Escherichia coli and Cronobacter sakazakii in ‘Tommy Atkins’ minimally processed mangos: Survival, growth and effect of UV-C and electrolyzed water

Food Microbiology 70 (2018) 49e54

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Escherichia coli and Cronobacter sakazakii in ‘Tommy Atkins’ minimally processed mangos: Survival, growth and effect of UV-C and electrolyzed water
lia Quintas a, * David Santo a, Ana Graça a, Carla Nunes b, Ce a

Universidade do Algarve, Instituto Superior de Engenharia, Campus da Penha and Centre for Mediterranean Bioresources and Food Campus de Gambelas, 8005-139, Faro, Portugal Agro-On, Centro Empresarial Gambelas, Pav. F-16, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2017 Received in revised form 9 August 2017 Accepted 11 September 2017 Available online 13 September 2017

These studies were aimed at assessing the growing capacity of Escherichia coli and Cronobacter sakazakii and the effectiveness of Ultraviolet-C (UV-C) radiation, acidic electrolyzed (AEW) and neutral electrolyzed (NEW) waters in the inhibition of these bacteria on minimally processed ‘Tommy Atkins’ mangoes (MPM). The fruits were contaminated by dip inoculation and kept 10 days at 4, 8, 12 and 20  C while enumerating bacteria. Contaminated mangoes were disinfected using UV-C (2.5, 5, 7.5 and 10 kJ/m2), AEW, NEW and sodium hypochlorite (SH) and the microorganisms were monitored. None of the enterobacteria grew at 4, 8 and 12  C regardless of having persisted during the 10-day period. At 20  C, E. coli and C. sakazakii grew, after adaption phases of 48 h and 24 h, to values of 8.7 and 8.5 log cfu/g at day eight, respectively. E. coli showed the highest reduction counts on the MPM washed with NEW and SH (2.2 log cfu/g). UV-C was more effective in reducing C. sakazakii (2.4e2.6 log cfu/g), when compared to AEW, NEW and SH (1.2e1.8 log cfu/g). The efficacy of decontamination technologies depends on microorganisms, highlighting the importance of preventing contamination at the primary production and of combining different methods to increase the safety of fresh-cut fruits. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Mango Escherichia coli Cronobacter sakazakii Ultraviolet Electrolyzed water

1. Introduction According to FAO (2016), the worldwide production of the most popular tropical fruit (mango, pineapple, avocado and papaya) is expected to grow by 3.0% annually over the next decade, which will result in about 116 million tons by 2024 compared with the 84 million tons attained in the period of 2012e2014. The total production of mangoes, which is the largest, followed by pineapple, papaya and avocado, is estimated to increase to 58 million tons by 2024, increasing at an annual rate of 2.8% over the next decade. The top ten mango producers will continue to contribute to about 80 percent of the world's supply, with Asia (60%), Africa (14%) and Latin America (25%), being the biggest producers. The increase in mango production resulted from the consumers' growing demand, driven by the excellent organoleptic characteristic of this fruit, namely the color, texture, succulence and unique

* Corresponding author. E-mail address: [email protected] (C. Quintas). http://dx.doi.org/10.1016/j.fm.2017.09.008 0740-0020/© 2017 Elsevier Ltd. All rights reserved.

flavor as well as its nutritional properties (Siddiq et al., 2013; Oliveira et al., 2016). The fruits are either used in the food industry for the fabrication of canned fruit, jams and concentrated pulps or may be eaten fresh or after minimal processing (fresh-cut fruits). Due to the fact that the production and post-harvest processing steps are different among the producers, and carried out based on various safety principles, the microbial quality of these fruits is a concern (Strawn et al., 2011; Penteado et al., 2004, 2014; Penteado, 2017). Human microbial pathogens can contaminate food of plant origin during the different phases of production: in the fields, during harvesting, processing, distribution, marketing and preparation for consumption. For example, in a multistate outbreak resultant from the consumption of cantaloupe, in the USA, the contamination was attributed to deficient agricultural practices and hygienic conditions (CDC, 2012). Regarding post-harvest procedures, Penteado et al. (2004) showed that ‘Tommy Atkins’ mangoes, can become contaminated with Salmonella enterica in case the washing water used during the hot treatment is contaminated. High temperatures of the washing/rinsing waters may contribute to the internalization of pathogens eventually present in

50

D. Santo et al. / Food Microbiology 70 (2018) 49e54

the water (Penteado, 2017). Thus, the microbial quality of waters used in the post-harvest phases is important. This procedure is often used by some producers/packers to eliminate flies' larvae. In all the steps, incorrect human handling is a significant source of contamination (Abadias et al., 2008a; Beuchat, 2002; Graça et al., 2015, 2017a). Salmonellosis is amongst the most common cases of outbreaks in the United States due to fresh produce consumption, of which some have been associated to mango contaminated with Salmonella Newport, in 1999 (Sivapalasingam et al., 2004) and S. Saintpaul in 2001, in the USA (Beatty et al., 2004), and Salmonella Braenderup in 2012 (CDC, 2012) in the USA and Canada. The second most relevant etiologic agent of food borne diseases are the pathogenic strains of E. coli. E. coli 0157:H7 was the casual agents of outbreaks resulting from the ingestion of other fruits such as Cantaloupe and pineapple (Sivapalasingam et al., 2004). In 2006 an outbreak caused by E. coli 0157:H7 in spinach, in the United States and Canada was also described (Calvin, 2007). Additionally, other members of the Enterobacteriaceae family may occur naturally on plant material, namely the genera Pantoea, Klebsiella, Pectobacterium (Leff and Fierer, 2013) and Cronobacter (Garbowska et al., 2015; Kucerova et al., 2011; Schmid et al., 2009; Vojkovska et al., 2016), which have been isolated from fruits and/or vegetables. Although naturally occurring bacteria could be epiphytic, their presence in fresh produce may be involved in spoilage processes, but also a possible threat for human infections (Kim and Beuchat, 2005). In the case of C. sakazakii, an emergent pathogen for neonates and immunocompromised adults, it has been isolated from various plant food origin, though not associated to outbreaks resulting from the consumption of fruits. However, C. sakazakii showed a significant growth in fresh-cut ‘Royal gala’ apple, ‘Rocha’ pear, and ‘Piel de sapo’ melon, at 12 and 20  C (Santo et al., 2016). Previous studies have reported that pathogenic enterobacteria can survive and grow in mango. Strawn and Danyluk (2010) describe that five different serovars of S. enterica were able to grow on ‘Tommy Atkins’ mangoes at 23  C and 12  C, as opposed to E. coli O157:H7 that could not grow. Ma et al. (2016), presented results showing that the sevorars of S. enterica tested on the Palmer cultivar were not able to grow at 28  C and 4  C. However, S. enterica Enteritidis was able to grow in pasteurized Palmer mango pulp, at 25  C, after an adaptation phase of 19 days (Penteado et al., 2014). In general, data on the behavior of human pathogens on tropical fruits remain limited, though they are necessary to increase the consumers' confidence and satisfaction and evaluate the potential microbial risks associated to them. Additionally, in the flowchart of minimally processed fruit production, a phase of disinfection is mandatory, and washing with solutions of sodium hypochlorite is the most common method. However, this method has been attributed to various drawbacks, namely its safety and the conse
n ~ ez quences of its application for man and environment (Castro-Iba et al., 2016; Pezzuto et al., 2016; Meireles et al., 2016; Ramos et al., 2013). Electrolyzed water (acid electrolyzed water and neutral electrolyzed water) and short wave Ultraviolet-C (UV-C) illumination (Bintsis et al., 2000; Huang et al., 2008; Ramos et al., 2013) are decontamination technologies that can be applied to fresh vegetables and fruits as alternatives to sodium hypochlorite solutions. The efficacy of electrolyzed water has been demonstrated on utensils and surfaces (Rahman et al., 2016) and various food products of plant origin, such as fresh-cut apple (Graça et al., 2011), blueberries (Kim and Hung, 2012), fresh-cut pear (Graça et al.,
ndez et al., 2015), cilantro (Hao 2017b), broccoli (Martínez-Herna et al., 2015), lettuce, carrot and endive (Abadias et al., 2008b) and tomatoes and lettuce (Pangloli and Hung, 2011). The efficacy of

electrolyzed water was also observed in animal origin food products such as pork (Rahman et al., 2013), chicken, and beef as well as fish (Al-Holy and Rasco, 2015). On the other hand, UV-C was also used as a substitution for sodium hypochlorite solutions in order to decrease microbial populations and/or lengthen shelf-life of apples ~o-da(Graça et al., 2013), apricots (Yun et al., 2013), kiwifruit (Beira Costa et al., 2014), mango and pineapple (George et al., 2015), melon (Manzocco et al., 2011), pear (Graça et al., 2017b) and
s-Herna
ndez et al., 2010). watermelon (Arte Therefore, the objective of the present work was to study the survival and growth of Escherichia coli and Cronobacter sakazakii on fresh-cut mango of the cultivar ‘Tommy Atkins’ at different temperatures. Additionally, the effect of the UV-C irradiation, acidic electrolyzed (AEW) and neutral electrolyzed (NEW) waters on the populations of the two Enterobacteriaceae species on contaminated fresh-cut mango was studied. 2. Methods 2.1. Mango preparation Imported ‘Tommy Atkins’ (cv) mangoes (Mangifera indica) were bought in local supermarkets and prepared on the day the study began. Fruits were washed in running tap water and disinfected by immersion and scrubbing with a sodium hypochlorite solution (0.5%) during 30 s. After drying at room temperature, mangoes were aseptically peeled and, with a sterile cork borer, cut in pieces of 1 g each (1 cm long and radius 0.6 cm) without the kernel and skin and used in the microbial growth experiments. Using a disinfected knife, 10 g of these unpeeled pieces were prepared to test the physical and chemical decontaminations. 2.2. Enterobacteria and inocula preparation The microorganisms used in this study were Escherichia coli (the non-toxicogenic strain of E. coli O157:H7 NCTC 12900, E. coli ATCC 25922 and E. coli ATCC 10536) and Cronobacter sakazakii [a strain isolated from a baby, ATCC BAA 894 and a strain isolated from soil, 4E (Universidade do Algarve) (Santo et al., 2016)], prepared in a mixture (cocktail). The enterobacteria were stored at 80  C and maintained on Tryptone Soy Agar (TSA) (Oxoid, Hampshire, UK) at 4 ± 1  C. To contaminate the fruit, the bacteria were grown on TSA at 37 ± 1  C for 24 ± 2 h, following a growth in 50 mL of Tryptic Soy Broth (TSB) (Biokar Diagnostics, Allonne, France) at 37 ± 1  C in an orbital shaker (VWR, Incubating Mini Shaker, USA) at 150 rpm, during 24 h. The bacterial cells were recovered by centrifugation (15 min at 9016 g) (Heraeus, Multifuge 1 L-R, Germany) originating a pellet that was resuspended in 50 mL of sterile saline peptone [8.5 g/L NaCl (Panreac, Barcelona, Spain) and 1 g/L peptone (Biokar)] to prepare suspensions with 107 cfu/mL which were used as fresh-cut mango inocula. The adjustments of the bacterial counts were made using a standard curve, measuring the transmittance at 420 nm in a spectrophotometer (Spectrophotometer UVeVis, 175 Shimadzu-UV160, USA). The bacterial concentration of the inocula were checked using the colony count method described by Miles and Misra (1938), inoculating droplets (20 mL) of ten-fold dilutions on the surface of the TSA medium, in triplicate, following an incubation of the plates during 24 ± 2 h at 37 ± 1  C. 2.3. Growth of E. coli and C. sakazakii on fresh-cut mangoes The mango portions (1 g) were contaminated by “dip inoculation” through immersion in 107 cfu/mL suspensions of E. coli and C. sakazakii separately during 3 min at 150 rpm in an orbital shaker. Next, the mango pieces were dried in a laminar flow hood (Bioquell,

D. Santo et al. / Food Microbiology 70 (2018) 49e54

Microflow, UK) during 30 min, packed in biaxially-oriented polypropylene (BOPP) (0.030 mm thick) bags and stored at 4 ± 0.5  C, 8 ± 0.5  C, 12 ± 0.5  C and 20 ± 0.5  C, during 10 days. The enterobacteria existing on the minimally processed mango were counted individually at days 0, 1, 2, 3, 6, 8 and 10 after the storage. The contaminated mango portions were diluted in 9 mL of sterile saline peptone, stomached during 2 min in a Stomacher (Model 400 Circulator, Seward, Norfolk, England). Fruit suspensions were then serially diluted in saline peptone and aliquots of 20 mL were surface plated in triplicate, using the Miles and Misra method (Miles and Misra, 1938), on Sorbitol MacConkey agar (Biokar Diagnostics) to count the number of E. coli and on DFI agar (Oxoid) to enumerate C. sakazakii. Following an incubation at 37 ± 1  C for 24 ± 2 h, the counts were expressed as colony forming units (cfu) per gram of mango. In each sampling point, four replications were made and the assays were repeated twice. 2.4. Mango disinfection treatments 2.4.1. Contamination of fresh-cut mango The contamination of fresh-cut mango portions (10 g) was done, submerging the fruit in the enterobacteria suspensions containing 107 cfu/mL, separately, in an orbital shaker (150 rpm) during 3 min following a 30 min air drying in a laminar flow hood. Then, the contaminated mango portions were allocated in 9 lots. Four of the lots were treated with UV-C at various doses (2.5, 5, 7.5 and 10 kJ/ m2). The other 4 lots were washed in 500 mL of acidic electrolyzed water (AEW), neutral electrolyzed water (NEW), sodium hypochlorite (SH) or distilled water (DW) during 5 min in an orbital shaker (150 rpm). After the disinfection treatments the mango pieces were drained and washed with DW during 3 min in an orbital shaker (150 rpm). The chemical decontaminated lots were air dried for 30 min in a laminar flow hood. Inoculated and untreated mango portions were used as control. After each treatment, the enterobacteria were counted in aliquots of decimal dilutions of the treated mangoes, previously stomached, on Sorbitol MacConkey agar (E. coli) and on DFI agar (C. sakazakii) using the Miles and Misra inoculation technique (Miles and Misra, 1938) as described previously (Graça et al., 2013). 2.4.2. UV-C The UV-C treatments of mango were done in an UV-C chamber described in Graça et al. (2013). In summary, the UV-C cabinet (100 cm  100 cm x 50 cm) possesses two groups of five unfiltered germicidal lamps (Philips, TUV 25W G25 T8 Longlife) on the top and bottom of the chamber, a ventilation system in the back and a sample tray placed equidistantly between the UV-C lamps, where the samples are placed to be irradiated. The mango samples were treated with the irradiation doses of 2.5, 5, 7.5 and 10 kJ/m2 which were reached by modifying the exposure time keeping intensity of the light constant. A radiometer (UVX Radiometer, UVP.Inc, California, USA) placed at the same distance as the samples (15 cm) was used to measure the intensity of the lamps (calculated as a mean of 20 measurements done in various places of the sample net). The UV-C lamps were turned on 30 min prior to treatments. Throughout the treatments there were no temperature changes were not registered. 2.4.3. Electrolyzed water The acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW) used to disinfect the mango pieces were produced using an electrolyzed water (EW) apparatus (Envirolyte EL-400, Envirolyte Industries International Ltd., Estonia). AEW and NEW were produced in the apparatus with a saturated sodium chloride solution and a current of 20e23 A. These waters were collected in

51

bottles and refrigerated (4  C) until use for up to a day. When ready to be used for disinfection, the free chlorine concentration was corrected to 100 mg/L with distilled water immediately before treatments and the pH as well as oxidation reduction potential (ORP) were measured. ORP and pH were measured with a pHmeter (Model GLP-21, Crison, Spain), using an ORP electrode (Crison 52e61) and a pH electrode (Crison 52-02), respectively. Free chlorine content were measured with a free and total chlorine photometer (HANNA Instruments, model HI9133, Woonsocket, RI, USA). The AEW and NEW used in the present study had the characteristics of pH, ORP and free chlorine described in Table 1. 2.5. Statistical analysis The decrease of the Enterobacteriaceae counts on minimally processed mango were calculated by deducting the population of contaminated non disinfected mango pieces from the bacteria numbers enumerated after the disinfection and incubation in the same conditions. The mean values obtained resulted from 2 different assays, comprising 4 replicates per treatment in each assay. Analysis of variance and Duncan's multiple range tests using SPSS v.22 software (SPSS Inc., USA) were used to treat the data obtained. Significant differences in enterobacteria declining were established by the least significant difference at the 0.05 level of significance. 3. Results and discussion The survival and growth of E. coli (Fig. 1A) and C. sakazakii (Fig. 1B) inoculated on fresh-cut ‘Tommy Atkins’ mangoes, at different temperatures (4, 8, 12 and 20  C) during a period of ten days are represented in Fig. 1. At 4  C, 8  C and 12  C the population of both enterobacteria decreased during the first or second day and then remained almost unchanged until the end of the assay. At 20  C, E. coli population inoculated at 5.4 log cfu/g declined during a period of two days, increasing on the third day by about 2.3 log cfu/ g before a stationary phase followed until the end of the assays where the population attained 7.6 log cfu/g. Strawn and Danyluk (2010) observed a similar behavior of E. coli O157:H7 on cut mangos and papayas at 23  C. However, at 12  C, E. coli, as observed in the present work, was not able to grow on mangoes but on papayas. The same authors reported that E. coli did not grow on the fruits at 4  C but were able to survive during 28 days. An exponential growth of E. coli O157:H7O at 20  C and 25  C on minimally processed peaches (various cultivars) (Alegre et al., 2010b), apples ‘Golden delicious’ (Alegre et al., 2010a), melon (Abadias et al., 2012) and fresh-cut orange (Salazar et al., 2015) was also reported. However, at 10  C this bacterium was able to grow at lower specific growth rates in peaches (Alegre et al., 2010b), apples (Alegre et al., 2010a), and in oranges after 10 day adaptation phase (Salazar et al., 2015) while at 5  C, no growth was observed. On ‘Rocha’ pear (Graça et al., 2017b) exponential growth was observed at 20, 10 and 8  C. However, on pineapple at 25  C and 5  C E. coli O157:H7 was not able to multiply (Abadias et al., 2012). In regards to C. sakazakii, the population inoculated at 4.6 log cfu/g decreased during the first day until 3.2 log cfu/g and then grew exponentially during two days reaching a population of 7.2 Table 1 Physicochemical properties of the AEW and NEW tested.

AEW NEW SH

pH

ORP

Free chlorine

2.87 ± 0.05 7.95 ± 0.06 6.8 ± 0.02

1113 ± 2 mV 757 ± 4 mV 597 ± 2 mV

102 ± 3 mg/L 101 ± 2 mg/L 101 ± 2 mg/L

52

D. Santo et al. / Food Microbiology 70 (2018) 49e54

10

A

Number of E. coli (Log CFU/g)

9 8 7 6 5 4 3 2 1 0 0

2

4

6

8

10

12

Time (day)

B

Number of C. sakazakii (Log UFC/g)

10 9 8 7 6 5 4

3 2 1 0 0

2

4

6

8

10

12

Time (day) Fig. 1. Growth of inoculated E. coli (A) and C. sakazakii (B) on fresh-cut mango kept at 4  C, 8  C, 12  C and 20  C, during 10 day. Values are the means of 2 experiments with 4 replicates each and bars indicate standard deviation (C4  C; , 8  C; D12  C; X 20  C).

log cfu/g, corresponding to an increase of 4 log cfu/g. Next, a stationary phase occurred followed by a decreasing of the bacterial population during the last day of the assay, when counts of 5.3 log cfu/g were enumerated. Kim and Beuchat (2005) studied the growth of C. sakazakii in fresh-cut apple (Red delicious cv), cantaloupe, strawberries, watermelon, cabbage, carrots, cucumbers, iceberg lettuce and tomatoes, reporting that at 25  C only the strawberries did not allow the growth of C. sakazakii. At 12  C, growth occurred in all the samples with the exception of strawberries, cabbage and carrots and at 4  C the number of C. sakazakii remained unchanged or gradually decreased. Santo et al. (2016) described that C. sakazakii was able to grow in fresh-cut apple (Royal gala, cv), pear (Rocha, cv) and melon (Piel de sapo, cv) at 8, 12 and 20  C, contrary to what happened in mango in which there was only growth at 20  C after an adaptation phase of almost 24 h. The results described in the present work show that fresh-cut ‘Tommy Atkins’ mango allows the growth of both Enterobacteriaceae at 20  C but not at 4  C, 8  C or 12  C, during the period studied. Penteado et al. (2014) also described that S. enterica was not able to growth in mango pulp at 4  C being only able to grow at

25  C. On the other hand, Ma et al. (2016) reported that S. enterica did not grow in mango at 4  C and at 28  C a poor growth was detected. The inability of E. coli and C. sakazakii to grow at 4  C is certainly related to the combination of lowest temperature for growth, which is inferior to 5  C in both species, and the characteristics/composition of the fruit (for example pH or others). Nevertheless, the inhibition of the enterobacteria in mango, held at 8  C and 12  C, may result from the physicochemical characteristics of mango matrix, such as its pH and composition. According to Ma et al. (2016), mango extracts inhibit the growth of S. enterica. Additionally, Engels et al. (2011) and Asif et al. (2016) in their recent review report that different parts of mango have anti-microbial activity. For example, mango gallotannins showed antimicrobial activities against Staphylococcus aureus, L. monocytogenes, E. coli and S. enterica (Engels et al., 2011). Furthermore, Ma et al. (2016) showed that the growth of S. enterica was limited in the presence of growing concentration of mango extracts, being almost completely inhibited when the media was constituted by 100% mango extract. Moreover, temperatures away from the optimum value can certainly potentiate the inhibitory effect of the antimicrobial mango constituents, that is, the low (infra-optimum) and high (supra-optimum) temperatures for growth may enhance the antimicrobial effect of the components of the mango fruit. The decontamination effect of UV-C irradiation (2.5, 5, 7.5 and 10 kJ/m2), electrolyzed acidic water (AEW) and neutral electrolyzed acidic water (NEW) (101 mg/L of free chlorine), on minimally processed mango previously contaminated with single cultures of E. coli and C. sakazakii is shown in Fig. 2. The antimicrobial activity obtained was compared with mango pieces disinfected with SH solutions (101 mg/L of free chlorine) and distilled water (DW). The application of the mentioned methods resulted in declining of E. coli and C. sakazakii populations. In the case of E. coli, the application of the physical method at different doses resulted in reductions of 1.49e1.96 log cfu/g, which were inferior to the decreases obtained with chemical methods. The application of the chemical methods resulted in significant higher microbial reductions of E. coli such as 2.19 and 2.17 log cfu/g obtained after the immersion in NEW and SH, respectively, where no statistical differences were found between them (p > 0.05). With regards to C. sakazakii, the highest reductions were achieved when the physical treatments were used at various doses: UV5 (2.72 log cfu/ g), UV7.5 (2.41 log cfu/g) and UV10 doses (2.70 log cfu/g) and no statistical differences among these values were detected (p > 0.05). The lowest bacterial decrease of 2.09 log cfu/g was caused by the lowest UV-C dose applied (2.5 kJ/m2) (Fig. 2). Concerning the utilization of the chemical treatments, the counts reductions of C. sakazakii population resulted from the washings with AEW (1.76 log cfu/g) and NEW (1.70 log cfu/g) were higher than those caused by the utilization of SH (1.20 log cfu/g), although all were inferior to the reductions attained with the physical methods of decontamination. DW washing was the treatment that resulted in the lowest reduction values of E. coli and C. sakazakii on the fresh-cut mangoes. The results obtained in the present study, regarding the effectiveness of disinfection methods in decreasing the populations of E. coli, are different from what has been obtained by other authors. The highest effectiveness of UV-C radiation, when compared with chemical treatments in the inactivation of E. coli and S. enterica, was observed on apples (Graça et al., 2013) and Rocha pear (Graça et al., 2017b) and of E. coli O157:H7 and different serotypes of S. enterica in apricots (Yun et al., 2013). Yaun et al. (2004) used UV-C radiation to inactivate the population of E. coli and S. enterica on lettuce, tomato and apple surfaces and observed that the UV-C was more effective against these microorganisms than SH (20e320 ppm). Kim and Hung (2012) also reported that UV-C treatments were more effective than EW inactivating E. coli O157:H7 in blueberries.

D. Santo et al. / Food Microbiology 70 (2018) 49e54

53

Enterobacteria reducƟon (Log CFU/g)

3.5 3 e

2.5 2 1.5

e d,e

d c,d

e d

c,d b

c

c

e c

1

b

0.5

a a

0 UV2.5

UV5

UV7.5

UV10

AEW

NEW

SH

DW

DecontaminaƟon methods Escherichia coli

Cronobacter sakazakii

Fig. 2. Reduction of E. coli (dark grey bars) and C. sakazakii (light grey bars) after treating mango portions with UV-C irradiation, acidic electrolyzed water (AEW), neutral electrolyzed water (NEW), sodium hypochlorite (SH) (100 mg/L of free chlorine) and with distilled water (DW). For each pathogen, columns with different letters indicate significant differences between treatments using Duncan multiple range test (P < 0.05%). Values are the means of 2 experiments with 4 replicates each and bars correspond to standard errors.

However, in mango, a contrary tendency was observed, as previously described, as in that fruit E. coli was less sensitive to UV-C than to chemical treatments. In the case of C. sakazakii (Santo et al., 2016), the effectiveness of UV-C radiation at 7.5 and 10 kJ/ m2 dosages was higher than AEW, NEW and SH in reducing the population of that bacteria on fresh-cut apple, pear and melon, a tendency that was also observed in mango. C. sakazakii produces a polysaccharide capsule and a yellow pigment (most strains), whose production is temperature dependent, that allows the bacterium to protect itself from some stressful conditions, namely to the disinfectants (Kucerova et al., 2011). Additionally, results obtained by Yan et al. (2016) suggest that different E. coli strains have different sensitivities to UV-C. The Enterobacteriaceae, used in the present study have certainly different strategies/capacities to protect themselves from the antimicrobial effects of both physical and chemical methods of decontamination, which were, certainly, dependent of the constituents and texture of the food matrix. 4. Conclusion E. coli and C. sakazakii were not able to grow on fresh-cut ‘Tommy Atkins’ mangoes held at temperatures of 4  C, 8  C and 12  C. However, growth was observed at 20  C with E. coli and C. sakazakii being able to increase exponentially their population in less than 72 h and 48 h, respectively. Although both species' behavior have a similar pattern of growth on mango, they were differently affected by the decontamination methods tested, with E. coli being more sensitive to the chemical methods and C. sakazakii to the physical method tested. The use of UV-C (5, 7.5 and 10 kJ/m2) resulted in C. sakazakii reductions higher than 2 log cfu/g, while AEW, NEW and SH resulted in inferior reductions. The contrary was observed for E. coli, whose population was more efficiently reduced, by 2 log cfu/g, when AEW, NEW and SH were used, rather than when the physical method was applied. The results highlight the importance of considering the differential survival strategies of the microorganisms to protect themselves as well as the importance of food matrixes when studying and testing different decontamination technologies. In addition, good agricultural and manufacturing practices and efficient refrigeration chains should never be neglected.

Acknowledgements ^ncia e Tecnologia This study was funded by Fundaç~ ao para a Cie (FCT), which belongs to the Ministry of Education and Science from Portugal, through the project no PTDC-PTDC/AGR-ALI/111687/ 2009. A. Graça was supported by a PhD grant from FCT (SFRH/BD/ 76745/2011). References ~ as, I., 2008a. Microbiological Abadias, M., Usall, J., Anguera, M., Solsona, C., Vin quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments. Int. J. Food Microbiol. 123, 121e129. ~ as, I., 2008b. Efficacy of neutral Abadias, M., Usall, J., Qliveira, M., Alegre, I., Vin electrolyzed water (NEW) for reducing microbial contamination on minimallyprocessed vegetables. Int. J. Food Microbiol. 123, 151e158. ~ as, I., 2012. Growth potential of Abadias, M., Alegre, I., Oliveira, M., Altisent, R., Vin Escherichia coli O157: H7 on fresh-cut fruits (melon and pineapple) and vegetables (carrot and escarole) stored under different conditions. Food Contr. 27, 37e44. ~ as, I., 2010a. Factors affecting Alegre, I., Abadias, M., Anguera, M., Oliveira, M., Vin growth of foodborne pathogens on minimally processed apples. Food Microbiol. 27, 70e76. ~ as, I., 2010b. Fate of Escherichia coli Alegre, I., Abadias, M., Anguera, M., Usall, J., Vin O157:H7, Salmonella and Listeria innocua on minimally-processed peaches under different storage conditions. Food Microbiol. 27, 862e868. Al-Holy, M.A., Rasco, B.A., 2015. The bactericidal activity of acidic electrolyzed water against Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes on raw fish, chicken and beef surfaces. Food Contr. 54, 317e321.
s-Herna
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