products. Currently, modi ed atmosphere packaging and refrigeration are the most utilized tools for improving the shelf-life of these foods to delay microbial growth and the physiological degradation of vegetable tissue ( Alegre et al., 2010 ). Decontamination methods are another tool for reducing the microbial cell loads of the raw materials and have been shown to have positive effects on product safety and shelf-life ( Manzocco et al., 2011; Ramos et al., 2013 ). Presently, chlorine is the most widely used washing and sanitizing agent among those available for fresh produce. However, numerous re- ports indicate that chlorine has limited antimicrobial ef cacy, allowing 1 e2 logarithmic reductions in the bacterial population of raw materials at the permitted concentrations, which have been

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

75

associated with the production of potentially toxic substances ( Abadias et al., 2008 ). Also disinfectants alternative to chlorine, such as hydrogen peroxide, organic acids and ozone, demon- strated their inability to completely eradicate or kill microorgan- isms on fresh produce, as well as their potential toxicity ( Alegre et al., 2013 ). The washing procedures are able to remove only a part of spoilage or pathogenic microbial cells from raw fruits and vegetables; the remaining part can survive the sanitizing agents that attach to the surfaces of the raw material ( Allende et al., 2008 ). Peeling, slicing, and shredding fresh produce stimulate growth among the surviving microorganisms and transfer them to both the inner tissues and the released nutrients ( Lanciotti et al., 2003 ). In addition, the reduction of the naturally occurring pop- ulation, due to washing and sanitization, can reduce the compe- tition for space and nutrients among human epathogenic species ( Schuenzel and Harrison, 2002 ). Consumer concern to chemical synthetic additives has stimulated research into alternative methods for reducing the decay of minimally processed fruits and vegetables and improving product safety ( Ayala-Zavala et al., 2008 ). The use of protective cultures has been proposed due to their potential bene ts for minimally processed fruits and vege- tables ( Rodgers, 2001 ). Protective cultures of lactic acid bacteria (LAB) to increase safety and shelf-life have been developed over the last few decades ( Vescovo et al., 1996; Leroy et al., 2003; Palmai and Buchanan, 2002 ). For example, Torriani et al. (1997) and Scolari and Vescovo (2004) demonstrated that a strain of Lactobacillus casei could be used to increase the safety of mini- mally processed vegetables due to the inhibition of A. hydrophila , Staph. aureus , E. coli and L. monocytogenes . Selected strains of Pseudomonas syringae , Pseudomonas graminis , Gluconobacter asaii , Candida spp., Dicosphaerina fagi and Metschnikowia pulcherrima have shown great potential as biocontrol agents for minimally processed fruits due to their ability to antagonize several food- borne pathogens under laboratory conditions ( Abadias et al., 2009; Trias et al., 2008; Alegre et al., 2013 ). However, the use of bio- protective cultures has been limited for commercial products at the industrial level because satisfactory outcomes under labora- tory settings are unable to guarantee success under real processing and distribution conditions ( Trias et al., 2008; Abadias et al., 2009 ). Even if, some authors have shown that microorganisms isolated from the same commercial type of product were able to successfully control spoilage and pathogenic microorganisms ( Vescovo et al., 1996; Reina et al., 2006; Rodgers, 2008 ). However, in our knowledge no previous reports regard the selection and use of lactic acid bacteria as biocontrol agents in minimally processed lamb's lettuce and apples. In addition, natural antimicrobials, such as essential oils (EOs) and some of their components, have been proposed as a means to increase the quality and safety of mini- mally processed fruit and vegetables ( Allende et al., 2008; Gutierrez et al., 2009; Vandekinderen et al., 2009; De Azeredo et al., 2011; Siroli et al., 2014 ). Based on numerous literature re- ports, citral, hexanal and 2-(E)-hexenal were shown to be the most effective for increasing the safety and shelf-life of ready-to-eat fruit ( Lanciotti et al., 1999, 2003; Corbo et al., 2000; Belletti et al., 2008; Siroli et al., 2014 ). Moreover, these molecules medi- ated an enhancement of the sensorial properties and a retention of the original color of sliced fresh apples packaged under a modi ed atmosphere ( Lanciotti et al., 1999; Corbo et al., 2000 ). In addition, Thymus vulgaris EO has been proposed as an interesting alternative to chlorine for minimally processed vegetables that is able to in- crease the safety and shelf-life of products without detrimental effects on their sensorial properties ( Gutierrez et al., 2009 ). However, in our knowledge no previous reports regard the use of lactic acid bacteria as biocontrol agents in combination with nat- ural antimicrobials in minimally processed lamb's lettuce and

apples. From this perspective, the main aims of this research were as follows: i) to isolate, identify and characterize LAB from mini- mally processed fruits and vegetables and select some of those strains for use as biocontrol agents in the same products; ii) to evaluate the effects of selected strains on the shelf-life and safety characteristics of minimally processed apples and lamb's lettuce; iii) to evaluate the combined effects of the most effective biocontrol agents and natural antimicrobials on minimally pro- cessed lamb's lettuce and apple in terms of safety, shelf-life and sensorial characteristics.

2. Materials and methods

2.1. Natural antimicrobials

Hexanal, 2-(E)-hexenal and citral were purchased from Sigma- e Aldrich (Milano, Italy). Thyme EO was obtained from Flora s.r.l. (Pisa, Italy).

2.2. Isolation and identi cation of LAB from minimally processed

apples and lamb's lettuce

Samples of commercially sliced apples and minimally processed lamb's lettuce were obtained from a local market. A 10-g portion of each vegetable was suspended in 90 ml of sterile saline solution (0.9% NaCl, w/v) and homogenized using a Stomacher for 2 min at room temperature. Serial dilutions were made, plated on de Man, Rogosa and Sharpe (MRS) agar (Oxoid Ltd., Basingstoke, United Kingdom), and incubated at 30 C for 48 e 72 h under anaerobic conditions, to isolate presumptive mesophilic LAB. For lamb's let- tuce, a 24 h enrichment in MRS broth at 30 C was necessary. Serial dilutions of the cultures were then plated on MRS agar. Multiple colonies, possibly with distinct morphologies, were isolated from the MRS plates. Gram-positive, catalase-negative, non-motile rods and cocci were cultivated in MRS broth at 30 C for 24 h and then re-streaked onto MRS agar. Stock cultures were stored at 20 C in MRS broth with 10% (v/v) glycerol. Genomic DNA was extracted from each strain of presumptive LAB using the InstaGene Matrix kit (Bio-Rad Laboratories, Milano, Italy). A total of 39 representative isolates were identi ed using RAPD-PCR (primer M13) and by sequencing the 16S rRNA region according to the protocol described by De Angelis et al. (2006) .

2.3. Phenotypic characterization and evaluation of antagonistic

activity of identi ed LAB

The identi ed strains were characterized based on their ability to grow under various environmental conditions, such as various temperatures (4, 8, 15 and 30 C), various levels of sodium chloride (2, 4 and 6%), high concentrations of sucrose (20%) and low pH values (3.5, 4.0 and 4.5). The strains were grown overnight after were inoculated at a level of approximately 5 log CFU/ml in tubes with 10 ml of MRS broth to evaluate growth at various tempera- tures or in MRS broth supplemented with the selected concentra- tions of NaCl or sucrose. Regarding the conditions at low pH values, glacial acetic acid was used to create the selected pH conditions. The inoculated tubes (5 repetitions for each condition) were stored at 30 C with the exception of the tubes used to evaluate the minimum growth temperatures. The growth of the strains was evaluated based on the optical density at 600 nm (OD 600 ) using a UV-1204 spectrophotometer (Shimadzu, Kyoto, Japan). If growth was not observed, the viability of the strains was veri ed by counting on MRS agar plates. To evaluate the ability of the identi ed LAB to antagonize the L. monocytogenes Scott A, E. coli 555 and Salmonella enteritidis E5

76

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

strains, the method reported by Schillinger and Lucke (1989) was used. The strains used as target microorganisms belonged to the Department of Agricultural and Food Sciences of Bologna University.

2.4. Preparation of minimally processed apples and lamb's lettuce for treatment with the selected LAB

Based on the results obtained in the rst experimental phase, the Lactobacillus paracasei M3B6 and Lactobacillus plantarum CIT3 strains were chosen as potential biocontrol agents for minimally processed apples, while the L. plantarum V7B3 and L. casei V4B4 strains were chosen as a potential biocontrol agents for minimally processed lamb's lettuce. The protocols used to prepare minimally processed apples and lamb's lettuce are reported in Fig. 1 A, B. The raw lamb's lettuce and apples were acquired from local retailers. The chosen LAB (which were previously pre-cultured in MRS broth at 37 C for 24 h) were inoculated at a level of approximately 7 log CFU/ml in dipping and washing solutions for apple and lamb's lettuce, respectively. Under certain conditions, L. monocytogenes Scott A and E. coli 555 were inoculated into the washing or dipping solutions at levels ranging from 2.5 to 3.5 log CFU/ml. Six distinct conditions were considered for apples, and seven conditions were considered for lamb's lettuce ( Table 1 ). After the treatments, apples were dried with paper, packaged under an active modi ed atmo- sphere with 7% O 2 and 0% CO 2 and then stored at 6 C. Following the various treatments, the lettuce samples were spin dried, packaged in arti cial ordinary atmosphere and then stored at 6 C.

2.5. Preparation of minimally processed apples and lamb's lettuce for treatment with the selected LAB in combination with natural antimicrobials

During this phase, the combination of natural antimicrobials and lactic acid bacteria were tested on apples and lamb's lettuce. The L. plantarum CIT3 and V7B3 strains were used on apples and lamb's lettuce, respectively, alone or in combination with 2-(E)-hexenal/ hexanal and citral/2-(E)-hexenal in the apple dipping solution and with thyme EO in the lamb's lettuce washing solution. Also during this phase, challenge tests with E. coli and L. monocytogenes were performed. As described by the experimental plan, the pathogenic microorganisms were inoculated into the washing or dipping solu- tion at levels ranging from 3.5 to 4.5 log CFU/ml. Supplementation of the biocontrol agents and/or natural antimicrobials and/or patho- gens was carried out in the dipping and washing solution for apples and lamb's lettuce, respectively. All of the conditions employed during this experimental phase, both for apples and lettuce, are reported in Table 2 . All samples were stored at 6 C and were pack- aged in active modi ed atmosphere with 7% O 2 and 0% CO 2 and in arti cial ordinary atmosphere for apples and lettuce, respectively. Apple and lamb's lettuce samples that were not deliberately inoculated with pathogenic species were subjected to a sensory evaluation by 30 untrained consumers. Two distinct panel tests were performed for sliced apples and lamb's lettuce after 7 and 5 days of storage, respectively. Flavor, smell, browning, rmness, crispness, sweetness, bitterness, acidity and juiciness were the quality parameters considered for the sliced apples. Smell, color,

parameters considered for the sliced apples. Smell, color, Fig. 1. Working protocol employed to prepare sliced
parameters considered for the sliced apples. Smell, color, Fig. 1. Working protocol employed to prepare sliced
parameters considered for the sliced apples. Smell, color, Fig. 1. Working protocol employed to prepare sliced

Fig. 1. Working protocol employed to prepare sliced apples (A) and minimally processed lamb's lettuce (B); the addition of lactic acid bacteria and/or natural antimicrobials was performed during the dipping phase (apple) or washing step (lettuce). The apple control samples were dipped only in citric and ascorbic acids, whereas samples washed with chlorine represented the minimally processed lamb's lettuce controls.

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

77

Table 1 Conditions tested on apples and lamb's lettuce; the inoculation of the selected LABs was at a level of 7 log CFU/mL in dipping or washing solutions; when provided by the experimental plan, the pathogenic microorganisms were inoculated at a level ranged between 2.5 and 3.5 log CFU/mL in dipping or washing solutions. The products were dipped (apples) or washed (lettuce) for two minutes.

Tested conditions

Apples

Lamb's lettuce

1. Dipping (0.5% ascorbic acid; 1.0% citric acid)

2. Dipping þ pathogens ( L . monocytogenes , E . coli )

3. Dipping þ L. casei (M3B6)

4. Dipping þ L. plantarum (CIT3)

5. Dipping þ L. plantarum (CIT3) þ pathogens ( L . monocytogenes , E . coli )

6. Dipping þ L. casei (M3B6) þ pathogens ( L . monocytogenes , E . coli )

1. Washing water þ L. casei (V4B4)

2. Washing water þ L. plantarum (V7B3)

3. Washing water þ pathogens ( L . monocytogenes , E . coli )

4. Washing water þ L. casei (V4B4) þ pathogens ( L . monocytogenes , E . coli )

5. Washing water þ L. plantarum

(V7B3) þ pathogens ( L . monocytogenes , E . coli )

6. Washing water þ Chlorine 120 ppm

7. Washing water þ Chlorine

120 ppm þ pathogens ( L . monocytogenes , E . coli )

browning, texture and avor were the quality attributes considered for the lamb's lettuce. The assessors were asked to rate each attri- bute on a 0 to 7 point scale. In addition, they were asked to rate each product in terms of overall acceptability.

2.6. Microbiological analyses

The growth of LAB, yeasts and mesophilic aerobic bacteria were evaluated using plate counting on i) MRS (Oxoid Ltd., Basingstoke, United Kingdom) with added cycloheximide (Sigma e Aldrich); ii) on Sabouraud Dextrose Agar (SAB, Oxoid Ltd., Basingstoke, United Kingdom) with added chloramphenicol (Sigma e Aldrich); iii) and on Plate Count Agar (PCA, Oxoid Ltd., Basingstoke, United Kingdom). Ten gram samples were diluted in 90 ml of saline so- lution (0.9% (w/v) NaCl). After homogenization, the samples were serially diluted in sterile saline solution. The LAB were incubated at 37 C for 48 h; yeast and mesophilic aerobic bacteria were incu- bated at 30 C for 48 h. The inoculated L. monocytogenes and E. coli pathogens was detected using plate counting on Listeria Selective Agar Base (LSO, Oxoid Ltd., Basingstoke, United Kingdom) with selective listeria supplement (Oxoid Ltd., Basingstoke, United Kingdom) and on Violet Red Bile Agar (VRBA, Oxoid Ltd., Basing- stoke, United Kingdom) with 4-methylumbelliferyl- b-D-glucuro- nide (Oxoid Ltd., Basingstoke, United Kingdom). Plates were incubated at 37 C for 24 h. For the apple samples, the control samples and those supplemented with LAB were analyzed imme- diately after treatments and after 2, 5, 7, 9, 13 and 16 days of storage, whereas for lamb's lettuce, analysis was performed immediately after treatments and after 2, 5, 7 and 9 days of storage. In the trials involving the combination of natural antimicrobials and the biocontrol agent, the analyses were performed immediately after treatments and after 3, 7, 10, 13, 15, 22, and 27 days of storage for apples and immediately after treatments and after 2, 5, 7, 12 and 15 days of storage for lamb's lettuce. The control samples not inoculated with the target pathogenic species were checked for the presence of naturally occurring L. monocytogenes ( McClain and Lee, 1988 ) and E. coli . This last was checked on Violet Red Bile Agar (VRBA, Oxoid Ltd., Basingstoke, United Kingdom) with 4-methylumbelliferyl- b-D-glucuronide (Oxoid Ltd., Basingstoke, United Kingdom).

Table 2 Conditions tested on apples and lamb's lettuce; the inoculation of the selected LABs were at a level of 7 log CFU/mL; the pathogenic microorganisms were inoculated at a level ranged between 3.5 and 4.5 log CFU/mL. Thyme EO was added at a concen- tration of 250 ppm; while 2-(E)-hexenal/hexanal and citral/2-(E)-hexenal were used at 125 ppm for each compound. LABs and/or antimicrobial compounds and/or pathogens were supplemented in the dipping and washing solution for apples and lamb's lettuce, respectively.

Tested conditions

Apples

Lamb's lettuce

1. Dipping (0.5% ascorbic acid; 1.0% citric acid)

2. Dipping þ 2-(E)-hexenal/hexanal

3. Dipping þ 2-(E)-hexenal/ hexanal þ pathogens

( L . monocytogenes , E . coli )

4. Dipping þ 2-(E)-hexenal/ hexanal þ pathogens

( L . monocytogenes , E . coli ) þ

L. plantarum (CIT3)

5. Dipping þ 2-(E)-hexenal/

hexanal þ L. plantarum (CIT3)

6. Dipping þ 2-(E)-hexenal/citral

7. Dipping þ 2-(E)-hexenal/ citral þ pathogens ( L . monocytogenes , E . coli )

8. Dipping þ 2-(E)-hexenal/ citral þ pathogens

1. Washing water þ Chlorine

120 ppm

2. Washing water þ Chlorine

120 ppm þ pathogens

( L . monocytogenes , E . coli )

3. Washing water þ L. plantarum

(V7B3)

4. Washing water þ L. plantarum

(V7B3) þ pathogens ( L . monocytogenes , E . coli )

5. Washing water þ L. plantarum

(V7B3) þ pathogens

( L . monocytogenes , E . coli ) þ thyme EO

6. Washing water þ L. plantarum (V7B3) þ thyme EO

7. Washing water þ thyme EO

8. Washing water þ pathogens

( L . monocytogenes , E . coli ) þ thyme EO

( L . monocytogenes , E . coli ) þ

L. plantarum (CIT3)

9. Dipping þ 2-(E)-hexenal/

citral þ L. plantarum (CIT3) 10. Dipping þ pathogens ( L . monocytogenes , E . coli )

9. Washing water þ pathogens

( L . monocytogenes , E . coli )

2.7. Statistical analysis

The preparation of minimally processed samples was performed in three different days and the data collected are the meaning of three repetitions. The data were analyzed using one-way ANOVA with the Sta- tistica statistical package for Windows. The ability to discriminate between samples was evaluated using post-hoc comparisons.

3. Results and discussion

3.1. Identi cation and characterization of lactic acid bacteria from

minimally processed apples and lamb's lettuce

Commercial apple and lamb's lettuce samples were analyzed to isolate LAB. Apple samples exhibited LAB cell loads that ranged from 2 to 4 log CFU/g, whereas the lamb's lettuce samples exhibited LAB counts lower than 1 log CFU/g. Consequently, an enrichment procedure was necessary to isolate LAB from the lettuce samples. A total of 15 and 55 strains were isolated from the lamb's lettuce and apple samples, respectively. Gram-positive, catalase-negative, non- motile cocci and rods that were oxidase negative and able to grow on MRS agar were randomly isolated from the highest plate dilu- tion of each product or enrichment and identi ed from partial sequencing of the 16S rRNA (data not showed). The following LAB species were identi ed on the minimally processed apples: Leu- conostoc mesenteroides (10 isolates), L . plantarum / pentosus (14 iso- lates), Weissella soli (2 isolates), L . casei / paracasei / rhamnosus (6 isolates), Lactococcus lactis (1 isolate). L . casei / paracasei / rhamnosus (1 isolate) and L. plantarum (5 isolates) were identi ed on the lamb's lettuce samples.

78

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

None of the identi ed strains demonstrated the ability to pro- duce bacteriocin. However, some strains were able to inhibit

S. enteritidis E5, L. monocytogenes Scott A and E. coli 555. As reported

in Table 3 , the most promising strains included two Ln. mesenter- oides strains (M5B7 and M19B25), two strains of L. plantarum (CIT3 and ESA3) and the L. paracasei M3B6 strain isolated from minimally

processed apples; among the strains isolated from minimally pro- cessed lamb's lettuce, two strains of L. casei (V4B4) and L. plantarum V7B3 and V4B5 showed the best performances. The results concerning the physiological characterization, re- ported in Table 4 , indicated that all strains grew at 30 C within

24 h, most grew at 15 C within 5 e 9 days and only 3 strains grew at

4 C within 7 days. None of the strains tested were inhibited by 2% or 4% NaCl or by 20% sucrose, attaining cell loads higher than 8

log CFU/ml within 48 h of incubation at 30 C. In the presence of 6% NaCl at 30 C, all strains, with the exception of three, grew within 5 days, attaining cell load levels that exceeded 8 log CFU/ml. Low pH values created the most stringent hurdles. In fact, none of the strains were able to grow at pH 3.5 within 20 days of incubation at

30 C. However, at pH 4.0 and 4.5, 15 strains grew within 48 h.

3.2. Effects of the selected LAB as biocontrol agents on the microbiological qualities of minimally processed apples and lamb's lettuce

The L. paracasei M3B6 and L. plantarum CIT3 strains and the

L. casei V4B4 and L. plantarum V7B3 strains were chosen as po-

tential biocontrol agents for minimally processed apples and lamb's lettuce, respectively. To assess the ability of the selected biocontrol agents to antag- onize pathogenic microorganisms in real products and under real processing conditions during the dipping or washing phase, L. monocytogenes and E. coli were inoculated under various conditions. The LAB were used as biocontrol agents and remained viable throughout the storage period considered, regardless of whether the deliberately inoculated pathogenic microorganisms were pre- sent. In fact, in the samples treated with the biocontrol agents, the LAB loads were always higher than 6 log CFU/g (data not shown). In contrast, in the control samples that were not treated with biocontrol agents, the LAB loads were less than 3 log CFU/g of product at the end of storage. Regarding the growth of yeast, no signi cant differences were observed with respect to the presence of biocontrol agents, with the exception of the samples inoculated with L. paracasei M3B6. In fact, the latter allowed maintenance of the yeast cell load at levels that were approximately 1 logarithmic cycle lower than those of the other samples ( p < 0.05) after 16 days of storage at 6 C. Under these conditions, the yeast loads remained below the threshold of spoilage, which is 6 log CFU/g for sliced apples (data not shown). The L. plantarum CIT3 strain achieved a signi cant ( p < 0.05) inhibition of E. coli and L. monocytogenes ( Fig. 2 A, B) in sliced apples. In fact, in the products inoculated with L. plantarum CIT3, the ki- netics of E. coli death accelerated signi cantly and was below the limit of detection after 7 days. Furthermore, this LAB strain inhibited the growth of L. monocytogenes until the end of the storage period. In addition, the L. paracasei M3B6 strain accelerated the kinetics of E. coli death, although to a lesser extent, and exerted an inhibitory effect on L. monocytogenes during the rst 9 days of storage at 6 C. However, the presence of biocontrol agents cannot guarantee the complete inactivation of L. monocytogenes when the bacteria are present at levels higher than the initial 1.5 log CFU/g. These levels are quite unusual under real processing conditions and in real products. However, it is well known that L. monocytogenes is a psychrotrophic microorganism that is able to grow in the absence

Table 3 Inhibitory activity of the identi ed strains on Escherichia coli , Salmonella enteritidis and Listeria monocytogenes .

Strain

Identi cation

Inhibition of indicator strains a

 

E. coli

Salmonella

Listeria

CE

þ CA 1

L. plantarum / L. pentosus

þþ

þ

þ

CE

þ CA 2

L. plantarum / L. pentosus

e

e

þþ

CE

þ CA 4

L. plantarum / L. pentosus

þ

e

þ

CE

þ CA 5

L. plantarum / L. pentosus

e

þ

e

CI

þ CE 2

L. pentosus / L. plantarum Lactobacillus plantarum L. plantarum / L. pentosus Lactobacillus plantarum L. plantarum / L. pentosus L. plantarum / L. pentosus Lactobacillus plantarum L. plantarum / L. pentosus Lactobacillus plantarum

þ

þ

e

CIT1

þþ

e

e

CIT2

þþ

þþ

e

CIT3

þþ

þþþ

þþþ

ESA þ T2 1 ESA þ T2 2

e

e

þ

þþ

þ

þ

ESA1

þ

þþ

e

ESA2

þþ

þ

þ

ESA3

þþ

þþþ

þþ

M10G19

Lactococcus lactis subsp. Lactis þ

e

þ

M11G8

Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Weissella soli Weissella soli Lactobacillus plantarum Lactobacillus paracasei Lactobacillus casei / paracasei Lactobacillus casei / paracasei Leuconostoc mesenteroides Leuconostoc mesenteroides Lactobacillus paracasei Lactobacillus casei / paracasei Lactobacillus casei / paracasei

ee

e

M16B21

e

þþþ

þ

M16B22

þþ

e

e

M17B23

þþ

þ

þ

M17G20

þ

e

e

M19B24

e

e

þ

M19B25

þ

þþþ

þþ

M2B2

e

þ

þ

M2G1

e

þ

þþ

M2G2

þþþ

þ

þþþ

M2P2

ee

e

M3B6

þ

þþþ

þþþ

M3G4

e

þþ

þþþ

M3G5

ee

e

M5B7

þþ

þþ

þþ

M5B8

e

þþ

þþþ

M7B16 1

e

þþ

þþþ

M7B16 2

þþ

þ

e

M8B18

e

e

þþ

V

7

Lactobacillus plantarum Lactobacillus casei Lactobacillus plantarum Lactobacillus plantarum / acidophilus Lactobacillus plantarum L. plantarum / L. pentosus

ee

e

V4B4

þ

þþþ

þþþ

V4B5

þþ

þþ

þþþ

V4B6

e

þþþ

þþ

V7B3

þþ

þþ

þþþ

V9

ee

e

a Symbols: e No inhibition zone; þ small inhibition zone (0.5 e 1 mm); þþ medium inhibition zone (1 e 2 mm); þþþ large inhibition zone ( > 2 mm).

of other obstacles and under refrigeration, although at a markedly

low growth rate. This rate can increase dramatically if the product is subjected to thermal abuse ( Beuchat, 2002 ). Therefore, the inves- tigated biocontrol agents, particularly L. plantarum CIT3, seem to create an effective hurdle for the multiplication of L. monocytogenes for at least 16 days of refrigerated storage. Moreover, the two investigated biocontrol agents seem to increase safety against

E. coli , which is another pathogen frequently associated with fresh-

cut products. Although the investigated strain of E. coli was not able to grow,

even in the control samples where it showed a slow loss of viability, adding the biocontrol agents, increased its death kinetics. The observed results are consistent with those in the literature ( Trias et al., 2008 ). In fact, similar effects on E. coli and L. monocytogenes were obtained under laboratory conditions using selected strains of

P. syringae , P. graminis , G. asaii , Candida spp. and M. pulcherrima

inoculated as biocontrol agents in fresh-cut fruits ( Beuchat, 2002; Trias et al., 2008 ). However, the literature data do not completely explain the effects of biocontrol agents on the spoilage microbiota and more generally on the shelf-life of products. It is well known that the shelf-life of fresh-cut fruits is mainly determined by changes in the color and texture of the product ( Soliva-Fortuny and Martín-Belloso, 2003 ). The data obtained indicated that the

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

79

Table 4 Time (days) necessary to the identi ed Lactic Acid Bacteria strains to grow at different conditions.

Strain

Identi cation

Temperature

 

NaCl a

pH b

Sucrose c

 

30

C

15 C

8 C

4 C

2%

4%

6%

4.5

4.0

3.5

20%

M2B4

Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Leuconostoc mesenteroides Lactobacillus casei / paracasei Lactobacillus casei / paracasei Lactobacillus casei / paracasei Lactobacillus casei / paracasei Lactobacillus paracasei Lactobacillus paracasei Lactococcus lactis subsp. Lactis Weissella soli Weissella soli Lactobacillus plantarum Lactobacillus plantarum Lactobacillus plantarum Lactobacillus plantarum Lactobacillus plantarum L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus L . plantarum / L . pentosus Lactobacillus plantarum / acidophilus Lactobacillus plantarum Lactobacillus plantarum Lactobacillus plantarum Lactobacillus casei

1

5

9

15

1

1

5

9

e

e

1

M5B7

2

5

9

15

2

5

5

eee

2

M5B8

2

5

9

15

2

2

5

eee

2

M16B22

1

2

5

12

1

1

2

eee

1

M19B24

1

2

5

7

1129

e

e

1

M19B25

1

2

5

7

1119

e

e

1

M17G20

1

2

5

12

1

1

1

eee

1

M11G8

1

2

5

15

1

1

2

9

e

e

1

M16B21

1

5

9

15

1

2

5

eee

1

M17B23

1

2

5

7

1129

e

e

1

M3G4

1

2

7

15

1

1

2

2

5

9

1

M7B16 2

1

5

9

15

2

2

5

5

5

9

1

M8B18

1

5

9

15

1

2

5

9

5

9

2

M3G5

1

2

5

15

1

1

2

2

5

9

1

M3B6

1

5

9

12

1

1

2

2

5

9

1

M7B16 1

1

5

9

15

1

1

2

2

5

9

1

M10G19

1

2

7

15

1

1

9

eee

1

M2G1

1

2

9

15

1

2

9

eee

1

M2G2

1

2

7

12

2

2

9

eee

2

M2P2

1

2

5

12

1

1

1

2

9

9

1

ESA1

1

2

9

15

1

1

1

2

5

9

1

ESA3

1

2

9

12

1

1

1

2

5

9

1

CIT1

1

2

9

12

1

1

1

2

5

9

1

CIT3

1

2

9

12

1

1

1

2

5

9

1

ESA2

1

2

9

12

1

1

1

2

5

9

1

CIT2

1

2

9

12

1

1

1

2

5

9

1

CED þ CARV1 CED þ CARV2 CED þ CARV4 CED þ CARV5 CIT þ CEDRO 2 ESA þ T21 ESA þ T2 2

1

2

5

15

1

1

1

2

9

9

1

1

2

5

15

1

1

1

2

9

9

1

1

2

5

9

1

1

1

2

99

1

1

2

5

15

1

1

1

2

9

e

1

1

2

5

15

1

1

1

2

15

e

1

1

5

5

15

1

1

1

2

15

9

1

1

5

5

15

1

1

1

2

15

e

1

V9

1

2

5

9

1112

9

e

1

V4B6

1

2

9

12

1

1

1

2

5

e

1

V7

1

2

2

15

1

1

1

2

e

e

1

V7B3

1

5

9

12

1

1

2

5

9

e

1

V4B5

1

2

9

1

1125

5

e

1

V4B4

1

2

9

12

1

1

1

2

5

e

1

a,b and c) The samples were incubated at 30 C.

investigated biocontrol agents (particularly L. plantarum CIT3) were able to signi cantly inhibit the growth of yeast but negatively affected the sensory characteristics of the product. The presence of biocontrol agents results in a premature browning of the products. However, the color in the samples inoculated with the biocontrol agents remained acceptable for up to 7 days of storage at 6 C (data not shown). L. casei V4B4 and L. plantarum V7B3 showed excellent adapt- ability to the minimally processed lamb's lettuce stringent condi- tions. In fact, under all of the conditions considered, LAB maintained cell loads similar to the inoculum levels throughout the storage period (data not shown). The two biocontrol agents reduced the levels of the total aerobic mesophilic population similarly to the reductions induced by chlorine in the samples that were not inoculated with pathogens. The strain of L. plantarum V7B3 produced a signi cant ( p < 0.05) reduction in the mesophilic bacteria in the samples inoculated with the investigated pathogenic microorganisms (at a rate of approxi- mately 1 logarithmic cycle). In contrast, the other samples inocu- lated with the investigated pathogenic microorganisms exhibited a higher level of the total aerobic mesophilic bacteria (data not shown). The differences observed immediately after washing, ac- cording to the investigated biocontrol agents and the presence or absence of pathogenic microorganisms inoculated, were attenu- ated during the storage period. After 9 days in all samples, the total aerobic mesophilic cell loads ranged between 6.5 and 7.5 log CFU/g

of product. The lowest values were recorded in the presence of the biocontrol agent L. plantarum V7B3, which was as effective against the spoilage micro ora as traditional disinfectants (data not shown). In fact, no signi cant differences were observed according to one-way ANOVA. The L. plantarum V7B3 strain also showed great potential for controlling the investigated pathogenic microorganisms ( Fig. 3 A, B). In fact, the presence of the L. plantarum V7B3 strain increased the E. coli death kinetics and reduced the viability of

L. monocytogenes , which was only able to grow (although very

slowly) in the samples washed only with water and in the pres-

ence of the biocontrol agent L. casei V4B4. In fact,

L. monocytogenes also increased its load by approximately 0.8 e 0.9

logarithmic cycles in the control samples washed with chlorine and inoculated with the target microorganisms. The inoculated strain of E. coli lost viability under all conditions, although at different kinetics. However, even after 9 days of storage, the cell load did not fall below 2 log CFU/g. Even in this case, the inoculum levels of the investigated pathogenic microorganisms exceeded those encountered under real conditions. Therefore, the obtained lamb's lettuce results clearly indicate the potential of the selected LAB strains (and in particular, the L. plantarum V7B3 strain) to control the spoilage micro ora and inhibit pathogenic microor- ganisms. In addition, the appearance and color of the products were not affected by the addition of the LAB. In fact, after 9 days of storage, the samples treated with the biocontrol agents exhibited

80

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

80 L. Siroli et al. / Food Microbiology 47 (2015) 74 e 84 Fig. 2. Growth

Fig. 2. Growth of Escherichia coli (A) and Listeria monocytogenes (B) during storage at 6 C in sliced apples relative to the addition of a lactic acid bacteria strain to the dipping solution.

the same turgidity observed in the controls treated with chlorine during the washing step (data not shown). Although the data regarding the control of pathogenic species are comparable to the data in the literature, the results indicate the possibility of transferring the conditions developed in this work to increase also the product shelf-life. In fact, other authors have focused their attention on the ability of biocontrol cultures to inhibit the pathogenic microorganisms frequently associated with fresh-cut products while avoiding effects on the shelf-life of the product. Regarding minimally processed vegetables, previous

data indicate the good potential of selected LAB strains (with particular emphasis on speci c strains of L. casei ) for the control of A. hydrophila , Staph. aureus , E. coli and L. monocytogenes ( Palmai and Buchanan, 2002; Torriani et al., 1997 ). Furthermore, Allende et al. (2007) and Trias et al. (2008) proposed the use of bacteriocin producer strains of L. plantarum , L. lactis , Ln. mesenteroides , Weissella cibaria and Pediococcus acidilactici to in- crease the safety of the products and speci cally inhibit

L. monocytogenes .

3.3. Effects of the selected LAB in combination with natural antimicrobials on the microbiological quality of minimally processed apples and lamb's lettuce

To assess the effects of the selected biocontrol agents,

L. plantarum CIT3 and V7B3 were added in combination with the

natural antimicrobials, selected based on previous results (data not

shown), to the dipping and washing solutions for apples and lamb's lettuce, respectively. The L. plantarum CIT3 strain was absolutely well adapted to the investigated system and was not affected by the presence of the natural antimicrobials used in the dipping step. In fact, the LAB loads remained constant and similar to the inoculum level (6 e 7 log CFU/g) without any signi cant differences ( p > 0.05) during the rst 10 days of storage in all samples inoculated with the biocontrol agent. In samples not treated with the biocontrol agent, the LAB load was less than 3 log CFU/g throughout the period of storage (data not shown). As shown in Fig. 4 A, E. coli did not grow on the sliced apples during the storage at 6 C. In fact, a loss of vitality was detected in all types of investigated products, but with more or less accelerated death kinetics relative to the conditions adopted in the dipping step. The presence of natural antimicrobials contributed toward accelerating the death kinetics of the target microorganisms. In the presence of mixtures of natural antimicrobials, E. coli cell loads decreased below the detection limit 24 h earlier compared to controls subjected to traditional dipping. Adding the biocontrol agent further increased the safety of the product, signi cantly decreasing ( p < 0.05) the cell loads of E. coli below the detection limit after 7 days. The growth of L. monocytogenes in sliced apples is shown in Fig. 4 B. This pathogenic species maintained a constant cell load during storage at 6 C. None of the conditions tested were able to effectively inactivate this microorganism. However, application of

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

81

L. Siroli et al. / Food Microbiology 47 (2015) 74 e 84 81 Fig. 3. Growth

Fig. 3. Growth of Escherichia coli (A) and Listeria monocytogenes (B) during storage at 6 C in the minimally processed lamb's lettuce relative to the addition of a lactic acid bacteria strain to the washing solution.

the 2-(E)-hexenal/citral mixture, and especially the combined use of this mixture with the biocontrol agent, resulted in a signi cant reduction ( p < 0.05) of the cell load of L. monocytogenes during the rst 10 days of storage compared to that of the controls. Yeast cell loads were not signi cantly affected ( p > 0.05) by the type of dipping during the rst 15 days of storage. However, the presence of citral in the dipping solution was able to prevent the sample from exceeding the spoilage threshold of yeast (6 log CFU/ g), even after 27 days of storage, with the exception of the samples added with pathogenic microorganisms. In all of the other samples, the levels of yeast exceeded the spoilage threshold after 27 days of storage, whereas the controls and the controls added with patho- gens exceeded this threshold after 22 days. Although the selected biocontrol agent was highly effective in the control of the inocu- lated pathogens, it failed to show any inhibitory effect against yeast. However, it is known that in matrices with high C/N ratios char- acterized by low pH and high sugar content, yeast is clearly more competitive compared to LAB ( Patrignani et al., 2013 ). Regardless, the yeast spoilage threshold was achieved when the product was already degraded in terms of color and texture (data not shown). The sensory assessment of sliced apples in relation to the adopted dipping condition is reported in Table 5 . The organoleptic properties were evaluated after 7 days of storage at 6 C while taking into account the usual shelf-life of this type of product. No signi cant differences with respect to the dipping solution were recorded for various descriptors, including smell, rmness, crisp- ness, juiciness and sweetness. In contrast, the samples dipped with 2-(E)-hexenal/citral solution received the lowest score for the browning descriptor, indicating the highest retention of the initial color compared to the other samples. However, the samples dipped with 2-(E)-hexenal/citral solution had the lowest score in terms of

overall quality, which was most likely due to the lowest score for avor and the highest values for acidity and bitterness. The samples dipped in the 2-(E)-hexenal/hexanal mixture received a signi cantly higher overall quality assessment compared to the other samples with the exception of samples dipped in the presence of the L. plantarum CIT3 that were added or not added to the same mixture of natural antimicrobials. However, according to the browning scores, the use of the selected biocontrol agent caused a decrease in the color parameters similar to those observed in the control samples. The negative effect of the LAB biocontrol agents on the fruit color parameters was also observed by several authors ( Trias et al., 2008; Leverentz et al., 2006 ). The addition of natural antimicrobials counteracted the wors- ening of the sample color induced by the biocontrol agent. The 2- (E)-hexenal/citral mixture was the most effective in preventing the browning in response to the biocontrol agent. However, previous work reported that six-carbon-atom alde- hydes, such as those considered in this work, can positively affect the initial color retention of sliced apples packaged in a modi ed atmosphere. In contrast, a cytotoxic effect of citral on fruit tissues was observed by Belletti et al. (2008) in fruit based salads. However, these con icting responses might be due to the different concen- trations and modality of supplementation adopted by Belletti et al. (2008) . In fact, these authors used higher concentrations of citral compared to the present work and supplemented the natural antimicrobial directly into the product syrup. Regarding the combined use of L. plantarum V7B3 and thyme EO, the strain showed excellent adaptability to the stringent conditions of the investigated system, as well as the presence of thyme EO. In fact, the loads of the added LAB remained nearly constant throughout the storage period at 6 C and were similar to the inoculation levels ( p > 0.05) (data not shown). In the control samples

82

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

82 L. Siroli et al. / Food Microbiology 47 (2015) 74 e 84 Fig. 4. Growth

Fig. 4. Growth of Escherichia coli (A) and Listeria monocytogenes (B) during storage at 6 C in sliced apples relative to the addition of a lactic acid bacteria strain and/or natural antimicrobials to the dipping solution.

and in the samples not supplemented with the biocontrol agent, LAB cell loads remained below the detection limit (1 log CFU/g). As shown in Fig. 5 A, E. coli did not grow throughout the storage period at 6 C. In fact, a loss of viability was clear in all product types, although with lower death kinetics compared to those observed in apples. Actually, in the control samples subjected to washing with water, the level of E. coli was decreased by 1 loga- rithmic cycle after 15 days. The addition of chlorine or thyme EO in combination with the biocontrol agent accelerated the death ki- netics of the investigated microorganism. In particular, the com- bination of the biocontrol agent with thyme EO produced an

inactivation of E. coli analogous to that of chlorine without signif- icant differences. In Fig. 5 B, the growth of L. monocytogenes is reported. As shown, the target microorganism maintained a constant load during stor- age at 6 C in the samples washed only with water. The addition of chlorine, thyme and/or the biocontrol agent produced a signi cant reduction ( p < 0.05) in the initial L. monocytogenes load (approxi- mately 1 logarithmic cycle). The biocontrol agent and thyme EO showed an effect comparable to that of chlorine. It is well known that chlorine is able to reduce the microbial loads to levels that never exceed 1 e2 logarithmic cycles ( Alegre et al., 2013 ).

Table 5 Sensory evaluation scores* of 7 day-old minimally processed sliced apples dipped in different solutions.

Sensory criteria

Apple type

Control

L. plantarum CIT3

2-(E)-Hexenal/citral

2-(E)-Hexenal/hexanal

2-(E)-Hexenal/citral þ

2-(E)-Hexenal/hexanal þ

 

L. plantarum CIT3

L. plantarum CIT3

Bitterness

1.76 ± 1.3 ab 3.13 ± 1.6 a 4.74 ± 1.4 bc 3.89 ± 1.5 a 3.63 ± 1.4 a 4.16 ± 1.2 a 3.32 ± 1.3 a 2.18 ± 1.3 a 3.79 ± 1.5 ab 3.63 ± 1.3 a

1.68 ± 1.2 a 3.00 ± 1.3 a 5.13 ± 1.1 c 3.68 ± 1.2 a 3.50 ± 1.5 a 4.42 ± 1.3 a 3.61 ± 1.4 a 2.92 ± 1.8 ab 3.95 ± 1.3 ab 3.79 ± 1.5 ab

2.47 ± 1.8 b 2.63 ± 1.6 a 2.18 ± 0.7 a 3.92 ± 1.5 a 3.58 ± 1.5 a 4.16 ± 1.3 a 3.08 ± 1.6 a 3.37 ± 2.1 b 2.68 ± 1.7 a 2.79 ± 1.6 a

1.42 ± 0.8 a 4.16 ± 1.7 a 3.87 ± 1.4 b 4.05 ± 1.3 a 3.79 ± 1.3 a 4.63 ± 1.4 a 3.63 ± 1.2 a 2.71 ± 1.5 ab 4.53 ± 1.4 b 4.63 ± 1.4 b

1.98 ± 1.3 ab 2.80 ± 1.5 a 3.80 ± 1.6 b 4.13 ± 1.2 a 3.93 ± 1.5 a 4.68 ± 1.3 a 3.30 ± 1.2 a 3.15 ± 1.8 ab 3.30 ± 1.5 ab 3.53 ± 1.7 a

1.50 ± 0.9 a 3.88 ± 1.4 a 4.13 ± 1.6 b 3.93 ± 1.3 a 3.70 ± 1.3 a 4.25 ± 1.4 a 3.75 ± 1.3 a 2.83 ± 1.7 ab 4.15 ± 1.3 ab 4.23 ± 1.3 ab

Smell

Browning

Firmness

Crispiness

Juiciness

Sweetness

Acidity

Flavor

Overall quality

*Values are means ± SE for analyses performed by 25 assessors. Within a raw, values with common superscript do not differ signi cantly ( p > 0.05). For each sensory criteria a seven-point sensory scale (from 0 ¼ low intensity to 7 ¼ high intensity) was employed.

L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

83

L. Siroli et al. / Food Microbiology 47 (2015) 74 e 84 83 Fig. 5. Growth

Fig. 5. Growth of Escherichia coli (A) and Listeria monocytogenes (B) during storage at 6 C in the minimally processed lamb's lettuce relative to the addition of a lactic acid bacteria strain and/or natural antimicrobials to the washing solution.

Furthermore, the antimicrobial ef cacy of chlorine is known to strictly depend on the organic matter content of the product; with increasing concentrations of the organic substance, the antimi- crobial effectiveness of chlorine decreases ( Gil et al., 2009 ). After 15 days, the L. monocytogenes loads in the samples washed with only water were similar to the initial load. However, the addition of thyme, the biocontrol agent and chlorine produced a reduction in the Listeria cell loads (approximately 1 logarithmic cycle) compared to the initial load. The total aerobic mesophilic bacteria, the main spoilage agents in this type of product, exceeded the threshold of spoilage (7 log CFU/g) after only 5 days of storage in the samples washed with water and inoculated with pathogens (data not shown). In the samples treated with chlorine, total mesophiles reached the threshold of spoilage after 7 days of storage, independent of the presence or absence of deliberately inoculated pathogens. The replacement of chlorine with thyme EO, with or without the biocontrol agent, exerted a positive effect on the shelf-life of the product, and the total mesophilic load remained below the threshold of spoilage (7 log CFU/g) even after 15 days of storage, independent of the presence of pathogens in the washing solution. The sensory assessment of minimally processed lamb's lettuce, which was performed after 5 days of storage, showed no signi cant differences with respect to the washing conditions for any of the investigated descriptors (smell, color intensity, browning, texture avor and overall quality). In contrast to sliced apples, the presence of the biocontrol agent did not negatively affect the color of the

minimally processed lamb's lettuce, most likely due to the distinct intrinsic features of the raw materials, their initial microbiota and their spoilage patterns. Concerning the EO used, no negative effects of thyme EO were observed by Gutierrez et al. (2009) on the sen- sory properties of minimally processed vegetables, such as lettuce and carrots.

4. Conclusions

The use of the L. plantarum CIT3 and V7B3 strains on apples and lettuce, respectively, provided encouraging results regarding the safety and shelf-life of the investigated minimally processed products. The obtained results are also more interesting because LAB are classi ed as GRAS (Generally Recognized As Safe) and are often found to have bene cial effects on consumer health. The re- sults also highlighted the importance of isolation and selection of biocontrol agents from commercial products of the same type. In fact, the superior performance of the strains used was not only against deliberately inoculated pathogens but also against spoilage microorganisms that are naturally present in the samples. These abilities have to be attributed to the capability of the strains to colonize the product and survive under the stringent conditions of refrigerated storage. In addition, the ability of biocontrol agents to not affect the quality indices of the product is important. Regarding the strain used in lamb's lettuce, it did not reduce the product quality, even when used in combination with thyme EO. Devel- oping alternatives to the use of chlorine (the most common

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L. Siroli et al. / Food Microbiology 47 (2015) 74e 84

sanitizer used to treat leafy vegetables) is important, and this research demonstrated the good potential of the L. plantarum V7B3 strain, alone or in combination with thyme EO, as an alternative to chlorine. In addition, despite its higher potential degradation of the product compared to L. plantarum V7B3, the L. plantarum CIT3 strain was shown to preserve the quality of treated fresh-cut apples for up to 9 days when used alone and up to 16 days when combined with natural antimicrobials. However, further analyses should be performed to con rm that the application of these strains as po- tential biocontrol agents does not alter the sensory properties of the nal products.

Acknowledgments

This experimental research was supported by the national project, AGER-STAY FRESH 2010 2370.

References

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