Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp.

International Journal of Food Microbiology 215 (2015) 179–186

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp. Baruzzi Federico ⁎, Loris Pinto, Laura Quintieri, Antonia Carito, Nicola Calabrese, Leonardo Caputo Institute of Sciences of Food Production, National Research Council of Italy,G. Amendola 122/O, 70126 Bari, Italy

a r t i c l e

i n f o

Article history: Received 18 May 2015 Received in revised form 7 September 2015 Accepted 27 September 2015 Available online 8 October 2015 Keywords: Antimicrobial peptides Tissue decay Spoilage bacteria Cold storage Vegetable shelf-life

a b s t r a c t The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfected fresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptides in controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolates were collected from ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonas media, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas putida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was found when Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarole chicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papain and rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability. As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using the peptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced (P ≤ 0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTE lettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable cell density in the inoculated wounds. This is the first paper providing direct evidence regarding the application of an antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Ready-to-eat (RTE) vegetables include fresh fruit or vegetables that have been handled (by peeling, trimming, washing and cutting) to obtain 100% of edible product and stored under different conditions in order to preserve their freshness (Lamikanra, 2002). Furthermore, the post-harvest handling of vegetables can damage tissues, resulting in the oxidation of phenolic compounds via polyphenol oxidase reaction and the appearance of undesirable brown, red, or black discoloration (Chisari et al., 2007). Unlike enzymatic vegetable browning, fluorescent pseudomonads, mainly responsible for vegetable tissue decay in RTE vegetables, have also proven to cause a rapid browning reaction on the cut surface of lettuce and more severe subsequent rot than those caused by other bacteria (Pascoe and Premier, 2000). Pseudomonas marginalis has been clearly correlated with the deterioration of RTE endive (Nguyen-the and Prunier, 1989), whereas P. cichorii causes a destructive disease in head lettuce characterized by shiny, dark-brown, firm necrotic spots in the inner parts of leaves (Grogan et al., 1977). The production of pectolytic enzymes such as pectate lyase in Pseudomonas viridiflava and Pseudomonas chlororaphis ⁎ Corresponding author. E-mail address: [email protected] (B. Federico).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.09.017 0168-1605/© 2015 Elsevier B.V. All rights reserved.

(Lee et al., 2013; Liao et al., 1988) negatively affects the visual quality of fresh-cut produce with tissue spoilage during cold storage. In order to keep the overall acceptability of RTE vegetables high, the control of psychrotrophic pseudomonads on leafy vegetables is a pivotal strategy for mitigating the spoilage of these products, often occurring in the proximity of cut surfaces throughout cold storage. Disinfection of vegetables is based on the application of chlorine-based compounds during washing steps (Gil et al., 2009). However, these compounds have a limited efficacy in reducing microbial load (Garcia et al., 2003; Kim et al., 2006; Pinto et al., 2015) and can affect the safety of the final product (Nieuwenhuijsen et al., 2000). Thus, the use of natural antimicrobial molecules has been recently proposed to increase their efficacy on vegetable spoilage microorganisms. Natural antimicrobial molecules include compounds of microbial (Cálix-Lara et al., 2014), plant (Roller and Seedhar, 2002) or animal (Poverenov et al., 2014) origin. Among animal-derived molecules, fermented whey or whey proteins, generally recognized as safe (GRAS), reduced the load of total aerobic bacterial cells, yeasts, and molds of tomato, strawberries and lettuce (Ahmed et al., 2011, 2012; Martin-Diana et al., 2006; Santos et al., 2015). There have been many studies on the antimicrobial activity of bovine lactoferrin (BLF) and its peptides against pathogenic bacteria; however, little evidence has been reported on their activity against spoilage microorganisms in cheese, wine, and meat (Del Olmo et al., 2009; Enrique et al.,

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B. Federico et al. / International Journal of Food Microbiology 215 (2015) 179–186

2009; Caputo et al., 2015; Quintieri et al., 2012). Pseudomonas growth was also controlled when LfcinB was deposited via a plasma process (PE-CVD) on a polyethylene surface (Quintieri et al., 2013b). To the best of our knowledge, the only experiment concerning BLF application on fruits was carried out by Wang et al. (2012), who reported that both BLF and its esterified derivative strongly inhibited spore germination and germ tube elongation in Penicillium expansum which is responsible for apple decay during storage. This work aimed at evaluating the antimicrobial efficacy of BLF hydrolysates (including the purified antimicrobial peptide LfcinB), against selected Pseudomonas strains responsible for spoilage in various RTE vegetables. 2. Material and methods 2.1. Isolation and identification of bacterial strains from ready-to-eat vegetables Presumptive spoilage pseudomonads were isolated from two samples of refrigerated ready-to-eat curly endive (Chicorium endivia L. var. crispum Lam.) and head lettuce (Lactuca sativa L. var. capitata L.), Trocadero type, purchased from a local market. 30 g of each vegetable were aseptically trasferred in a sterile stomacher bag containing 120 ml of sterile peptone water 0.1%; (Becton Dickinson Difco, Milan, Italy) and homogenized in a stomacher (BagMixer, Interscience, St. Nom., France) for 2 min. Serial decimal dilutions of each homogenate were prepared in sterile saline solution (0.9% NaCl), plated on Pseudomonas agar base enriched with Pseudomonas CFC selective supplement (PSA, BioLife Italiana srl, Milan, Italy) and incubated at 30 °C for 24 h. After incubation, 48 well-isolated colonies were picked up from PSA plates. Pure cultures of each isolate were used for DNA extraction using the Wizard Genomic DNA Purification kit (Promega Italia Srl, Milan, Italy) following the manufacturer's instructions. DNA quantity was determined using the spectrophotometer Nanodrop 1000 (Thermo Scientific, Waltman MA). Biotyping of isolates was performed following the two-step RAPD-PCR method as previously reported (Baruzzi et al., 2000; Ricelli et al., 2007; Baruzzi et al., 2012a). Pseudomonas fluorescens DSM 50090 was used as outgroup strain for comparison purposes. One single bacterial isolate for each cluster was identified by amplifying and sequencing the 16S rRNA gene (Wiesburg et al., 1991); in addition, and only for Pseudomonas fluorescens strains, a more affordable taxonomic position was achieved by sequencing the rpoB gene (1247 bp) (AitTayeb et al., 2005). Taxonomic strain identification was performed by comparing the sequences of the biotypes with those present in the Basic BLAST Search, as described by Altschul et al. (1997), against nucleotide collection (nr/nt). The identified biotypes, representative of each RAPD cluster, were stored in Nutrient Broth (NB; Becton Dickinson Difco) with 20% glycerol at −80 °C in the bacterial collection at the Institute of Sciences of Food Production.

Besides, each strain was spotted on unwounded leaves that were also inoculated or not with sterile saline solution (Controls 3 and 4, respectively). Control and treated samples were kept under a laminar flow hood for 5–10 min before being stored at 4 °C. Each sample (50 wounds/sample) was performed in triplicate (N = 3). The number of spoiled wounds (displaying tissue browning sometimes followed by softening) on the leaf samples was recorded for four days of cold storage in air and expressed as percentage of spoiled wounds out of those examined. Strains showing the highest spoilage percentage were further assayed on Trocadero lettuce (L. sativa L.), celery (Apium graveolens subsp. Dulce Mill., Pers), Belgian endive (Cichorium intybus L.) and escarole chicory (C. endivia L. var. latifolium Lam.) for six days of cold storage; in the case of celery, viable cells of different strains were inoculated on the cross-section of the petiole. 2.3. Lactoferrin hydrolysis The following enzymes were used for the hydrolysis of bovine lactoferrin (BLF, NZMP lactoferrin 7100, Fonterra, Boulogne-Billancourt, France): porcine pepsin (EC 3.4.23.1; 250 units/mg solid, Sigma-Aldrich), papain (EC 3.4.22.2; 3 units/mg Sigma-Aldrich), rennin from calf stomach (EC 3.4.23.4; ~20 units/mg; Sigma-Aldrich). BLF (5% w/v) was hydrolysed with rennin and pepsin according to Quintieri et al. (2012) and Elbarbary et al. (2010), respectively. Hydrolysis with papain, was performed as reported by Ou et al. (2010). After incubation, each reaction was stopped by heating at 90 °C and, after centrifugation (10.000 × g, 20 min, 4 °C), neutralized supernatants were quantified for protein concentration (Bradford, 1976). All BLF hydrolysates (LFHs) were freeze-dried and stored at −20 °C. Each LFH (10 μg of protein) was resolved on Tricine SDS-PAGE using the Mini Protean System (Bio-Rad Laboratories,Milan, Italy), as previously reported (Schägger, 2006) in order to check the complete digestion of BLF, confirmed by the absence of the related 80 kDa band on the electrophoretic pattern. Electrophoresis was carried out for 30 min at 30 V and then for 180 min at 90 V. After the run, gels were stained with 0.125% Comassie Brilliant Blue G-250 (Sigma–Aldrich) according to the method of Neuhoff et al. (1988). 2.4. Antimicrobial in vitro assays In order to determine the useful concentration of BLF and LFHs, antimicrobial assays were carried out by inoculating spoiler pseudomonads (3 log CFU/ml) in mPCB amended with 0, 12.5, 25, 50 and 100 mg/ml of BLF. Bacterial growth was monitored by measuring optical density every 10 min with the Varioskan Flash (ThermoFischer Scientific) spectrofluorimeter at a wavelength of 600 nm up to 48 h. Each antimicrobial assay was performed in triplicate. At first, the antimicrobial activity was checked by calculating the Inhibition Index turbidity ratio (IITR) as follows:

2.2. Evaluation of spoilage by Pseudomonas spp. strains on vegetables All strains were evaluated for their ability to cause spoilage on Iceberg lettuce (L. sativa L. var. capitata L.), purchased from a local market. Undamaged leaves from lettuce heads were individually washed under tap water, disinfected for 5 min in 200 ppm sodium hypochlorite solution, rinsed in sterile distilled water at 4 °C and dried on blotting paper sheets for 30 min. Then, the disinfected lettuce leaves were cut into small pieces (midrib length of 5–6 cm) that were transferred to sterile Petri dishes. Each leaf piece was cross-wounded along the rib using a sterile scalpel, resulting in a total number of 50 wounds for replicate. Each strain was cultured in mPCB for 24 h at 30 °C to reach 0.325 ± 0.05 OD600nm, corresponding to ca. 8 log CFU/ml; bacterial cells, recovered after centrifugation at 7000 g for 10 min, were diluted in sterile saline solution to 5 log CFU/ml and inoculated on wounded leaf (10 μL/wound). Control wounds supplemented or not with sterile saline solution (Controls 2 and 1, respectively) were also performed.

IITR ¼ 1

ðNtx=NtoÞ ðNcx=NcoÞ

where the values of OD600 readings (N) of the control (c) or treated (t) samples are considered as a change between the beginning (time zero) and the time when control samples began the stationary phase (time x) (Giannuzzi and Zaritzky, 1996). The II values ranging from 0 to 1 indicated the absence of inhibition and the permanence of the microorganism in the lag phase, respectively. The concentration of BLF able to produce at least an inhibition index of 0.5, on average, was considered enough to be used in the subsequent assays. Besides, the latter were carried out by a standard plate count method that avoids interference caused by cell death; such interference cannot be excluded by spectrophotometric measurements. Thus, fresh cell cultures of the target Pseudomonas strains were inoculated (3 log CFU/ml) in mPCB amended with 50 mg/ml of BLF or each LFH and incubated at 30 °C up to 30 h; mPCB without

B. Federico et al. / International Journal of Food Microbiology 215 (2015) 179–186

hydrolysates was also included in the assay as a control sample. Decimal dilution of each culture was enumerated on Plate Count Agar (PCA; Becton Dickinson Difco) seeded with mPCB decimal dilutions after 5, 24 and 30 h of incubation at 30 °C. In this case II values were calculated as the logarithmic value of the ratio between the number of microorganisms at different times (Giannuzzi and Zaritzky, 1996). Inhibition indexes calculated with OD readings were named IITR, whereas those calculated with cell counts were named II, as reported by Giannuzzi and Zaritzky (1996). For Pseudomonas cultures without any apparent microbial growth after 30 h of incubation, and in the absence of countable colonies on Petri dishes, the antimicrobial activity of LFH was checked by inoculating 50 ml of LFH-free PCB medium with 1% of 30 h-old treated cultures; for samples with visible microbial growth after incubation (30 °C for 24 h), the assigned II value was N 1.0, while for samples without growth the assigned II value was 1.5. In the light of these results, a single hydrolysate and the purified antimicrobial peptides were chosen for in vivo experiments. 2.5. Evaluation of BLF derived peptides for the control of lettuce leaf microbial spoilage Undamaged leaves of Trocadero lettuce (L. sativa L.) were washed, cut and treated as reported in Section 2.2. Freeze-dried powders of LFH from pepsin digestion and its peptide lactoferricin B (Lfcin B), purified by applying a two-step preparative chromatography, coupled with LC/MS Orbitrap-based technology analysis, as previously described by Quintieri et al. (2012), were dissolved in the sterile saline solution at the final concentration of 50 mg/ml and 3 mg/ml, respectively. Then, the filter-sterilized (0.22 μm) solutions were supplemented with fresh bacterial cell cultures (5 log CFU/ml) of each selected strain. Four treatments were represented by wounded leaves and treated with 10 μL of sterile saline solution (treatment 1), 10 μL sterile saline solution amended with LFH or LFcin B (treatment 2), 10 μL of cell suspensions (treatment 3; 3 log CFU/wound) and 10 μL of cell suspensions containing LFH or LFcin B (treatment 4). Percentage of spoiled wounds was recorded over cold storage for 6 days as reported above. In order to verify the viability of spoiler pseudomonads in treated leaves, the number of viable cells of a single target Pseudomonas strain was evaluated during cold storage in comparison to the untreated samples. The experiment was conducted as described above. Pseudomonas spp. counts and the percentage of spoilage in wounded lettuce leaves were recorded after 3, 6, 10 and 13 days of cold storage at 4 °C in air. Viable cells of inoculated spoiler strain, as well as those from naturally contaminating microorganisms, were recovered from wounds by three subsequent washing steps with 150 μl of sterile saline solution, subsequent decimal dilution and seeding on PSA. In addition, the Pseudomonadaceae population contaminating the lettuce leaves was determined as reported in Section 2.1. The resulting viable cell concentration was expressed as log CFU/g or as log CFU/wound. The trial was carried out in triplicate (3 leaves for each treatment and for each sampling time, N = 3). 2.6. Statistical analysis Results related to the percentage of infected wounds and the inhibition index values were subjected to a square root arcsin transformation (Sokal and Rohlf, 1995) in order to meet the homogeneity-of-variance assumptions according to Levene's test. The univariate General Linear Model (GLM) procedure, applying a one-way ANOVA (P b 0.05), performed on SPSS software release 8 (SPSS, Inc., Chicago, IL), was used to examine the effect of the cold storage period on the percentage of infected wounds per strain. Two-way ANOVA was carried out to evaluate the independent and interaction effects of treatments with enzyme hydrolysates as well as purified peptide solutions and extension of cold storage on in vitro inhibition index values and on spoiled wounds for each selected strain. Multiple comparisons among individual means

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for each assayed strain were performed by Fisher's least significant difference (LSD) multiple range test at the 95% confidence interval. The level of significance was determined at P b 0.05. 3. Results and discussions 3.1. Isolation of bacteria from fresh-cut vegetables In the present work, a Pseudomonas spp. load higher than 5 log CFU/g was found in two cold-stored unspoiled ready-to-eat (RTE) vegetables (curly endive and lettuce leaves), using count methods suggested by López-Gálvez et al. (2010) on disinfected lettuce samples. Forty-eight isolates collected were typed by RAPD-PCR, resulting in 17 biotypes. Taxonomic identification (Table 1) showed that four biotypes from lettuce belonged to P. fluorescens, whereas 13 strains out of 40 isolates from curly endive were distributed among seven Pseudomonas species (10), Acinetobacter calcoaceticus (1) and Aeromonas media (2). The need to collect microbial strains potential responsible for vegetable tissue decay in RTE leafy vegetables led us to isolate, type and identify all biotypes occurring on PSA plates enumerated as the dominant Pseudomans spp. population, even though culture-independent techniques showed that natural microflora occurring on these vegetables is composed by several species from different genera (Handschur et al., 2005; Randazzo et al., 2009; Rudi et al., 2002). The isolation of strains from cold-stored unspoiled RTE vegetables was carried out in order to collect some Pseudomonas strains from microbial populations surviving chlorine-based washing steps and the cold-storage environment. The isolation of spoilers from heavily spoiled tissues (Lee et al., 2013) could have no relationship with RTE processing. As survival following RTE processing and cold storage is not evidence of spoilage ability, the Pseudomonas strains isolated were further characterized for their role in tissue decay on various vegetables. 3.2. Spoilage ability of Pseudomonas bacteria on some vegetables Previous studies had highlighted the isolation and the different spoilage abilities of Pseudomonas species from various fresh vegetables and fresh-cut products (Jacxsens et al., 2003; Lee et al., 2013; Pascoe and Premier, 2000); thus, in this work, we evaluated the spoilage ability of 7 species of Pseudomonas on Iceberg lettuce, well known to be subject to microbial spoilage under refrigeration (Li et al., 2001). Table 1 Taxonomic identification of Pseudomonas spp. strains isolated from fresh-cut vegetables. Source of isolation

Number Target 16S rDNA sequence of Isolates

Species

Strain

Trocadero lettuce

2 2 2 2 3 4 4 4 4 4

Pseudomonas fluorescens P. fluorescens

AB724288.1 locus_tag: PS417_095152 locus_tag: PflA506_01141 NR_102835.1 FM209480.1 AY206685.1

L1A L1C L2A L2B I3C I2A I2B I3B I3A I1C

2

GU078446.1

Curly endive

JQ974027.1 locus_tag: PflA506_01141

Pseudomonas cichorii P. simiae P. fluorescens P. fluorescens Pseudomonas jessenii P. jessenii Pseudomonas koreensis

I4C

5 2

locus_tag:PputGB1_R0001

Pseudomonas putida

I1B I2C

3

HM190229.1

Pseudomonas viridiflava

I1A

3

1 Sequences from Pseudomonas fluorescens strain A506, complete genome, accession number CP003041. 2 Sequences from Pseudomonas simiae strain WCS417 complete genome, accession number CP007637. 3 Sequences from Pseudomonas putida strain GB-1 complete genome, accession number CP000926.1.

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Table 2 Percentages of spoiled wounds (as average of three replicates ± standard deviation) of Pseudomonas strains on fresh cut iceberg lettuce after 2, 3 and 4 days of storage at 4 °C. Species

Untreated wounds Wounds amended with SS P. fluorescens

P. viridiflava P. jessenii P. cichorii P. putida P. koreensis

Strain

– L1A L2A L2B L1C I3B I1A I3A I3C I1B I4C

Days of storage 2

3

4

6.7 ± 1.2 14.3 ± 2.5 60.0 ± 3.0 6.0 ± 2.0 25.7 ± 2.5 44.7 ± 1.1 53.0 ± 4.0 68.0 ± 4.0 25.7 ± 2.5 68.0 ± 3.0 60.0 ± 4.0 20.0 ± 2.0

8.8 ± 1.1 28.0 ± 3.5 80.0 ± 5.0 16.0 ± 5.0 28.0 ± 4.0 74.7 ± 4.2 64.0 ± 5.0 70.0 ± 3.0 32.0 ± 3.0 82.0 ± 3.6 66.0 ± 3.0 28.0 ± 2.0

13.5 ± 0.7 30.7 ± 1.2 96.0 ± 1.0 82.0 ± 3.0 78.0 ± 3.0 99.0 ± 1.0 80.0 ± 5.0 72.7 ± 3.5 35.7 ± 1.5 96.0 ± 4.0 68.0 ± 2.0 38.7 ± 4.7

SS: saline solution (0.9% NaCl). All mean values shown are different each other for the least significant difference comparison. LSD (95% interval confidence level): ±10.80%.

Depending on the strain inoculate in wounded lettuce leaves, discoloration ranging from red to brown, sometimes associated with tissue softening, was found throughout cold storage. Table 2 reports the percentages of spoilage caused by Pseudomonas strains on wounds in Iceberg lettuce midrib leaves after 2, 3 and 4 days of cold storage. An example of spoilage caused by one of the Pseudomonas strains is shown in Fig. S1. As shown in Table 2, after 2 days of cold storage the highest percentage of spoiled wounds was detected for the strains P. viridiflava I1A, P. cichorii I3C, P. putida I1B and P. fluorescens L1A and I3B (61.8%, on average). After 4 days of cold storage, spoilage was observed on almost all samples inoculated with all P. fluorescens, P. cichorii I3C, P. viridiflava I1A and P. putida I1B strains, whereas the lowest percentages (35.7 and 38.7%, respectively) of spoiled wounds were found on samples inoculated with P. jessenii I3A and P. koreensis I4C. Similar spoilage symptoms were found by Pascoe and Premier (2000) on lettuce inoculated with P. fluorescens, even though these authors, differently from our data, did not observe any spoilage symptoms on control leaves. Our results are in accordance with those of other studies demonstrating P. cichorii's ability to cause tissue browning and bacterial rot in lettuce (Grogan et al., 1977; Hikichi et al., 1996; Pauwelyn et al., 2011). Besides, other pseudomonads have been found responsible for RTE endive spoilage during cold storage (Nguyen-the and Prunier, 1989). Among 14 Pseudomonas strains isolated from RTE lettuce and endive, four (I2A, I2B, I1C, I2C) were unable to cause spoilage, showing the same percentages (close to 30%) of spoiled wounds, calculated for wounded leaves supplemented with sterile saline (control 2); the percentage of spoiled wounds on Iceberg lettuce, without addition of sterile saline solution (control 1) reached ca. 13% at the fourth day of storage (Table 2). The percentage of spoiled wounds in control 2 compared with that of control 1 could be due to the occurrence of autochthonous bacteria; they can penetrate the host through cut surfaces or tissues damaged by microbial pectolytic enzymes promoting browning (Jay et al., 2005). No spoilage was observed on inoculated and unwounded leaves throughout six days of cold storage (controls 3 and 4); conversely, spoilage was found only when microbial cells colonized wounded vegetable tissues, as previously reported for other Pseudomonas species (Ferrante and Scortichini, 2009; Sisto et al., 2002). In the present work, the percentage of spoiled wounds increased on leaves inoculated with all strains, except for I1A and I1B, and this was correlated with the length of cold storage. No spoilage was observed on celery and endive. Among all strains, P. fluorescens L1C and L1A, P. cichorii I3C, P. viridiflava I1A and P. putida I1B spoiled over 65% of inoculated wounds after 3 days of cold storage at 4 °C (Table 2). These strains

were assayed for their spoilage ability on Trocadero lettuce and escarole chicory. The lowest percentage of spoiled wounds was found at each sampling day on Trocadero lettuce inoculated with P. fluorescens L1A (Table 3); spoiled wounds (ca 75%) were found only at the 5th day of cold storage. A different spoilage ability was observed when this latter strain was inoculated on escarole chicory (Table 3). A more severe spoilage ability was displayed by the P. fluorescens L1C on both escarole chicory and Trocadero lettuce (Table 3). P. putida I1B was the strain with the highest rate of spoiled wounds in both vegetables, whereas strains I3C and I1A displayed an intermediate behavior on escarole chicory. However, the ability of strains to induce spoilage was influenced by the vegetable tissue: a higher percentage of spoiled wounds (by at least ca. 85%) was observed, at the end of cold storage, on Trocadero lettuce than on escarole chicory, inoculated with all Pseudomonas spp. Wounded and uninoculated escarole chicory and Trocadero lettuce leaves registered an increase in the percentage of spoiled wounds from 16.4 ± 1.9 (average values ± standard deviation) to 47.1 ± 2.7 and from 0 to 38.2 ± 3.2, respectively, during cold storage. These results were significantly different from those recorded for inoculated wounds for all strains but only starting from the fifth day of cold storage (Table 3). These data suggest that part of the spoilage recorded for each Pseudomonas strain could derive from autochthonous bacteria naturally occurring on vegetables and not killed by chlorine treatment. The spoilage pattern displayed by the five vegetables assayed suggests that the appearance of off-colors depends on the Pseudomonas strains inoculated, on the type of vegetable tissue as well as on their interactions. Browning response caused by Pseudomonas strains could be related to the phenolic composition of tissue, thus the spoilage ability of strains could depend on their enzymatic activities (Lee et al., 2013) as well as on differing plant tissue reactions to microbial colonization among fresh-cut vegetables. In the light of these data, subsequent experiments were carried out on Trocadero lettuce choosing P. cichorii I3C, P. putida I1B, P. viridiflava I1A and P. fluorescens L1C strains as target microorganisms.

3.3. Lactoferrin hydrolysis and antimicrobial in vitro assays In order to control spoilage-related bacteria using antimicrobial peptides, spoiler strains were evaluated for their sensitivity to lactoferrin and its peptides released by three proteolytic enzymes (porcine pepsin, rennin from calf stomach and papain from Carica papaya). Tricine SDSPAGE analysis revealed that each enzyme was able to digest bovine lactoferrin after 4 h of incubation, showing the complete disappearance of the corresponding protein band (data not shown) in accordance with previously reported data (Elbarbary et al., 2010; Quintieri et al., 2012; Tomita et al., 1991). The resulting faint peptide bands, found in porcineand papain-digested lactoferrin, displayed molecular mass ranging from 6.0 to 14.4 kDa, whereas the electrophoretic pattern of rennin hydrolysate was similar to that found for pepsin; both showed six bands with apparent molecular weights ranging from 2.5 to 14.0 kDa. The preliminary assay carried out to define a useful concentration of BLF and its LFH showed that antimicrobial activity increased with the increase in BLF concentration; even though some strain-dependant resistances to antimicrobial activity were found at the same BLF concentration; broth supplemented with 50 mg/ml BLF produced an inhibition index (IITR) of 0.6 ± 0.27, on average (see Table S1). This method was used to choose the concentration for the subsequent experiments. The antimicrobial activity of BLF and its peptide mixtures, monitored up to 30 h by plate counting, was appreciable starting from the 24th h of incubation at 30 °C, the usual growth temperature for these strains. After 30 h of growth in LFH-free mPCB, the selected target strains increased in viable cell loads, reaching values close to 9 log CFU/ml. Table 4 shows the IIs recorded for all LFH against each selected strain after 5, 24 and 30 h of incubation.

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Table 3 Percentages of spoiled wounds (as average of three replicates ± standard deviation) caused by Pseudomonas strains on trocadero lettuce and escarole chicory during cold storage. Days of storage Vegetable

Strains

Trocadero lettuce

Untreated wounds Wounds amended with SS P. fluorescens L1A P. fluorescens L1C P. chicorii I3C P. viridiflava I1A P. putida I1B Untreated wounds Wounds amended with SS P. fluorescens L1A P. fluorescens L1C P. chicorii I3C P. viridiflava I1A P. putidaI1B

Escarole chicory

2

3

5

6

0.0 ± 0.0 14.7 ± 2.5 0.0 ± 0.0 10.7 ± 2.1 30.7 ± 1.2 44.7 ± 3.5 40.7 ± 1.2 16.4 ± 1.9 13.6 ± 2.1 33.7 ± 3.2 55.7 ± 2.1 64.3 ± 2.1 40.7 ± 1.2 50.3 ± 2.5

0.0 ± 0.0 25.7 ± 3.5 1.0 ± 1.0 10.7 ± 2.1 30.7 ± 1.2 44.7 ± 3.5 40.7 ± 1.2 16.4 ± 1.9 13.6 ± 2.1 34.7 ± 2.5 55.7 ± 2.1 64.7 ± 2.5 70.3 ± 3.5 90.3 ± 3.5

17.6 ± 1.2 30.7 ± 2.1 74.7 ± 3.5 95.3 ± 3.5 75.3 ± 3.5 95.3 ± 2.5 100.0 ± 0.0 27.6 ± 3.1 25.7 ± 3.1 40.7 ± 1.2 70.3 ± 2.5 64.7 ± 2.5 70.3 ± 3.5 95.7 ± 1.2

38.2 ± 3.2 32.3 ± 2.5 84.7 ± 3.5 95.3 ± 3.5 84.7 ± 3.5 95.3 ± 2.5 100.0 ± 0.0 47.1 ± 2.7 41.0 ± 2.6 45.3 ± 1.5 70.3 ± 2.5 70.3 ± 2.5 80.3 ± 2.5 100.0 ± 0.0

SS: saline solution. LSD trocadero lettuce (95% interval confidence level): ±7.92%. LSD escarole chicory (95% interval confidence level): ±7.84%.

The two-way ANOVA examined the effect of enzyme and incubation time on IIs. The native bovine lactoferrin inhibited only the growth of P. putida I1B, confirming data reported by several authors showing the higher antibacterial activity of its peptides (Quintieri et al., 2012; Tomita et al., 1991; Wakabayashi et al., 2003). The viability assay carried out after 30 h of incubation showed the bactericidal effect of BLF, pepsin- and rennin-LFH against this strain (Table 4); conversely, no antimicrobial effect was produced by papain-LFH. As concerns the I1A strain, a statistically significant interaction was found between the effects of the enzyme and incubation time on antimicrobial activity [F (6, 24) = 2.0966; p = 0.026]. After 30 h of incubation, rennin- and pepsin-digested BLF showed the highest II scores (F (3, 24) = 133.005; p = 4.36 × 10−15). Conversely, papain-LFH and the undigested BLF did not show any antimicrobial effect on the strain (p N 0.219). At the end of incubation, in comparison with the unamended control, bovine lactoferrin rennin hydrolysate reduced the growth of

Table 4 Antimicrobial activity, expressed as inhibition index (II; average of three replicates ± standard deviation), of lactoferrin (BLF) and its LFH (50 mg/ml) obtained with different enzymes against spoiler Pseudomonas strains incubated at 30 °C in mPCB (Difco™, Becton Dickinson). Inhibition index(II) Strains

Treatment

P. fluorescens L1C

BLF Papain-LFH Pepsin-LFH Rennin-LFH BLF Papain-LFH Pepsin-LFH Rennin-LFH BLF Papain-LFH Pepsin-LFH Rennin-LFH BLF Papain-LFH Pepsin-LFH Rennin-LFH

P. viridiflava I1A

P. cichorii I3C

P. putida I1B

5h

24 h

30 h

0.00 ± 0.00 1.00 ± 0.00 0.24 ± 0.07 0.25 ± 0.01 0.01 ± 0.00 0.11 ± 0.01 0.37 ± 0.17 0.53 ± 0.02 0.09 ± 0.00 0.49 ± 0.11 0.27 ± 0.09 0.26 ± 0.10 1.00 ± 0.00 0.02 ± 0.02 1.00 ± 0.00 0.00 ± 0.01

0.03 ± 0.01 1.00 ± 0.00 0.01 ± 0.02 0.07 ± 0.01 0.00 ± 0.00 0.02 ± 0.03 0.34 ± 0.05 0.54 ± 0.01 0.02 ± 0.02 0.01 ± 0.01 0.49 ± 0.01 0.03 ± 0.04 1.00 ± 0.00 0.27 ± 0.01 1.00 ± 0.00 1.00 ± 0.00

0.02 ± 0.03 1.50 ± 0.00 0.01 ± 0.01 0.07 ± 0.12 0.01 ± 0.01 0.02 ± 0.02 0.47 ± 0.06 0.40 ± 0.12 0.01 ± 0.02 0.00 ± 0.00 0.43 ± 0.01 0.02 ± 0.03 1.50 ± 0.00 0.00 ± 0.00 1.50 ± 0.00 1.50 ± 0.00

The least significant difference comparison values (LSD, 95% interval confidence level) were calculated among the different treatments of each strain: L1C, ± 0.14; I1A, ± 0.22; I3C, ± 0.18; I1B, ± 0.02. II = 0 absence of antimicrobial activity, 0 ≤ II ≤ 1 different levels of bacteriostatic activity. II N 1 bactericidal activity (occurrence of microbial growth after viability assay). II = 1.5 bactericidal activity (absence of microbial growth after viability assay).

P. viridiflava I1A, by 5.7 log CFU/ml and a complete inhibition of P. putida I1B. This result is in accordance with that found by Elbarbary et al. (2010) assaying a lactoferrin hydrolysate prepared in the same way against E. coli and B. subtilis. The release of the peptide lactoferrampin (f 178–285) in this BLF hydrolysate (Elbarbary et al., 2010) could be responsible for the antimicrobial activity that we found against spoilage pseudomonads, as previously demonstrated against P. aeruginosa (van der Kraan et al., 2004). A bactericidal effect was displayed only against P. fluorescens L1C by papain-LFH up to 30 h of incubation, whereas a lower antimicrobial activity of both pepsin- and rennin-LFH (0.24, on average) was found within 24 h of incubation (p b 6.376 × 10− 5). No inhibition was shown by BLF. Starting from 5 h and up to the end of incubation, pepsin-LFH proved to inhibit significantly (P b 0.05) the growth of 3 out of 4 selected strains; indeed, P. cichorii I3C and P. viridiflava I1A counts decreased by ca. 4.8 log cycle, on average, in comparison with the untreated cultures, whilst no cells were enumerated for P. putida I1B. Recently, the efficacy of pepsin-digested hydrolysate (LFH) was tested in vitro against Pseudomonas spp. isolated from cold-stored Mozzarella cheese (Quintieri et al., 2013a); these bacteria were able to grow and promote caseinolytic activities in the governing liquid at 4 °C (Baruzzi et al., 2012b). The different growth patterns shown by pseudomonads with different enzymes suggest that there is a strain-specific sensitivity to the antimicrobial peptides released from lactoferrin. However, the highest antimicrobial activity (calculated as the average of inhibition indexes) was ascribed to the peptides released by pepsin hydrolysis, followed by that released from rennin. 3.4. Antimicrobial activity assay on Trocadero lettuce leaves On the basis of the previous results, bovine lactoferrin pepsin hydrolysate was selected in order to be tested against the spoilage Pseudomonas spp., on incised and inoculated lettuce leaves. Its application in uninoculated lettuce wounds showed the appearance of bronzing spots, putatively attributed to iron content in the native protein (ca 15% of Fe3+ ion; Chung and Kenneth, 1993) and resembling the bronzing symptoms caused by FeSO4 (Peng and Yamauchi, 1993). Recently, it has been demonstrated that the antibacterial activity of lactoferrin pepsin hydrolysate against several psychrotrophic Pseudomonas spp. strains (Quintieri et al., 2012) was mainly correlated to the release of the peptide lactoferricin B (LfcinB 17–42). For these reasons, here we applied the purified peptide on lettuce wounds as it did not produce any bronzing signs. As shown in Table 5, the treatment of lettuce wounds with this

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Table 5 Effects of LfcinB (3 mg/mL) on wound decays of lettuce cv ‘trocadero’ leaves infected by Pseudomonas strains up to day 6 of cold storage (4 °C). Strain P. fluorescens L1C

P. viridiflavaI1A

P. chicorii I3C

P. putida I1B

Treatment1 SS LfcinB CS CS+ LfcinB SS LfcinB CS CS+ LfcinB SS LfcinB CS CS+ LfcinB SS LfcinB CS CS+ LfcinB

Spoiled wounds (%) at different days 2 3

4

5

6

31.7 ± 2.1 21.3 ± 2.1 51.0 ± 2.6 51.0 ± 1.7 29.3 ± 2.1 39.0 ± 1.0 63.3 ± 4.7 29.0 ± 1.0 22.7 ± 1.5 19.3 ± 1.2 48.0 ± 1.0 31.0 ± 2.6 31.0 ± 1.0 23.7 ± 1.5 53.3 ± 2.9 24.7 ± 1.5

31.3 ± 1.5 28.3 ± 2.5 77.3 ± 6.1 99.3 ± 1.2 48.3 ± 3.2 38.3 ± 1.5 68.3 ± 4.5 44.0 ± 5.0 31.0 ± 1.7 28.3 ± 2.3 88.0 ± 4.4 44.7 ± 0.6 28.3 ± 2.9 29.3 ± 2.3 94.0 ± 5.3 33.3 ± 2.5

33.7 ± 2.5 40.3 ± 2.5 97.3 ± 4.6 99.7 ± 0.6 48.3 ± 3.2 41.7 ± 3.8 66.3 ± 4.0 45.0 ± 3.6 37.7 ± 4.0 36.3 ± 2.9 95.3 ± 4.5 44.0 ± 3.6 35.0 ± 3.6 31.7 ± 2.9 92.7 ± 9.5 45.0 ± 3.6

52.0 ± 3.5 53.3 ± 6.5 94.7 ± 6.1 92.3 ± 7.1 47.7 ± 2.1 50.3 ± 4.0 91.0 ± 6.2 61.3 ± 2.9 48.7 ± 3.2 49.3 ± 5.5 92.0 ± 7.2 54.3 ± 5.1 54.3 ± 3.8 54.3 ± 5.7 98.0 ± 3.5 65.7 ± 1.5

1 Treatment SS: saline solution (0.9% NaCl); CS: bacterial cell suspension (3 log cfu/ wound). 2 The least significant difference comparison values (LSD, 95% interval confidence level) were calculated among the different treatments of each strain: L1C, ±13%; I1A, ±12%; I3C, ±13%; I1B, ±12%.

peptide inoculated with P. viridiflava I1A, P. chicorii I3C and P. putida I1B caused a significant (P b 0.001) reduction in spoilage, under cold storage, by a mean of 27.4, 37.3 and 42.3%, respectively. In the case of lettuce wounds inoculated with I1A and I1B, a significant (P b 0.05) reduction in the efficacy of LfcinB was found from day 5 of cold storage; however, wounds inoculated with these strains and not supplemented with LfcinB displayed higher and significant (P b 0.05) differences in percentage of spoiled wounds compared with inoculated and treated ones (Table 5). In order to verify whether the reduction in spoilage percentage was related to viable cell concentrations, an additional in vivo assay was carried out with P. putida I1B. Even though LfcinB controlled the spoilage produced by P. putida I1B and P. cichorii I3C to the same degree, the I1B strain was chosen because this bacteria caused high spoilage rates on different vegetables (Tables 2 & 3) and, at the same time, was the most sensitive strain (Table 4) to pepsin-LFH peptides in the in vitro assay. The Pseudomonas spp. population, evaluated by using whole leaf samples, carried out immediately after the beginning of cold storage, was found to be ca. 1 log CFU/g higher in the inoculated leaves than in the uninoculated ones; this difference disappeared within three days of cold storage (data not shown). Based on some preliminary calculations, the cell recovery rate of the wound-washing method was estimated to be close to 80% (data not shown). In contrast to the whole leaf sample method, the wound-washing method was able to distinguish

naturally-occurring pseudomonads from the target strain in all samples and at each sampling time. As shown in Table 6, wounds treated with sterile saline solution (treatment 1) and sterile LfcinB solution (treatment 2) showed a complete absence of pseudomonads until day 13 of cold storage. Thus, microbial cell counts registered on the inoculated wounds (treatments 3 and 4) were presumably represented only by the P. putida I1B strain, that increased during the cold storage period, reaching an average value of 4.76 ± 0.26 log CFU/wound. The twoway ANOVA analyses did not show any significant difference between the average viable cell concentration value from treatments 3 and 4, over the whole storage period. As previously found, some spoiled wounds were found even in the absence of microbial inoculum (treatments 1 and 2). In these cases, the spoiled wounds never exceeded 15%. Significant differences between uninoculated (treatments 1 and 2) and inoculated samples (treatments 3 and 4) were found starting from day 6 and were ascribed to the occurrence of viable cells of the P. putida I1B strain (Table 6). The percentage of spoiled wounds increased with the extension of storage time; starting from day 6 of storage its values in inoculated leaf samples unamended with LfcinB (treatment 3) was always significantly higher than the remaining samples; indeed, the addition of LfcinB to inoculated samples (treatment 4) significantly halved (95% interval confidence level) the percentage of spoiled wounds. The ability of Pseudomonas strains to cause spoilage in leafy vegetables depends on several factors among which lipopeptide production has been found to be involved in plant pathogenicity, antimicrobial activity, regulation of attachment and detachment to and from surfaces, and motility (Raaijmakers et al., 2006) while they can also facilitate the access of cell-wall-degrading enzymes to the plant surface (Hildebrand et al., 1998). When lettuce leaves were inoculated with a lipopeptide-deficient mutant, as in the case of P. cichorii SF1-54, a strain responsible for midrib butterhead lettuce spoilage (Pauwelyn et al., 2011), the extent of rotten midribs was significantly lower than that found on lettuce plants inoculated with the wild type, although both strains grew vigorously and reached similar cell densities (Pauwelyn et al., 2013). Also, when the quorum sensing system of Erwinia carotovora was inactivated by lactonase AiiA (Dong et al., 2000) the severity of spoilage symptoms on various vegetables changed significantly in the absence of significant differences in viable cell count. In the absence of specific experiments, our results suggest that the addition of LfcinB diverted metabolic pathways involved in phytotoxic activity or hydrolytic enzyme production, reducing the extent of spoilage symptoms independently from its antimicrobial activity. For this reason, after the chlorine-based washing steps carried out before packaging, the application of antimicrobial peptides can be considered a supplementary tool for the control of microbial spoilers of RTE leafy vegetables over cold storage.

Table 6 Mean values (log cfu/wound ± SD) of Pseudomonas spp. counts and percentages of spoiled wounds in ‘trocadero’ lettuce leaves inoculated with P. putida I1B and treated with LfcinB, during 13 days of cold storage at 4 °C.

Pseudomonas spp. Spoiled wounds (%)

Treatment

Days of cold storage1

SS LfcinB P. putida I1B P. putida I1B + LfcinB SS LfcinB P. putida I1B P. putida I1B + LfcinB

0 0.00 0.00 1.45 ± 0.05 1.54 ± 0.05 0.0 0.0 0.0 0.0

3 0.00 0.00 3.13 ± 0.16 2.82 ± 0.24 7.4 ± 6.4 7.4 ± 6.4 18.5 ± 6.4 22.2 ± 0.0

6 0.00 0.00 3.90 ± 0.46 4.31 ± 0.12 7.4 ± 6.4 7.4 ± 6.4 66.7 ± 11.1 29.6 ± 6.4

10 0.00 0.00 5.03 ± 0.21 4.43 ± 0.14 14.8 ± 6.4 11.1 ± 0.0 70.4 ± 6.4 33.3 ± 11.1

13 0.00 0.00 4.97 ± 0.11 4.54 ± 0.15 14.8 ± 6.4 14.8 ± 6.4 74.0 ± 12.8 37.0 ± 6.4

SS = sterile saline solution (0.9% NaCl). Data represent means ± standard deviations (N = 3). LSD (95% interval confidence level): Pseudomonas spp., 0.68 log CFU/wound; percentage of spoiled wounds, ±25.58%.

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2015.09.017. Acknowledgments This work was carried out under the research activities under the Project: “High-Convenience Fruits and vegetables: New Technologies for Quality and New Products” (OFRALSER), PON01_01435, financed by the Italian Ministry of Education, University and Research. The authors thank Mr. Gaetano Stea for his technical help in performing molecular analyses, and Dr. Maria Morea and Dr. Anthony Green for their useful suggestions regarding manuscript improvement. References Ahmed, L., Martin-Diana, A.B., Rico, D., Barry-Ryan, C., 2012. The impact of delactosed whey permeate treatment on shelf-life and antioxidant contents of strawberries. Int. J. Food Sci. Technol. 47, 1430–1438. Ahmed, L., Rico, D., Martin-Diana, A.B., Barry-Ryan, C., 2011. Optimization of application of delactosed whey permeate treatment to extend the shelf life of fresh-cut tomato using response surface methodology. J. Agric. Food Chem. 59, 2377–2385. AitTayeb, L., Ageron, E., Grimont, F., Grimont, P.A.D., 2005. Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates. Res. Microbiol. 156, 763–773. Altschul, S.F., Thomas, L.M., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Baruzzi, F., Cefola, M., Carito, A., Vanadia, S., Calabrese, N., 2012a. Changes in bacterial composition of zucchini flowers exposed to refrigeration temperatures. Sci. World J., 127805 (6 pp.). Baruzzi, F., Lagonigro, R., Quintieri, L., Morea, M., Caputo, L., 2012b. Occurrence of non-lactic acid bacteria populations involved in protein hydrolysis of cold-stored high moisture mozzarella cheese. Food Microbiol. 30, 37–44. Baruzzi, F., Morea, M., Matarante, A., Cocconcelli, P.S., 2000. Changes in the Lactobacillus community during ricotta forte cheese natural fermentation. J. Appl. Microbiol. 89, 807–814. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cálix-Lara, T.F., Rajendran, M., Talcott, S.T., Smith, S.B., Miller, R.K., Castillo, A., Sturino, J.M., Taylor, T.M., 2014. Inhibition of Escherichia coli O157:H7 and Salmonella enterica on spinach and identification of antimicrobial substances produced by a commercial lactic acid bacteria food safety intervention. Food Microbiol. 38, 192–200. Caputo, L., Quintieri, L., Bianchi, D.M., Decastelli, L., Monaci, L., Visconti, A., Baruzzi, F., 2015. Pepsin-digested bovine lactoferrin prevents mozzarella cheese blue discoloration caused by Pseudomonas fluorescens. Food Microbiol. 46, 15–24. Chisari, M., Barbagallo, R.N., Spagna, G., 2007. Characterization and role of polyphenol oxidase and peroxidase in browning of fresh-cut melon. J. Agric. Food Chem. 56, 132–138. Chung, T.D., Kenneth, N.R., 1993. Lactoferrin: the role of conformational changes in its iron binding and release. J. Am. Chem. Soc. 115, 6765–6768. Del Olmo, A., Morales, P., Nunez, M., 2009. Bactericidal activity of lactoferrin and its amidated and pepsin-digested derivatives against Pseudomonas fluorescens in ground beef and meat fractions. J. Food Prot. 72, 760–765. Dong, Y.H., Xu, J.L., Li, X.Z., Zhang, L.H., 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. PNAS 97, 3526–3531. Elbarbary, H.A., Abdou, A.M., Park, E.Y., Nakamura, Y., Mohamed, H.A., Sato, K., 2010. Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique. Int. Dairy J. 20, 646–651. Enrique, M., Manzanares, P., Yuste, M., Martinez, M., Valles, S., Marcos, J.F., 2009. Selectivity and antimicrobial action of bovine lactoferrin derived peptides against wine lactic acid bacteria. Food Microbiol. 26, 340–346. Ferrante, P., Scortichini, M., 2009. Identification of Pseudomonas syringae pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit (Actinidia chinensis planchon) in central Italy. J. Phytopathol. 157, 768–770. Garcia, A., Mount, J.R., Davidson, P.M., 2003. Ozone and chlorine treatment of minimally processed lettuce. J Food Sci. 68, 2747–2751. Giannuzzi, L., Zaritzky, N.E., 1996. Effect of ascorbic acid in comparison to citric and lactic acid on Listeria monocytogenes inhibition at refrigeration temperatures. LWT – Food Sci. Technol. 29, 278–285. Gil, M.I., Selma, M.V., López-Gálvez, F., Allende, A., 2009. Fresh-cut product sanitation and wash water disinfection: problems and solutions. Int. J. Food Microbiol. 134, 37–45. Grogan, R.G., Misaghi, I.J., Kimble, K.A., Greathead, A.S., Ririe, D., Bardin, R., 1977. Varnish spot, destructive disease of lettuce in California caused by Pseudomonas cichorii. Phytopathology 67, 957–960. Handschur, M., Pinar, G., Gallist, B., Lubitz, W., Haslberger, A.G., 2005. Culture free DGGE and cloning based monitoring of changes in bacterial communities of salad due to processing. Food Chem. Toxicol. 43, 1595–1605. Hikichi, Y., Saito, A., Suzuki, K., 1996. Infection sites of Pseudomonas cichorii into head leaf of lettuce. Ann. Phytopathol. Soc. Jpn. 62, 125–129.

185

Hildebrand, P.D., Braun, P.G., McRae, K.B., Lu, X., 1998. Role of the biosurfactant viscosin in broccoli head rot caused by a pectolytic strain of Pseudomonas fluorescens. Can. J. Plant Pathol. 20, 296–303. Jacxsens, L., Devlieghere, F., Ragaert, P., Vanneste, E., Debevere, J., 2003. Relation between microbiological quality, metabolite production and sensory quality of equilibrium modified atmosphere packaged fresh-cut produce. Int. J. Food Microbiol. 83, 263–280. Jay, J.M., Loessner, M.J., Golden, D.A., 2005. Vegetable and fruit products. In: Jay, J.M., Loessner, M.J., Golden, D.A. (Eds.), Modern Food Microbiology. Spring Science Business Media Inc., New York, pp. 125–147. Kim, H., Ryu, J.H., Beuchat, L.R., 2006. Survival of Enterobacter sakazakii on fresh produce as affected by temperature, and effectiveness of sanitizers for its elimination. Int. J. Food Microbiol. 111, 134–143. Lamikanra, O. (Ed.), 2002. Fresh-cut Fruits and Vegetables: Science, Technology, and Market. CRC Press, Boca Raton, Florida, USA. Lee, D.H., Kim, J.B., Kim, M., Roh, E., Jung, K., Choi, M., Heu, S., 2013. Microbiota on spoiled vegetables and their characterization. J. Food Prot. 76, 1350–1358. Li, Y., Brackett, R.E., Shewfelt, R.L., Beuchat, L.R., 2001. Changes in appearance and natural microflora on iceberg lettuce treated in warm, chlorinated water and then stored at refrigeration temperature. Food Microbiol. 18, 299–308. Liao, C.H., Hung, H.Y., Chatterjee, A.K., 1988. An extracellular pectatelyase is the pathogenicity factor of the soft-rotting bacterium Pseudomonas viridiflava. Mol. Plant-Microbe Interact. 1, 199–206. López-Gálvez, F., Allende, A., Truchado, P., Martínez-Sánchez, A., Tudela, J.A., Selma, M.V., Gil, M.I., 2010. Suitability of aqueous chlorine dioxide versus sodium hypochlorite as an effective sanitizer for preserving quality of fresh-cut lettuce while avoiding byproduct formation. Postharvest Biol. Technol. 55, 53–60. Martin-Diana, A.B., Rico, D., Frias, J., Mulcahy, J., Henehan, G.T.M., Barry-Ryan, C., 2006. Whey permeate as a bio-preservative for shelf life maintenance of fresh-cut vegetables. Innovative Food Sci. Emerg. Technol. 7, 112–123. Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., 1988. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis 9, 255–262. Nguyen-the, C., Prunier, J.P., 1989. Involvement of Pseudomonads in deterioration of ready-to-use salads. Int. J. Food Sci. Technol. 24, 47–58. Nieuwenhuijsen, M.J., Toledano, M.B., Eaton, N.E., Fawell, J., Elliott, P., 2000. Chlorination disinfection byproducts in water and their association with adverse reproductive outcomes: a review. Occup. Environ. Med. 57, 73–85. Ou, K., Liu, Y., Zhang, L., Yang, X., Huang, Z., Nout, M.J., Liang, J., 2010. Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by caco-2 cells. J. Agric. Food Chem. 58, 4894–4900. Pascoe, S., Premier, R., 2000. Fluorescent pseudomonads-contributors to rots and browning in lettuce. Practical Hydroponics and Greenhouses, pp. 36–39 (Nov./Dec.). Pauwelyn, E., Huang, C.J., Ongena, M., Leclère, V., Jacques, P., Bleyaert, P., Budzikiewicz, H., Schäfer, M., Höfte, M., 2013. New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation. Mol. Plant-Microbe Interact. 26, 585–598. Pauwelyn, E., Vanhouteghem, K., Cottyn, B., De Vos, P., Maes, M., Bleyaert, P., Höfte, M., 2011. Epidemiology of Pseudomonas cichorii, the case of lettuce midrib rot. J. Phytopathol. 159, 298–305. Peng, X.X., Yamauchi, M., 1993. Ethylene production in rice bronzing leaves induced by ferrous iron. Plant Soil 149, 227–234. Pinto, L., Ippolito, A., Baruzzi, F., 2015. Control of spoiler Pseudomonas spp. on fresh cut vegetables by neutral electrolyzed water. Food Microbiol. 50, 102–108. Poverenov, E., Danino, S., Horev, B., Granit, R., Vinokur, Y., Rodov, V., 2014. Layer-by-layer electrostatic deposition of edible coating on fresh cut fruit model: anticipated and unexpected effects of alginate–chitosan combination. Food Bioprocess Technol. 7, 1424–1432. Quintieri, L., Caputo, L., Monaci, L., Deserio, D., Morea, M., Baruzzi, F., 2012. Antimicrobial efficacy of pepsin-digested bovine lactoferrin on spoilage bacteria contaminating traditional mozzarella cheese. Food Microbiol. 31, 64–71. Quintieri, L., Caputo, L., Morea, M., Baruzzi, F., 2013a. Control of mozzarella spoilage bacteria by using bovine lactoferrin pepsin-digested hydrolysate. In: Méndez-Vilas, A. (Ed.), Worldwide Research Efforts in the Fighting Against Microbial Pathogens: From Basic Research to Technological Developments. BrownWalker Press, Boca Raton, USA, pp. 118–122. Quintieri, L., Pistillo, B.R., Caputo, L., Favia, P., Baruzzi, F., 2013b. Bovine lactoferrin and lactoferricin on plasma-deposited coating against spoilage Pseudomonas spp. Innovative Food Sci. Emerg. Technol. 20, 215–222. Raaijmakers, J.M., de Bruijn, I., de Kock, M.J., 2006. Cyclic lipopeptide production by plantassociated pseudomonas spp.: diversity, activity, biosynthesis, and regulation. Mol. Plant-Microbe Interact. 19, 699–710. Randazzo, C.L., Scifò, G.O., Tomaselli, F., Caggia, C., 2009. Polyphasic characterization of bacterial community in fresh cut salads. Int. J. Food Microbiol. 128, 484–490. Ricelli, A., Baruzzi, F., Solfrizzo, M., Morea, M., Fanizzi, F.P., 2007. Biotransformation of patulin by Gluconobacter oxydans. Appl. Environ. Microbiol. 73, 785–792. Roller, S., Seedhar, P., 2002. Carvacrol and cinnamic acid inhibit microbial growth in freshcut melon and kiwifruit at 4° and 8 °C. Lett. Appl. Microbiol. 35, 390–394. Rudi, K., Flateland, S.L., Hanssen, J.F., Bengtsson, G., Nissen, H., 2002. Development and evaluation of a 16S ribosomal DNA array-based approach for describing complex microbial communities in ready-to-eat vegetable salads packed in a modified atmosphere. Appl. Environ. Microbiol. 3, 1146–1156. Schägger, H., 2006. Tricine-SDS-PAGE. Nat. Protoc. 1, 16–22. Santos, M.I.S., Martins, S.R., Pedroso, L., Sousa, I., Ferreira, M.A.S.S., 2015. Potential bio-activity of whey fermented extract as sanitizer of organic grown lettuce. Food Control 50, 477–481.

186

B. Federico et al. / International Journal of Food Microbiology 215 (2015) 179–186

Sisto, A., Baruzzi, F., Palumbo, G., Morea, M., 2002. Differentiation of Pseudomonas syringae subsp. savastanoi strains isolated from various host plants by restriction fragment length polymorphism. Phytopathol. Mediterr. 41, 63–71. Sokal, R.R., Rohlf, F.J., 1995. Biometry: the principles and practice of statistics in biological research. In SokalBiometry: The Principles and Practice of Statistics in Biological Research. WH. Freeman& Co., San Francisco. Tomita, M., Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K., 1991. Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J. Dairy Sci. 74, 4137–4142. van der Kraan, M.I., Groenink, J., Nazmi, K., Veerman, E.C., Bolscher, J.G., Nieuwamerongen, A.V., 2004. Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides 25, 177–183.

Wakabayashi, H., Takase, M., Tomita, M., 2003. Lactoferricin derived from milk protein lactoferrin. Curr. Pharm. Des. 9, 1277–1287. Wang, J., Shi, X.-G., Wang, H.-Y., Xia, X.-M., Wang, K.-Y., 2012. Effects of esterified lactoferrin and lactoferrin on control of postharvest blue mold of apple fruit and their possible mechanisms of action. J. Agric. Food Chem. 60, 6432–6438. Wiesburg, G.W., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703.