Inactivation of Escherichia coli O157:H7, Listeria ...

Food Microbiology 27 (2010) 24–28

Contents lists available at ScienceDirect

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

Inactivation of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves by X-rayq Barakat S.M. Mahmoud a, *, Gary Bachman a, Richard H. Linton b a b

Coastal Research & Extension Center, Mississippi State University, 3411 Frederic St., Pascagoula, MS 39567, USA Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, IN 47907-2009, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 May 2009 Received in revised form 30 June 2009 Accepted 12 July 2009 Available online 17 July 2009

Several recent foodborne disease outbreaks associated with leafy green vegetables, including spinach, have been reported. X-ray is a non-thermal technology that has shown promise for reducing pathogenic and spoilage bacteria on spinach leaves. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves using X-ray at different doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) was studied. The effect of X-ray on color quality and microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated spinach was also determined. A mixture of three strains of each tested organism was spot inoculated (100 ml) onto the surface of spinach leaves (approximately 8–9 log ml1), separately, and air-dried, followed by treatment with X-ray at 22 C and 55–60% relative humidity. Surviving bacterial populations on spinach leaves were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). More than a 5 log CFU reduction/leaf was achieved with 2.0 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on spinach leaves and inherent levels were significantly (p < 0.05) lower than the control sample throughout refrigerated storage for 30 days. Treatment with X-ray did not significantly affect the color of spinach leaves, even when the maximum dose (2.0 kGy) was used. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Inactivation E. coli O157:H7 L. monocytogenes Foodborne pahogens S. enterica Shigella flexneri Spinach leaves X-ray

1. Introduction Fruits and vegetables are an essential part of healthy eating. The WHO, FAO and USDA recommend that consumers include more fruits and vegetables in their diet (five servings or more a day) in an effort to decrease the risk of cardiovascular disease and cancer (Allende et al., 2006). As a result, the US consumption of fresh fruit and vegetables increased from 83.1 and 164.1 pounds per capita in 1996 to 100.4 and 220.2 pounds per capita in 2003, for fruits and vegetables, respectively (Cook, 2004). As produce consumption has increased, a significant increase in the number of foodborne disease outbreaks and illnesses, associated with fresh produce, has also been reported (Beuchat and Brackett, 1990; Brackett, 1999; Lang et al., 2004; Lee et al., 2004; Corbo et al., 2006). During the time period of 1992–2004, fresh spinach consumption increased by 180% (from less than 1 lb/capita to almost 2.5 lb/capita) (USDA-ERS,

q This paper is journal article of the Mississippi Agriculture and Forestry Experiment Station. * Corresponding author. Tel.: þ1 228 762 7783x304. E-mail address: [email protected] (B.S.M. Mahmoud). 0740-0020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2009.07.004

2006). The impact of the 2006 bagged spinach outbreak was recognized with a marked decrease in consumption of spinach and related products. From August 24, 2006 to February 24, 2007, the consumption of bagged spinach decreased by 43%, bagged salad containing spinach decreased by 42%, and bagged salad not containing spinach decreased by 8% (FMI, 2007). Microbial contamination has been associated with many sources including irrigation water, animal manure and by food handler contact (Wei et al., 1995; Cherry, 1999; FDA, 2001; Lee et al., 2004; Martinez-Sanchez et al., 2006). In the 1990’s, the Centers for Disease Control and Prevention (CDC) estimated that at least 12% of foodborne-outbreak-associated illnesses were linked to fresh produce items (FDA, 2004). Many recent bacteria-related outbreaks have been associated with leafy green vegetables, including contamination by Escherichia coli O157:H7, Salmonella spp., and Shigella flexneri (FDA, 2001). In the 2006 spinach outbreak, 204 people became sick, over 100 were hospitalized, 31 had hemolytic uremic syndrome (HUS) disease and 3 deaths were reported after eating contaminated green leaf spinach with E. coli O157:H7 (FDA, 2007). Special attention and research related to microbial interventions have been dedicated to the microbial safety and quality of fresh produce. Washing with chlorinated water (up to 200 ppm) is

B.S.M. Mahmoud et al. / Food Microbiology 27 (2010) 24–28

routinely applied to reduce microbial contamination in fresh produce (Pirovani et al., 2001; Mermelstein, 1998). However, washing with chlorinated water can only achieve a 1–2 log CFU reduction (Zhang and Farber, 1996; Cherry, 1999). Moreover, chlorine reacts with organic materials and can produce harmful byproducts such as chloramines and trihalomethanes (Richardson et al., 1998; Zhang and Farber, 1996). Other aqueous sanitation treatments that use hydrogen peroxide, peroxyacetic acid, trisodium phosphate, ozone, chlorine dioxide, and their combinations have also been studied. However, for most industrial applications, these treatments have been found to be minimally effective at reducing pathogens (less than a 3 log CFU reduction) on produce surfaces (Sapers and Sites, 2003; Zhang and Farber, 1996; Lukasik et al., 2003; Cherry, 1999). Chlorine dioxide gas has reported to have a good antimicrobial effect against pathogenic bacteria on fresh produce (Mahmoud et al., 2007, 2008 and Mahmoud and Linton, 2008), but, it also has some negative quality-related impacts (i.e. discoloration of leafy green vegetables) (Mahmoud and Linton, 2008). More effective sanitation technologies and more applicable industry-based technologies are needed for the food industry to decrease contamination that lead to safer and higher quality fruit and vegetable products. Ionizing radiation has the potential to ensure the safety and quality of leafy green vegetables (Han et al., 2004; Niemira and Fan, 2006). X-ray is a novel technology for produce decontamination, which has not yet been studied in much detail. In August 2008, FDA allowed using ionizing irradiation for leafy green vegetables namely, iceberg lettuce and spinach. Our previous studies demonstrated that X-ray sanitation technology can result in very high microbial efficacy (>6 log reduction) for pathogens on various food products. On oysters, more than a 6 log reduction of Vibrio vulnificus and Vibrio parahaemolyticus was achieved after treatment with 3–5 kGy X-ray (Mahmoud and Burrage, 2009; Mahmoud, 2009a). More than a 6 log reduction of E. coli O157:H7, Salmonella enteric, S. flexneri and V. parahaemolyticus was achieved on shrimp after treatment with 2–3 kGy X-ray (Mahmoud, 2009b). Greater than a 7.0 log reduction of Eenterobacter sakazakii were observed with 4.0, 5.0, 6.0, 6.0 and 6.0 kGy X-ray in the tryptic soy broth, skim milk, 1% fat milk, 2% fat milk and 3.5% fat milk (Mahmoud, 2009c). The main objectives of this study were to: (a) investigate the reduction of E. coli O157:H7, Listeria monocytogenes, Salmonella enterica and S. flexneri on spinach leaves (to achieve a 5 log reduction which is recommended by FDA) using X-ray treatments, and, (b) study the effect of X-ray on produce quality (visual color and inherent microflora counts) of spinach leaves during storage at 4 C for 30 days. 2. Material and methods 2.1. Spinach (Spinacia oleracea L.) Spinach (Spinacia oleracea L.) leaves (Fresh Express Inc., Salinas, CA) were purchased at a local supermarket (Pascagoula, MS) the day before the experiment and stored at 4 C until use. Fresh unblemished (from the same lot with the same expiration date) leaves, all leaves showing decay, cuts, or bruises were removed, were selected. 2.2. Bacterial strains and growing conditions Four different bacteria were used including: (1) A 3-strain mixture of E. coli O157:H7 (C7927, EDL933 and 204P), (2) A 3-strain mixture of L. monocytogenes (Scott A, F5069 and LCDC 81-861), (3)

25

A 3-serotype mixture of S. enterica (S. Enteritidis, S. Javiana and S. Montevideo) and (4) A 2-strain mixture of S. flexneri (ATCC 9199 and ATCC 12022). These strains were selected based on their prevalence in vegetable and fruits and for their resistance to antimicrobial treatments. All bacterial strains were obtained from ATCC or from our personal culture collection. Bacterial strains were grown in tryptic soy broth with 0.6% yeast extract (TSBYE, Difco, Becton Dickinson) and inoculated at 37 C for 24 h prior to use. Three or two strains of each bacterium were mixed with an equal volume to give approximately 108–9 CFU ml1. 2.3. Inoculation of spinach leaves A spot-inoculation method was used to inoculate the pathogenic bacteria on spinach leaves (Mahmoud et al., 2007). Briefly, 100 ml of each mixture (a suspension in TSBYE) culture was spotted (10 drops) onto the surface of spinach leaf. Afterwards, the spinach leaves were air-dried at 22 C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. 2.4. Description of RS 2400 radiator and generation of X-ray Specific irradiation doses (0.1, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) were generated using the RS 2400 industrial cabinet X-ray irradiator (Rad Source Technologies, 2007) according to Mahmoud (2009a). Briefly, at higher currents (mA), more electrons leave the filament. The electrons gather energy, equal to the potential difference; the higher the potential difference, the more energy the electrons gather. When the electrons reach the gold target plated inside the inner tube, they interact with the gold atoms and emit photons called X-rays. The X-ray doses in the treatment chamber were determined using a dosimeter (Rad Sources Technology. Inc., GA). 2.5. Treatment of inoculated spinach leaves with X-ray Inoculated spinach leaves were placed in sterile plastic bags [one leaf in each bag; two bags for each treatment (each treatment was repeated three times)] inside the exposure chamber (samples were placed in same distance around the generator X-ray tube). Samples were treated with 0.1, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy X-ray [The delivered doses were confirmed by placing the dosimeter (RadCal 9010 w/0.6 cc chamber; Radcal Co., Monrovia, CA) at the same place and same conditions, as treated samples, inside the Xray chamber] at 22 C and 55–60% relative humidity. At each examined dose, samples were pulled from the exposure chamber for microbial enumeration to determine surviving cell populations. 2.6. Microbial enumeration Two controls were used, 1) uninoculated and untreated control, to determine background microflora and pathogen levels (if they were present), and 2) inoculated and untreated control for comparisons with inoculated-treated samples. Both controls, as well as the inoculated and treated spinach samples, were mixed with 100 ml of 0.1% sterilized peptone water in a sterile 200 ml stomaching bag (Fisher Scientific, Pittsburgh, NJ) and homogenized for 2 min using a Stomacher 80 Lab-blender (Stomacher 400, Seward, London, UK). Serial 10-fold dilutions were prepared in 0.1% peptone water (Difco – Becton Dickinson, Sparks, MD, USA). Surviving bacterial populations on spinach leaves were evaluated using a nonselective medium (tryptic soy agar) for 6 h with the appropriate selective medium overlay for each bacteria; cefixmetellurite sorbital MacConkey (CT-SMAC) (Difco, Becton Dickinson) agar for E. coli O157:H7, modified oxford agar (MOA; using DIFCO Modified Oxford Antimicrobic Supplement) for L. monocytogenes

26


Table 1 Standardized color chart for spinach leaves. Color intensity

Red Green Blue

Scale 1

2

3

4

5

6

7

8

9

10

84 200 93

84 190 93

84 180 93

84 170 93

84 160 93

84 150 93

84 140 93

84 130 93

84 120 93

84 110 93

Where 1 is the lightest and 10 is the darkest, with 5 and 6 being the optimal colors for fresh spinach leaves.

and xylose lysine desoxycholate (XLD) (Difco, Becton Dickinson) for S. enterica and S. flexneri. Plates were then incubated for an additional 18 h at 37 C (according to Mahmoud et al., 2008). Colonies were counted and the results expressed as log CFU/spinach leaf. 2.7. D-value determination A first-order kinetic model (linear model) was used to analyze the data for log of surviving organisms per treatment dose (Mahmoud et al., 2007). The D-values (X-ray dose required for a 90% reduction) were determined using survival data for 0, 0.1, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy X-ray during treatment. D-values analyses were performed using Microsoft Excel (Microsoft Windows XP). 2.8. Effect of X-ray on the quality (visual color and microflora counts) of spinach leaves

0.00 0.10 0.20 0.30 0.50 0.75 1.00 1.50 2.00

a a b c d e e

0

A

f

g 2

4

6

2.9. Statistical analysis All experiments were replicated three times using two spinach samples per experiment for a total of six data points per treatment. Data were pooled and the mean values and standard deviations were determined. Differences between samples were determined using a Student’s t-test, with Microsoft Excel (Microsoft Windows XP), and were considered to be significant when p < 0.05. 3. Results and discussion 3.1. Inactivation of E. coli O157:H7, L. monocytogenes, S. enterica and Shigella flexneri The inactivation of inoculated of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri on spinach leaves by 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5, and 2.0 kGy X-ray at 22 C and 55–60% relative humidity is shown in Fig. 1A–D, respectively. The inactivation effect of X-ray against tested pathogens increased with increasing X-ray

X-ray doses (kGy)

X-ray doses (kGy)

Fresh spinach leaves (25 g) were used for this quality and microflora counts study. The untreated (control) leaves and the leaves treated with the lowest (0.1 kGy) and highest (2.0 kGy) X-ray were packaged separately into plastic clamshell containers (Monte Package Company, Riverside, MI, USA), wrapped in PVC film (AEP industries Inc. South Hackensack, NJ, USA), and stored at 4 C for 30 days. Samples were withdrawn from the refrigerator at 0, 3, 6, 9, 12, 20 and 30 days, and several microbial-related parameters and color measurements were evaluated. The microbiological analyses for mesophilic bacteria, psychrotrophic bacteria and for yeast and mold were also examined,

according to Mahmoud et al. (2007). For each determination, 25 g samples of spinach leaves were homogenized for 2 min using a Stomacher 80 Lab-blender (Stomacher 400, Seward, London, UK) with 225 ml of sterilized 0.1% peptone water (Difco Laboratories, Sparks, MD). Serial dilutions (101–106) were prepared from the homogenate with 0.1% sterilized peptone water. For mesophilic counts, 0.1/1.0 ml of each dilution was plated onto TSA and incubated at 37 C for 24 h. For psychrotrophic counts, 0.1/1.0 ml of each dilution was plated onto TSA and incubated at 5 C for 10 days. For yeast and mold counts, 0.1/1.0 ml of each dilution was plated onto acidified potato dextrose agar (PDA) (Difco – Becton Dickinson, Sparks, MD, USA) and incubated at 25 C for 5 days. Viable counts were expressed as log CFU g1. The color was measured visually (by four judges; two men and two women) using a standardized color chart for spinach leaves, including numerical values from 1 to 10 where 1 is the lightest and 10 is the darkest, with 5 and 6 being the optimal colors for fresh spinach leaves (Table 1), according to Mahmoud and Linton, 2008.

0.00 0.10 0.20 0.30 0.50 0.75 1.00 1.50

c c d e

2

X-ray doses (kGy)

X-ray doses (kGy)

a b b c d e

C

f g

h 0

2

4

6

Log (CFU/leaf)

4

6

8

10

Log (CFU/leaf)

Log (CFU/leaf) 0.00 0.10 0.20 0.30 0.50 0.75 1.00 1.50 2.00

B

f g 0

10

8

a b

8

10

0.00 0.10 0.20 0.30 0.50 0.75 1.00 1.50

a b c d e f

D

g h 0

2

4

6

8

10

Log (CFU/leaf)

Fig. 1. Inactivation of inoculated E. coli O157:H7 (1A), L. monocytogenes (1B), S. enterica (1C), and S. flexneri (1D) on spinach leaves by X-ray. Different lower case letters for each treatment are significantly different (P < 0.5).


27

Table 2 Changes in the mesophilic bacterial counts (Log CFU g1) of treated spinach leaves with X-ray during storage at 4 C for 30 days.

Table 4 Changes in the yeast and mold counts (Log CFU g1) of treated spinach leaves with X-ray during storage at 4 C for 30 days.

Storage time (days)

Storage time (days)

Treatments

0 3 6 9 12 20 30

4.2 4.5 6.3 6.8 7.3 7.7 8.2

Treatments Control

0 3 6 9 12 20 30

4.6 4.9 5.1 5.7 6.1 7.0 7.5

0.01a 0.05a 0.23a 0.05a 0.28a 0.66a 0.11a

0.1 kGy 3.3 3.5 3.9 4.3 4.8 5.3 5.7

0.01b 0.15b 0.20b 0.60b 0.37b 0.11b 0.15b

2.0 kGy NDc NDc NDc NDc NDc 1.2 0.34c 2.5 0.49c

Control

0.15a 0.05a 0.05a 0.10a 0.17a 0.25a 0.23a

0.1 kGy 3.4 3.7 4.2 4.9 5.2 5.6 6.7

0.05b 0.15b 0.10b 0.30b 0.20b 0.20b 0.26b

2.0 kGy NDc NDc NDc 1.0 1.6 1.8 2.9

0.00b 0.06b 0.20c 0.34c

Mean values with different letters in same row are significantly different (p < 0.05). ND ¼ no detectable survivors (less than 1.0 log CFU g1).


doses, as expected. Approximately 0.2, 0.9, 0.6 and 1.2 log CFU reduction of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri per spinach leaf were achieved by treatment with 0.1 kGy X-ray, respectively. While, 3.5, 5.4, 3.4 and 5.2 log CFU reduction per spinach leaf were achieved by treatment with 1.0 kGy X-ray, respectively. Furthermore, the populations of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri were reduced to less than the detectable limit (2 log CFU/spinach leaf) by treatment with 2.0, 1.5, 2.0 and 1.5 X-ray, respectively. The calculated D-values (kGy) of inoculated E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri on spinach leaves were 1.1, 1.0, 1.2 and 0.96 kGy X-ray, respectively. Our results are similar to those reported by Gomes et al. (2008). In their study using electron beam irradiation, doses ranging from 0.9 to 1.2 kGy resulted in a 5 log CFU reduction of E. coli O157:H7 on baby spinach. Neal et al. (2008) found that treatment of inoculated baby spinach by 0.4 kGy e-beam radiation resulted in a reduction in population of E. coli O157:H7 and Salmonella by 3.7 and 3.4 log CFU reductions, respectively. While at 0.70 kGy, both pathogens were reduced by a 4 log CFU reduction. Lee et al. (2006), reported that 1.0 kGy of gamma radiation, resulted in a 4-log reduction of E. coli inoculated on spinach leaves. Salmonella was more resistant to X-ray treatments than other pathogens. Our results are in agreement with those obtained by (Goularte et al., 2004; Murano, 1996; Mahmoud et al., 2007). X-ray has shown a stronger inactivation effect on tested pathogens than other agents and technologies, on spinach leaves. Lee and Baek (2008) reported that treatment of spinach leaves with chlorine dioxide and sodium hypochlorite significantly decreased levels of E. coli O157:H7 by 2.6 and 1.1 log CFU g1, respectively. Pirovani et al. (2001) reported that treatment of spinach leaves with chlorinated water (125 ppm free chlorine) reduced Salmonella by a 1.4 log CFU reduction. Izumi (1999) found that reductions in population ranged from 0.4 to 2.3 log cycles, with the maximum reduction occurring on the surface of spinach leaves treated by rinsing with electrolyzed water (20 ppm available chlorine).

3.2. Effect of treatment with X-ray on the quality of spinach leaves The treatment with X-ray could negatively affect the quality of spinach leaves after treatment and during storage. To investigate this possibility, microflora counts and color of untreated and treated spinach leaves with X-ray (0.1 and 2.0 kGy) were evaluated during storage at 4 C and 90% RH, for 30 days (the shelf life of spinach at 4 C is approximately two weeks, however, in this investigation the shelf life study was extended to 30 days as the inherent microbial counts for treated samples were very low until the end of the storage period). Treatment with 0.1 KGy was significantly (p < 0.05) reduced the initial populations of mesophilic bacteria, psychrotrophic bacteria, and yeast and mold counts on spinach leaves from 4.6, 4.8, and 4.2 log CFU g1 to 3.3, 3.4 and 3.4 log CFU g1, respectively. Meanwhile, treatment with 2.0 kGy significantly (p < 0.05) reduced the initial populations of mesophilic bacteria, psychrotrophic bacteria, and yeast and mold to less than the detectable limit (1 log CFU g1), as shown in Tables 2–4, respectively. During storage, the microflora on spinach leaves increased gradually for untreated and treated samples; however, treated samples maintained microbial populations significantly at a lower level compared to the untreated control. Treatment with 2.0 kGy X-ray maintained the population of mesophilic, psychrotrophic, and yeast and mold under the detectable limit (1.0 log CFU g1) for 12, 6, and 6 days storage, respectively. These results are in agreement with those obtained by Babic et al. (1996). No significant (p > 0.05) influences on the color of spinach leaves were observed by the judges, after treatment with X-ray and during storage (Table 5). These results are in agreement with those obtained by Gomes et al. (2008) who reported that the sensory characteristics (color) of spinach leaves were not affected by ionizing radiation up to 2 kGy. Also, X-ray has been reported to be effective at inactivating spoilage and pathogenic bacteria with no negative impact on the quality of ready to eat vacuum-packaged mullet (Robertson et al., 2006).

Table 3 Changes in the psychrotrophic bacteria counts (Log CFU g1) of treated spinach leaves with X-ray during storage at 4 C for 30 days.

Table 5 Changes in the visual quality (color) of treated spinach leaves with X-ray during storage at 4 C for 30 days.

Storage time (days)

Storage time (days)

Treatments

0 3 6 9 12 20 30

6.3 5.4 5.1 5.3 5.7 5.2 5.7

Treatments Control

0 3 6 9 12 20 30

4.8 5.0 6.2 6.4 6.8 7.6 8.1

0.05a 0.15a 0.11a 0.06a 0.05a 0.35a 0.20a

0.1 kGy 3.4 3.7 4.3 4.9 5.3 5.7 6.4

0.11b 0.15b 0.70b 0.36b 0.15b 0.20b 0.60a

2.0 kGy NDc NDc NDc 1.6 1.8 1.9 2.3

0.55b 0.17b 0.15c 0.64a


Control

1.09a 1.08a 1.27a 0.45a 1.17a 1.04a 0.47a

0.1 kGy 6.0 5.3 5.2 5.4 5.7 5.0 5.1

0.94a 0.47a 0.99a 0.79a 1.33a 0.96a 0.84a

2.0 kGy 6.4 5.1 5.1 5.1 5.3 5.3 5.2

0.90a 0.15a 1.07a 0.59a 0.99a 0.95a 0.87a

No significant differences (p > 0.05) between samples (in the same raw) were detected (p > 0.05).

28


In conclusion, this is the first report that describes inactivation of inoculated E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri on spinach leaves by X-ray. This study indicated that, the X-ray treatment was effective against the tested pathogenic bacteria and inherent microflora on spinach leaves and did not adversely affect color. X-ray treatment which could be a good alternative to other preservative techniques that are currently being used by the produce industry and, the use of this technology warrants further study. References Allende, A., Tomas-Barberan, F.A., Gil, M.I., 2006. Minimal processing for healthy traditional foods. Trends Food Sci. Technol. 17, 513–519. Babic, I., Royb, S., Watada, A.E., Werginb, W.P., 1996. Changes in microbial populations on fresh cut spinach. Int. J. Food Microbiol. 31, 107–119. Beuchat, L.R., Brackett, R.E., 1990. Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. J. Food Sci. 55, 755–758. Brackett, R., 1999. Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biol. Technol. 15, 305–311. Cherry, J.P., 1999. Improving the safety of fresh produce with antimicrobials. Food Technol. 53, 54–57. Cook, R., 2004. Us per capita fruit vegetable consumption 2003. http://postharvest. ucdavis.edu/datastorefiles/234-66.pdf. Corbo, M.R., Del Nobile, M.A., Sinigaglia, M., 2006. A novel approach for calculating shelf life of minimally processed vegetables. Int. J. Food Microbiol. 106, 69–73. FDA, 2001. Analysis and evaluation of preventive control measures for the control and reduction/elimination of microbial hazards on fresh and fresh-cut produce: chapter IV. Outbreaks associated with fresh and fresh-cut produce: incidence, growth, and survival of pathogens in fresh and fresh-cut produce. http://www. cfsan.fda.gov/wcomm/ift3-4a.html. FDA, 2004. Produce safety from production to consumption: 2004 action plan to minimize foodborne illness associated with fresh produce consumption. http:// www.cfsan.fda.gov/_dms/prodpla2.html. FDA, 2007. FDA finalizes report on 2006 spinach outbreak. http://www.fda.gov/bbs/ topics/NEWS/2007/NEW01593.html. Food Marketing Institute (FMI), 2007. Perishables group: facts, figures and the future. http://www.factsfiguresfuture.com/archive/april_2007.htm. Gomes, G., Moreira, R.G., Castell-Perez, M.E., Kim, J., Da-Silva, P., Castillo, A., 2008. EBeam irradiation of bagged, ready-to-eat spinach leaves (Spinacea oleracea): an Engineering approach. J. Food Sci. 73, E75–E102. Goularte, L., Martins, C., Morales-Aizpurua, I., Destro, M., Franco, B., Vizeu, D., Hutzler, B., Landgraf, M., 2004. Combination of minimal processing and irradiation to improve the microbiological safety of lettuce (Lactuca sativa, L.). Radiat. Phys. Chem. 71, 157–161. Han, J., Gomes-Feitosa, C.L., Castell-Perez, M.E., Moreira, R.G., Silva, P.F., 2004. Quality of packaged romaine lettuce hearts exposed to low-dose electron beam irradiation. Lebensm-Wiss Technol. 37, 705–715. Izumi, H., 1999. Electrolyzed water as a disinfectant for fresh-cut vegetables. J. Food Sci. 64, 536–539. Lang, M.M., Harris, L.J., Beuchat, L.R., 2004. Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water. J. Food Prot. 67, 1092–1103. Lee, S.Y., Baek, S.Y., 2008. Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach. Food Microbiol. 25, 582–587.

Lee, S.Y., Michael, C., Dong-Hyun, K., 2004. Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves. J. Food Prot. 67, 1371–1376. Lee, N., Jo, C., Shin, D., Kim, W., Byun, M., 2006. Effect of irradiation on pathogens inoculated into ready-to-use vegetables. Food Microbiol. 23, 649–656. Lukasik, J., Bradley, M.L., Scott, T.M., Dea, M., Koo, A., Wei, Yea-Hsu, Bartz, J.A., Farrah, S.R., 2003. Reduction of poliovirus 1, bacteriophages, Salmonella Montevideo, and Escherichia coli O157:H7 on strawberries by physical and disinfectant washes. J. Food Prot. 66, 188–193. Mahmoud, B.S.M., 2009a. Reduction of vibrio vulnificus in pure culture, half shell and whole live oysters (Crassostrea virginica) by X-ray. Int. J. Food Microbiol. 130, 135–139. Mahmoud, B.S.M., 2009b. Effect of X-ray on Inoculated E. coli O157:H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus on ready-to-eat Shrimp. Food Microbiol. 26, 860–864. Mahmoud, B.S.M., 2009c. Inactivation effect of X-ray treatments on E nterobacter sakazakii in pure culture, skim milk, low -fat milk and whole-fat milk. Lett. Appl. Microbiol. 49, 562–567. Mahmoud, B.S.M., Burrage, D.D., 2009. Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray. Lett. Appl. Microbiol. 48, 572–578. Mahmoud, B.S.M., Bhagat, A.R., Linton, R.H., 2007. Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica on strawberries by chlorine dioxide gas. Food Microbiol. 24, 736–744. Mahmoud, B.S.M., Vaidya, N.A., Corvalan, C.M., Linton, R.H., 2008. Inactivation kinetics of inoculated Escherichia coli O157:H7, Listeria monocytogenes and Salmonella ponna on whole cantaloupe by chlorine dioxide gas. Food Microbiol. 25, 857–865. Mahmoud, B.S.M., Linton, R.H., 2008. Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas. Food Microbiol. 25, 244–252. Martinez-Sanchez, A., Allende, A., Bennett, R.N., Ferreres, F., Gil, M.I., 2006. Microbial, nutritional and sensory quality of rocket leaves as affected by different sanitizers. Postharvest Biol. Technol. 42, 86–97. Mermelstein, N.H., 1998. Minimal processing of produce. Food Technol. 52, 84–86. Murano, E., 1996. Food Irradiation: a Sourcebook. Iowa State University Press, Ames. Neal, J., Carera-Diaz, E., Lez, M.R.G., Maxim, J., Castillo, A., 2008. Reduction of Escherichia coli O157:H7 and Salmonella on baby spinach, using electron beam radiation. J. Food Prot. 71, 2415–2420. Niemira, B.A., Fan, X., 2006. Low-dose irradiation of fresh and fresh-cut produce: safety, sensory and shelf life. In: Sommers, C.H., Fan, X. (Eds.), Food Irradiation Research and Technology. Iowa: Blackwell Publishing and the Institute of Food Technologists, Ames, pp. 169–181. Pirovani, M.E., Guemes, D.R., Di Pentima, J.H., Tessi, M.A., 2001. Survival of Salmonella hadar after washing disinfection of minimally processed spinach. Lett. Appl. Microbiol. 31, 143–148. Richardson, S.D., Thruston, A.D., Caughran, T.V., Collete, T.W., Patterson, K.S., Lynkins, B.W., 1998. Chemical by-products of chlorine and alternative disinfectants. Food Technol. 52, 58–61. Robertson, C.B., Andrews, L.S., Marshall, D.L., Coggins, P., Schilling, M.W., Martin, R.E., Collette, R., 2006. Effect of X-Ray irradiation on reducing the risk of Listeriosis in ready-to-eat vacuum-packaged Smoked mullet. J. Food Prot. 69, 1561–1564. Sapers, G.M., Sites, J.E., 2003. Efficacy of 1% hydrogen peroxide wash in decontaminating apples and cantaloupe melons. J. Food Sci. 68, 1793–1797. USDA-ERS, 2006. Vegetables and melons Situation and Outlook Yearbook. United States Department of Agriculture-Economic Research Service, pp. 191. Wei, C.I., Huang, T.S., Kim, J.M., Lin, W.F., Tamplin, M.L., Bartz, J.A., 1995. Growth and survival of Salmonella montevideo on tomatoes and disinfection with chlorinated water. J. Food Prot. 58, 829. 836. Zhang, S., Farber, J.M., 1996. The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiol. 13, 311–321.