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Decontamination and survival of Enterobacteriaceae on shredded iceberg lettuce during storage
Food Microbiology 73 (2018) 129e136
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Decontamination and survival of Enterobacteriaceae on shredded iceberg lettuce during storage Tareq M. Osaili a, *, Akram R. Alaboudi b, Heba N. Al-Quran a, Anas A. Al-Nabulsi a a b
Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan Department of Veterinary Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, 22110, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 3 February 2018
Enterobacteriaceae family can contaminate fresh produce at any stage of production either at pre-harvest or post-harvest stages. The objectives of the current study were to i) identify Enterobacteriaceae species on iceberg lettuce, ii) compare the decontamination efficiency of water, sodium hypochlorite (free chlorine 200 ppm), peroxyacetic acid (PA 80 ppm; Kenocid 2100®) or their combinations and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce and iii) determine the survival of Enterobacteriaceae post-treatment storage of shredded iceberg lettuce at 4, 10 and 25 C, for up to 7 days. Klebsiella pneumonia spp. pneumonia, Enterobacter cloacae, Klebsiella oxytoca, Pantoea spp., Leclercia adecarboxylata and Kluyvera ascorbate were identified on iceberg lettuce. No significant difference (P 0.05) among Enterobacteriaceae survival after washing with water or sanitizing with sodium hypochlorite or Kenocid 2100® (reduction 0.6 log CFU/g) were found. Combined sanitizer treatments were more effective against Enterobacteriaceae than single washing/sanitizing treatments. Sanitization of iceberg lettuce with combined washing/sanitizing treatments reduced Enterobacteriaceae by 0.85e2.24 CFU/g. Post-treatment growth of Enterobacteriaceae during storage on samples sanitized with sodium hypochlorite and Kenocid 2100® was more than on samples washed with water. The D10-value of Enterobacteriaceae on shredded iceberg lettuce was 0.21 KGy. The reduction of Enterobacteriaceae populations on iceberg after gamma radiation (0.6 KGy) was 3 log CFU/g, however, Enterobacteriaceae counts increased post-irradiation storage by 4e5 log CFU/g. Therefore, washing shredded iceberg lettuce with combined sanitizing treatment (sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, or Kenocid 2100®/Kenocid 2100®) for total time of 6 min or exposing it to gamma irradiation (0.6 KGy) can decrease the risk of Enterobacteriaceae (reduction 2 log). Post-washing storage of sliced iceberg lettuce (4, 10, 25 C) could increase the risk of Enterobacteriaceae as their counts increased during storage even at low temperatures. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Enterobacteriaceae Decontamination Sodium hypochlorite Peroxyacetic acid Ionizing radiation
1. Introduction Consumption of fresh leafy green vegetables that are eaten raw or with minimal processing has increased because of healthy lifestyle patterns. Contamination of these products with microorganisms may be a health hazard to consumers and cause foodborne illness outbreaks. Leafy green vegetables may be contaminated with pathogenic and non-pathogenic bacteria at several points throughout the pre-harvest and post-harvest systems from irrigation water, feces, dust, soil, human handlings, storage facilities,
* Corresponding author. Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan. E-mail address: [email protected] (T.M. Osaili). https://doi.org/10.1016/j.fm.2018.01.022 0740-0020/© 2018 Elsevier Ltd. All rights reserved.
distribution systems, processing equipment and poor hygienic conditions (Beuchat, 1996). These bacteria can attach to the surface of leafy green vegetables and survive for extended periods time (Brandl and Amundson, 2008; Delaquis et al., 2007). Iceberg lettuce is one of the produce commodities most susceptible to contamination. Doyle and Erickson (2008) noted that part of the plant closest to the soil contained higher concentration of the microbes. Enterobacteriaceae is classified as large family of bacteria with common characteristics comprise of Gram-negative, rod-shaped, facultative anaerobic, non-spore forming, either capsulated or noncapsulated in addition of being motile or non-motile. Presence of pathogenic and antimicrobial resistant members of Enterobacteriaceae on fresh produce at retail constitute severe threats to consumers. Pathogenic or antimicrobial resistant members of
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Enterobacteriaceae have been isolated from green leafy vegetables (Al-Kharousi et al., 2016; Al-Holy et al., 2013; Hassan et al., 2011). Al-Holy et al. (2013) and Hassan et al. (2011) isolated Klebsiella pneumoniae, Enterobacter sp., Escherichia coli, Citrobacter sp., Acinetobacter, Shigella flexneri and other Enterobacteriaceae members that are resistant to antibiotics from green leafy vegetables sold in Saudi Arabia. Many methods have been used to decontaminate bacteria on fresh produce. Sodium hypochlorite (form of chlorine) has been widely used to reduce microbial contamination on fresh produce including lettuce. Escudero et al. (1999) reported that exposure of shredded lettuce to 100 and 300 ppm chlorine for 10 min reduced the populations of Yersinia enterocolitica 2 to 3 log CFU/g. However, other studies have found that chlorine (<200 ppm) are not effective at reducing microbial populations on lettuce (Li et al., 2001a; Taormina and Beuchat, 1999). Peroxyacetic acid (PA) has also been widely used to reduce microorganisms on fresh produce. Masson (1990) found that 90 ppm PA reduced total counts and fecal coliforms on cut salad mixtures by 2 log CFU/g. However, Davidson et al. (2013) showed that 50 ppm PA (Tsunami® 100), and 50 ppm of mixed peracid (Tsunami® 200) were not significantly more effective than water at reducing of E. coli O157:H7 on iceberg lettuce. Ionizing radiation maybe considered one of the most promising technology to improve the safety of fresh produce. In 2008, U.S. Food and Drug Administration (FDA) (2008) conferred its acceptance to use irradiation for killing pathogens on iceberg lettuce. Niemira (2007) compared between the effect of sodium hypochlorite wash and irradiation against E. coli O157:H7 internalized in romaine lettuce leaves. The author reported that the D10-value of E. coli O157:H7 in lettuce leaves was 0.39 KGy, while sodium hypochlorite (chlorine 300 and 600 ppm) resulted in <1 log reduction and water wash was ineffective. No studies were found in the literature on the effect of washing/ sanitizing solutions (water, sodium hypochlorite, Kenocid 2100® (PA) or their combination) and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce during post treatment storage. Thus, the objectives of the current study were to i) identify Enterobacteriaceae species found on iceberg lettuce, ii) compare the decontamination efficiency of washing/sanitizing solutions (water, sodium hypochlorite, Kenocid 2100® (PA) or their combination) and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce and iii) determine post-treatment survival of Enterobacteriaceae on shredded iceberg lettuce during storage at 4, 10 and 25 C for up to 7 days. 2. Materials and methods 2.1. Preparation of iceberg lettuce Iceberg heads samples were purchased from a local market, at the day of each experiment. Damaged leaves were removed and only intact leaves were used in the experiments. Iceberg leaves samples of each experiment were prepared from five iceberg heads. The leaves were shredded manually (ca 1 3 cm) by sanitized knife and cutting board. The shredded leaves were mixed manually in a sterile bag to be used in the experiments (25 g quantities). 2.2. Determination of aerobic plate count and Enterobacteriaceae count on iceberg samples Each sample (25g) was placed into sterile Stomacher bags (Seward, UK) under aseptic condition and combined with 225 ml quantities of peptone water (0.1%; Oxoid, UK), and then was homogenized in the Stomacher (AES-Chemunex, France) for 2 min.
The samples were serially diluted in 9 ml of sterile peptone water (0.1%) and volumes of 0.1 ml of suitable dilutions were plated in duplicate on Nutrient agar (Oxoid) to enumerate total plate counts and in Violet Red Bile Glucose agar (VRBGA, Oxoid) by pour plate method to enumerate Enterobacteriaceae. The plates were incubated aerobically at 37 C for 24 h. The typical Enterobacteriaceae colonies were counted expressed as log (CFU/g). 2.3. Identification of Enterobacteriaceae on the iceberg samples To reveal Enterobacteriaceae members on iceberg lettuce, thirty morphological different Enterobacteriaceae colonies grown on/in Violet Red Bile Glucose agar (VRBGA) from different shredded iceberg samples were identified using the VITEK 2 automated rieux S.A., Marcy L'Etoile, France). The ID-GNB card system (bioMe was used for Enterobacteriaceae identification. 2.4. Decontamination of iceberg lettuce 2.4.1. Washing/sanitation-single treatment In this method, water washing (sterile), sodium hypochlorite and Kenocid 2100® were used separately to sanitize the shredded iceberg samples. Commercial sodium hypochlorite (NaOCl) was purchased from the local market. Free chlorine concentration was determined using the method described by Willson (1935). Sodium hypochlorite solution was freshly prepared prior to each experiment by diluting 5 ml NaOCl (free chlorine 4%) with 1000 ml of 0.05M potassium phosphate buffer (pH 6.8 at 25 C) to achieve free chlorine concentration of 200 ppm (Lang et al., 2004). Kenocid 2100® (Ieper, Belgium), a peroxyacetic acid-based sanitizer, was obtained from the local market. It contains hydrogen peroxide (H2O2 20%), peracetic acid (PA 5%) and acetic acid (AA 10%). The solution was freshly prepared prior to each experiment to obtain PA concertation of 80 ppm. The solution was prepared by adding 1.6 ml of the Kenocid 2100® to 1000 ml sterile distilled water. Shredded iceberg leaves (25g) were placed into sterile Stomacher bag and 100 ml of distilled water (at room temperature, 21±1 C), sodium hypochlorite (chlorine 200 ppm) or Kenocid 2100® (PA 80 ppm) was added to the bag. After mixing thoroughly for 3 min, the solution was drained out from the bag and the leaves were dried by tissue paper to remove the remaining solution on the samples. 2.4.2. Washing/sanitation-combined treatment In this part of the study, combinations of treatments were used [(washing with water followed by washing with water), (washing with water followed by sanitizing with sodium hypochlorite), (washing with water followed by sanitizing with Kenocid 2100®), (sanitizing with sodium hypochlorite followed by sanitizing with sodium hypochlorite), (sanitizing with sodium hypochlorite followed by sanitizing with Kenocid 2100®) and (sanitizing with Kenocid 2100® followed by sanitizing with Kenocid 2100®)]. Shredded lettuce sample (25g) was firstly treated with the first solution (distilled water, sodium hypochlorite or Kenocid 2100®) for 3 min. Then, the solution was drained out from the bag and the shredded lettuce was kept at room temperature for 5 min. Consequently, the second solution was added to the bag and mixed with the shredded lettuce thoroughly for 3 min. After that, the solution was drained out from the bag and the shredded lettuce samples were dried by tissue paper without water rinse to shorten the washing/sanitizing process. 2.4.3. Ionizing radiation Quantities of 25 g of shredded lettuce was placed in sterile Stomacher bags, pressed by hand to remove air and sealed. The
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samples were transmitted to the research gamma unit (Gammafacility PX-g -30, Issledovatelj, Techsnabexport, Moskivia) at Jordan Atomic Energy Commission, Amman, Jordan by ice box. The instrument is spherically shaped and has a sample capacity of 4400 ml. The irradiation source is Co60; the current activity and dose rate of the source are 768.27Ci and 480.19 Gy/h, respectively. Dosimetry was performed using 2 ml ethylene chlorobenzene (ECB) dosimeters (Institute of Isotopes and Chemical Research Centre, Hungarian Academy of Sciences, Budapest, Hungary), and the dosimeter response was measured with oscillotitrator OK-302/ 1 (Radelkis, Budapest, Hungary). These dosimeters were calibrated against an international standard (RISØ, Roskilde, Denmark). The samples were exposed to irradiation doses of 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 KGy in order calculate D10-value of Enterobacteriaceae. Other samples prepared for storage study were exposed to irradiation dose of 0.6 KGy. 2.5. Storage of iceberg lettuce To determine the effect of storage on the survivals of Enterobacteriaceae on shredded iceberg lettuce post treatment (washing/ sanitation and ionizing radiation), the samples were kept in sterile bags at 4 and 10 C for 7 days and at 25 C for 4 days. Preliminary study showed that shredded iceberg lettuce deteriorated after 7 days of storage at 10 C and 4 days of storage at 25 C. 2.6. Microbiological analysis Shredded iceberg samples were analyzed to enumerate Enterobacteriaceae on the samples at 0, 1, 2, 3, 4, 5, 6 and 7 days of storage at 4 and 10 C and at 0, 1, 2, 3 and 4 days of storage at 25 C. The samples were placed into sterile Stomacher bags (Seward) under aseptic condition and combined with 225 ml quantities of peptone water (0.1%), and then were mixed in the Stomacher for 2 min. The samples were serially diluted by adding 1 mle9 ml of sterile peptone water (0.1%) and volumes of 0.1 ml of suitable dilutions were plated in duplicate in VRBGA by pour plate method. The plates were incubated aerobically at 37 C for 24 h. The typical colonies counted and the numbers were expressed as log (CFU/g). 2.7. Statistical analysis The statistical analysis was performed using the Statistical Package for Social Sciences software (SPSS, version 19, Chicago. Inc). The survivals of Enterobacteriaceae on shredded iceberg lettuce after treatments and during storage were compared. A P-value of < 0.05 was considered the level for statistical significance. One way ANOVA was used to examine the effect of treatments and storage time on the survivals of Enterobacteriaceae on shredded iceberg samples. Duncan post-hoc was conducted to determine the difference among variables groups. Data are expressed as mean and standard deviation. Each experiment was replicated three times. 3. Results 3.1. Total plate count and identification of Enterobacteriaceae on iceberg lettuce The total plate count and Enterobacteriaceae count on fresh iceberg lettuce samples were 7.03 ± 0.30 log CFU/g and 6.73 ± 0.26 log CFU/g, respectively. Enterobacteriaceae isolates (n ¼ 30) from iceberg lettuce samples were identified as Klebsiella pneumonia spp. pneumonia (13 isolates) that was mostly isolated from our samples followed by Enterobacter cloacae (11 isolates) and Klebsiella oxytoca (3 isolates), while Pantoea spp, Leclercia adecarboxylata and
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Kluyvera ascorbata were identified from only three samples. 3.2. Effect of washing/sanitation single treatments and storage on the populations of Enterobacteriaceae on shredded iceberg lettuce Fig. 1 shows the survival of Enterobacteriaceae on shredded iceberg lettuce after treatment with water, sodium hypochlorite or Kenocid 2100® for 3 min followed by storage for up to 7 days at 4, 10 and 4 days at 25 C. Water, sodium hypochlorite and Kenocid 2100® reduced the initial level of Enterobacteriaceae on the samples (6.73 log CFU/g) immediately (time 0) by 0.3, 0.6 and 0.5 log CFU/g, respectively. The differences among the survivals of Enterobacteriaceae on shredded iceberg lettuce after treatment immediately or during storage at 4 C were < 0.7 log CFU/g. Post-treatment storage (4 C) of iceberg lettuce did not change Enterobacteriaceae populations on the samples significantly regardless of the washing/ sanitizing solution. Although, there was a significant (P < 0.05) drop in the cell counts on the second day of storage, the populations increased significantly during the subsequent storage period to reach levels no significantly (P 0.05) different than the original levels. At storage temperature of 10 C, there were no significant differences (P 0.05) among the survivals of Enterobacteriaceae on the lettuce regardless of storage times. Enterobacteriaceae populations on samples washed with water did not change significantly during storage, while the populations on samples sanitized with Kenocid 2100® and sodium hypochlorite increased significantly (P < 0.05) after the third and fourth day of storage, respectively. At storage temperature of 25 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on the lettuce after 2, 3, and 4 days of storage; however, the differences were <0.7 log CFU/g. Enterobacteriaceae populations on the samples increased significantly (P < 0.05) after the first day of storage at 25 C regardless of the washing/sanitizing solutions. At the fourth day of storage, the populations on samples treated with water, sodium hypochlorite and Kenocid 2100® increased by 1.5, 2.2 and 2.4 log CFU/g, respectively. 3.3. Effect of washing/sanitation combined treatments and storage on the populations of Enterobacteriaceae on iceberg lettuce Combinations of the treatments reduced the initial level of Enterobacteriaceae on the shredded iceberg lettuce (6.73 log CFU/g) by 0.85, 1.65, 1.30, 2.24, 2.09 or 1.99 log CFU/g immediately after treatment with water/water, water/sodium hypochlorite, water/ Kenocid 2100®, sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, or Kenocid 2100®/Kenocid 2100® respectively, (Fig. 2). There were significant differences (P < 0.05) among the survivals on the samples after treatment immediately and after 1, 2 and 3 days of storage at 4 C. The survivals on the samples washed with water-included treatments were higher than the survivals on the samples sanitized with non-water-included solution. In addition, the survivals on samples sanitized with sodium hypochlorite-included treatment were lower than the survivals on samples sanitized with Kenocid 2100®-included treatment. On the fourth day until the end of the storage period, there were no significant differences (P 0.05) among the survivals on the samples regardless of treatments. Post-treatment storage of lettuce reduced Enterobacteriaceae populations on the samples non-significantly in the first two days of storage regardless of the treatment, but after that, the populations increased significantly (P < 0.05) during storage. At a storage temperature of 10 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on lettuce after treatments on day 1, 2, 3 and 5 of storage (Fig. 3). Similar to the results obtained from the samples stored at 4 C, during the
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ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
A 9 8 7 6 5 4 3 0
1
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7
STORAGE TIME (DAY) Water
Sodium hypochlorite
Kenocid 2100®
B ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
9 8 7 6 5 4 3 0
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STORAGE TIME (DAY) Water
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Kenocid 2100®
ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
C 9 8 7 6 5 4 3 0
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STORAGE TIME (DAY) Water
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Fig. 1. Survival of Enterobacteriaceae on iceberg lettuce after washing with water, sodium hypochlorite or Kenocid 2100® and storage at 4 C (a), 10 C (b) and 25 C (c) for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
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9 8 7 6 5 4 3 0
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STORAGE TIME (DAY) W/W
W/S
W/K
S/S
S/K
K/K
ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
Fig. 2. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 4 C for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
9 8 7 6 5 4 3 0
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STORAGE TIME (DAY) W/W
W/S
W/K
S/S
S/K
K/K
Fig. 3. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 10 C for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
first 3 days of storage, the survivals on the samples treated with water-included solutions were higher than the survivals on the samples treated with non-water-included solutions. Furthermore, the survivals on the samples sanitized with sodium hypochloriteincluded treatments were non-significantly lower than the survivals on the samples sanitized with Kenocid 2100®-included treatments. After the fifth day of storage, there were no significant differences (P 0.05) among counts on samples. Enterobacteriaceae on lettuce increased significantly (P < 0.05) at the third day of storage. At the end of storage period, the populations on samples increased by 0.9e2.5 log CFU/g. At storage temperature of 25 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on the lettuce after day 1 and 2 of storage (Fig. 4). The survivals on the samples washed with water alone were lower than the survivals on the samples treated with sanitizer-included solutions. The
populations on the samples increased significantly after 1 day of storage; the populations on the samples increased by 2.0e3.8 log CFU/g. 3.4. Effect of gamma radiation and storage on the populations of Enterobacteriaceae on shredded iceberg lettuce Irradiation reduced Enterobacteriaceae on lettuce (6.65 ± 0.06 log CFU/g) by 0.44, 1.10, 1.53, 2.14, 2.53 or 2.84 log CFU/g at 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 KGy, respectively. The D10-value of Enterobacteriaceae on shredded iceberg lettuce was 0.21 KGy (r ¼ 0.996). Fig. 5 shows that the level of Enterobacteriaceae on lettuce after irradiation was 3.70 ± 0.35 log CFU/g (3 log reduction). Enterobacteriaceae populations on the samples increased on the first day of storage and during the subsequent storage periods (P < 0.05). The survivals on samples stored at 25 C were higher than those on
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ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
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9 8 7 6 5 4 3 0
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7
STORAGE TIME (DAY) W/W
W/S
W/K
S/S
S/K
K/K
ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
Fig. 4. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 25 C for up to 4 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
9 8 7 6 5 4 3 0
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2
3
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7
STORAGE TIME (DAY)
Fig. 5. Growth of Enterobacteriaceae on iceberg lettuce after exposure to 0.6 KGy irradiation followed by storage for 7 days at 4 C and 10 C, and 4 days at 25 C.
samples stored at 4 or 10 C (P < 0.05). At the end of storage period, the populations on samples stored at 4, 10 and 25 C increased by 3.67, 4.29 and 5.17 log CFU/g, respectively. 4. Discussion The current study showed that iceberg samples were highly contaminated with Enterobacteriaceae family members, they were found at high levels on the surface of the samples. Our results showed that Enterobacteriaceae count of 6.73 log CFU/g on fresh iceberg lettuce samples was in the range of that reported in other studies for lettuce and other green vegetables (Al-Holy et al., 2013; €der et al., 2007). The attachment of Oliveira et al., 2010; Fro Enterobacteriaceae on the plant surfaces may occur by several
mechanisms including extracellular polymeric substances, cell surface hydrophobicity, bacterial surface charge, divalent cationic bridges and may depend on presence or absence of fimbriae (Hassan et al., 2011; Ukuku and Fett, 2002; Fratamico et al., 1996; Junkins and Doyle, 1992). In the current study, the identified Enterobacteriaceae genera and species on iceberg lettuce were similar to those isolated from iceberg lettuce and other green vegetables in other studies (Al-Holy et al., 2013; Johannessen et al., 2002). Predominantly, Klebsiella spp. and Enterobacter spp. have been isolated from soil, water, vegetation and intestinal contents of animals and humans. The link between these two bacterium and fresh produce was considered as an indicator for the contact between soil and iceberg (Doyle and Erickson, 2008; De Roever, 1998). E. coli and other enteric
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foodborne pathogens were not isolated from our samples; this suggests that iceberg lettuce was not contaminated by feces. Washing iceberg lettuce with sodium hypochlorite (free chlorine 200 ppm) for 3 min reduced Enterobacteriaceae by 0.6 log CFU/ g. Similar observation was reported by Takeuchi and Frank (2000) who found that washing lettuce with 200 ppm chlorine for 5 min reduced the E. coli O157:H7 populations between 0.7 and 1 log CFU/ g. Similarly, Garg et al. (1990) reported that dipping lettuce in chorine solution (100 ppm) for 5 min reduced E. coli population by l log CFU/g. However, Nascimento et al. (2003) reported that washing lettuce with chlorine (200 ppm) for 15 min reduced aerobic mesophilic and total coliform by 2.63 and 1.91 log CFU/g, respectively. These reductions might be explained by the metabolic inhibition of chlorine as it attaches the amino groups of the cell protein, prevents cell metabolism and then leads to cell death (Denyer and Stewart, 1998). Washing iceberg lettuce with Kenocid 2100® (80 ppm PA) for 3 min reduced Enterobacteriaceae by 0.5 log CFU/g. Other studies have reported higher effect of PA against bacteria than numbers reported in the present study. Hilgren and Sailverda (2000) showed that washing cabbage and celery with 80 ppm PA for 30 s reduced aerobic count by 0.84 and 1.07 log CFU/g, respectively. Ruiz-Cruz et al. (2007) showed that washing shredded carrots with 40 ppm PA for 2 min reduced Salmonella and E. coli O157:H7 populations by 2.1 and 1.24 log CFU/g, respectively. Furthermore, Nascimento et al. (2003) reported that 80 ppm AA (Tsunami 100®) for 15 min reduced aerobic mesophilic and total coliform on lettuce by 1.85 and 1.44 log CFU/g, respectively. PA is a very strong oxidizing agent, which can lead to bacterial cell membrane destabilization. The damage of bacterial membrane lipid and protein can cause loss of its solidity and its ability of attachment to the thio group of proteins which inhibits several essential enzymes necessary for protein synthesis (Denyer and Stewart, 1998). Sodium hypochlorite was more effective than water and slightly more effective than Kenocid 2100® in reducing the populations of Enterobacteriaceae from the surface of iceberg lettuce. Although Kenocid 2100® solution used in the current study contained H2O2, it was not more effective against Enterobacteriaceae than sodium hypochlorite. Unlike our findings, Alasri et al. (1993) found that addition of H2O2 to PA caused a synergistic antimicrobial effect with strong correlation between increases the concentration of H2O2 and microbial reduction. Strain variation, sanitizer concentration, exposure time, presence of soil and foreign materials on the surface of the vegetables, and type of fresh produce may explain the variation among the studies. Our results showed that combined treatments were more effective than single treatments. Washing iceberg lettuce with sodium hypochlorite two times (3 min each) was the most effective in Enterobacteriaceae decontamination compared to the other washing treatments; it reduced the population of Enterobacteriaceae up to 2.2 log CFU/g. This reduction could be explained by the increases in contact time between washing solution and attached bacteria on the surface of iceberg lettuce. Similar finding was reported by Pirovani et al. (2001) who found that increasing washing time of fresh-cut spinach with chlorine from 2 to 8 min increased the reduction in total microbial count from 2.8 to 3.5 log CFU/g. Vandekinderen et al. (2009) also reported that increasing washing time with PA from 1 to 10 min increased the reduction of microflora on of fresh-cut produce from 0.5 to 1.5 log CFU/g. Washing with water and sanitizers did not eliminate the entire bacteria on the surface of iceberg lettuce. This could be explained by the presence of Enterobacteriaceae cells in inaccessible sites in lettuce leaf such as pores, punctures and damage fraction that may prevent the direct contact between the washing solutions and attached bacteria (Burnett et al., 2000; Takeuchi and Frank, 2000;
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Seo and Frank, 1999). Seo and Frank (1999) found that E. coli O157:H7 can attach to the surface, trichomes, stomata, and cut edges of lettuce leaves. The pathogen gathered inside the stomata and attached preferentially to cut edges. Strong attachment of the bacteria to the tissue and formation of biofilms can also reduce the efficacy of the washing solution against the microorganisms (Doyle and Erickson, 2008). Enterobacteriaceae populations on sliced iceberg lettuce increased during post-washing storage at 4, 10 and 25 C. Slicing of iceberg lettuce released intracellular liquids, which may also enhanced growth of inaccessible bacteria that have been protected in small niches on the leaves. According to Wachtel et al. (2002) survival of E. coli O157:H7 on lettuce tissue from heads dropped 6 feet was higher than in undamaged tissue after storage at room temperature for 4 h and then stored at 4 C for 48 h. Similar to our findings, previous researchers reported that Enterobacteriaceae are able to grow at refrigeration temperatures. Li et al. (2001b) found that Enterobacteriaceae counts increased on chlorine-sanitized lettuce during storage at 5 or 15 C for 18 or 7 days, respectively. Prakash et al. (2000) reported that the populations of aerobic bacteria on diced celery increased through storage for 22 days at 5 C after washing with acidified solution (vinegar 5% AA), chlorinated solution (200 ppm) for 5 min and water. The increase was 6.2, 2.9 and 1.9 log CFU/g, respectively. Growth of Enterobacteriaceae during storage at 25 C was higher than 10 and 4 C. Decreasing the temperature (refrigerate temperature) minimizes microbial growth, though, psychrotrophic species are able to grow. Samples that were stored at 25 C deteriorated more rapidly than at 10 and 4 C. Qualitative changes occurred at high temperature may cause deleterious effect on fresh produce; this may enhance microbial growth (Allwood et al., 2004). The comparison among the washing solutions in our study showed that Enterobacteriaceae growth during storage on samples washed with Kenocid 2100® and with sodium hypochlorite was more than on samples washed with water. This could be explained by reduction of competitive background microflora (other than Enterobacteriaceae) that had been reduced by washing treatment. This inturn allows Enterobacteriaceae to grow under the specified storage condition (Carlin et al., 1996). Also, presence of inactive sanitizer residues on the leaves could be utilized by bacteria to support their growth. In addition, sanitizers may have caused damage to vegetable tissues; this may enhance microbial proliferation on vegetable too (Park and Lee, 1995). This study found that low irradiation dose (0.21 KGy) achieved 90% (1 log) reduction in the populations of Enterobacteriaceae on shredded iceberg lettuce. Similar findings were previously reported for members in the Enterobacteriaceae family. Niemira (2007) found that the D10-values of E. coli O157:H7 internalized in romaine lettuce leaves was 0.39 KGy. Martins et al. (2004) found that D10-values of Salmonella spp. on watercress ranged from 0.29 to 0.43 KGy. Treating iceberg lettuce with 0.6 KGy ionizing radiation reduced Enterobacteriaceae by ca 3 log CFU/g. Similar values were reported by Niemira et al. (2002) who found that irradiation dose of 0.5 KGy reduced E. coli O157:H7 populations by 3.6e3.8 log CFU/g on different types of lettuce. Irradiation sensitivity of microorganisms is affected by type of fresh produce, type of bacteria, and microbial attachment on the surface of the produce (Niemira et al., 2002). The increase in Enterobacteriaceae populations on sliced iceberg lettuce post-irradiation storage could be explained by the released of intracellular liquids which enhanced growth of survival bacteria on the leaves and by the ability of injured cells to recovery during storage. According to Prakash et al. (2000) the populations of aerobic bacteria on celery increased during storage at 5 C for 22 days after irradiation with 1.0 or 0.5 KGy. The use of 0.6 KGy in the current study is safe and did not change the texture and quality of
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iceberg lettuce samples. Niemira et al. (2002) found that irradiation dose up to 0.5 KGy did not change the quality of different types of lettuce. 4.1. Conclusion Enterobacteriaceae members are found on the surfaces of iceberg lettuce leaves at high levels. Klebsiella pneumonia and Enterobacter cloacae were mostly isolated from our samples. Enterobacteriaceae populations on sliced iceberg lettuce decreased < 1 log after washing with water, sodium hypochlorite, or Kenocid 2100® for 3 min but combined sanitizing treatments (sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, and Kenocid 2100®/Kenocid 2100®) for total time of 6 min decreased the populations by 2 log. During post-washing storage of sliced iceberg lettuce, Enterobacteriaceae counts increased (4 < 10 < 25 C), the populations were higher on samples treated with sanitizers. Gamma radiation improved the microbial safety and enhanced quality of leaves, however, Enterobacteriaceae counts increased at post-irradiation storage. Acknowledgements The authors thank the Deanship of Scientific Research at Jordan University of Science and Technology for funding this project (231/ 2015). References Alasri, A., Valverde, M., Roques, C., Michel, G., Cabassud, C., Aptel, P., 1993. Sporocidal properties of peracetic acid and hydrogen peroxide, alone and in combination, in comparison with chlorine and formaldehyde for ultrafiltration membrane disinfection. Can. J. Microbiol. 39, 52e60. Al-Holy, M., Osaili, T., El-Sayed, S., Alshammari, E., Ashankyty, I., 2013. Microbiological quality of leafy green vegetables sold in the local market of Saudi Arabia. Ital. J. Food Sci. 25, 446e452. Al-Kharousi, Z.S., Guizani, N., Al-Sadi, A.M., Al-Bulushi, I.M., Shaharoona, B., 2016. Hiding in fresh fruits and vegetables: opportunistic pathogens may cross geographical barriers. Int. J. Microbiol., 4292417 https://doi.org/10.1155/2016/ 4292417, 14 pages. Allwood, P.B., Malik, Y.S., Hedberg, C.W., Goyal, S.M., 2004. Effect of temperature and sanitizers on the survival of feline calicivirus, Escherichia coli, and F-specific coliphage MS2 on leafy salad vegetables. J. Food Protect. 67, 1451e1456. Beuchat, L.R., 1996. Pathogenic microorganisms associated with fresh produce. J. Food Protect. 59, 204e216. Brandl, M.T., Amundson, R., 2008. Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157: H7 and Salmonella enterica. Appl. Environ. Microbiol. 74, 2298e2306. Burnett, S.L., Chen, J., Beuchat, L.R., 2000. Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy. Appl. Environ. Microbiol. 66, 4679e4687. Carlin, F., Nguyen-the, C., Da Silva, A.A., Cochet, C., 1996. Effects of carbon dioxide on the fate of Listeria monocytogenes, of aerobic bacteria and on the development of spoilage in minimally processed fresh endive. Int. J. Food Microbiol. 32, 159e172. Davidson, G.R., Buchholz, A.L., Ryser, E.T., 2013. Efficacy of commercial produce sanitizers against nontoxigenic Escherichia coli O157: H7 during processing of iceberg lettuce in a pilot-scale leafy green processing line. J. Food Protect. 76, 1838e1845. De Roever, C., 1998. Microbiological safety evaluations and recommendations on fresh produce. Food Contr. 9, 321e347. Delaquis, P., Bach, S., Dinu, L.,D., 2007. Behavior of Escherichia coli O157: H7 in leafy vegetables. J. Food Protect. 70, 1966e1974. Denyer, S.,P., Stewart, G.S.A.B., 1998. Mechanisms of action of disinfectants. Int. Biodeterior. Biodegrad. 41, 261e268. Doyle, M.P., Erickson, M.C., 2008. Summer meeting 2007ethe problems with fresh produce: an overview. J. Appl. Microbiol. 105, 317e330. n, A.M.S., 1999. EffectiveEscudero, M.E., Vel azquez, L., Di Genaro, M.S., De Guzma ness of various disinfectants in the elimination of Yersinia enterocolitica on fresh lettuce. J. Food Protect. 62, 665e669. Food and Drug Administration (FDA), 2008. Code of Federal Regulations 21 CFR Part 179: irradiation in the production, processing and handling of food FDA-1999-F-
2405, 1999F-5522. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/ CFRSearch.cfm?fr¼179.26. (Accessed 1 March 2017). Fratamico, P.M., Schultz, F.J., Benedict, R.C., Buchanan, R.L., Cooke, P.H., 1996. Factors influencing attachment of Escherichia coli O157: H7 to beef tissues and removal using selected sanitizing rinses. J. Food Protect. 59, 453e459. €der, H., Martins, C.G., de Souza, K.L.O., Landgraf, M., Franco, B.D., Destro, M.T., Fro 2007. Minimally processed vegetable salads: microbial quality evaluation. J. Food Protect. 70, 1277e1280. Garg, N., Churey, J.J., Splittstoesser, D.F., 1990. Effect of processing conditions on the microflora of fresh-cut vegetables. J. Food Protect. 53, 701e703. Hassan, S.A., Altalhi, A.D., Gherbawy, Y.A., El-Deeb, B.A., 2011. Bacterial load of fresh vegetables and their resistance to the currently used antibiotics in Saudi Arabia. Foodb. Pathog. Dis. 8, 1011e1018. Hilgren, J.D., Salverda, J.A., 2000. Antimicrobial Efficacy of a peroxyacetic/octanoic acid mixture in fresh-cut-vegetable process waters. J. Food Sci. 65, 1376e1379. Johannessen, G.S., Loncarevic, S., Kruse, H., 2002. Bacteriological analysis of fresh produce in Norway. Int. J. Food Microbiol. 77, 199e204. Junkins, A.D., Doyle, M.P., 1992. Demonstration of exopolysaccharide production by enterohemorrhagic Escherichia coli. Curr. Microbiol. 25, 9e17. Lang, M.M., Harris, L.J., Beuchat, L.R., 2004. Evaluation of inoculation method and inoculum drying time for their effects on survival and efficiency of recovery of Escherichia coli O157: H7, Salmonella, and Listeria monocytogenes inoculated on the surface of tomatoes. J. Food Protect. 67, 732e741. Li, Y., Brackett, R.E., Chen, J., Beuchat, L.R., 2001a. Survival and growth of Escherichia coli O157: H7 inoculated onto cut lettuce before or after heating in chlorinated water, followed by storage at 5 or 15 C. J. Food Protect. 64, 305e309. Li, Y., Brackett, R.E., Shewfelt, R.L., Beuchat, L.R., 2001b. Changes in appearance and natural microflora on iceberg lettuce treated in warm, chlorinated water and then stored at refrigeration temperature. Food Microbiol. 18, 299e308. Martins, C.G., Behrens, J.H., Destro, M.T., Franco, B.D.G.M., Vizeu, D.M., Hutzler, B., Landgraf, M., 2004. Gamma radiation in the reduction of Salmonella spp. inoculated on minimally processed watercress (Nasturtium officinalis). Radiat. Phys. Chem. 71, 89e93. Masson, R.B., 1990. Search for new disinfectants for 4th range products. In: Proc. Congress Products of 4th Range and 5th Range. Nascimento, M.S., Silva, N., Catanozi, M.P.L.M., Silva, K.C., 2003. Effects of different disinfection treatments on the natural microbiota of lettuce. J. Food Protect. 66, 1697e1700. Niemira, B.A., 2007. Relative efficacy of sodium hypochlorite wash versus irradiation to inactivate Escherichia coli O157: H7 internalized in leaves of romaine lettuce and baby spinach. J. Food Protect. 70, 2526e2532. Niemira, B.A., Sommers, C.H., Fan, X., 2002. Suspending lettuce type influences recoverability and radiation sensitivity of Escherichia coli O157: H7. J. Food Protect. 65, 1388e1393. Oliveira, M., Usall, J., Vinas, I., Anguera, M., Gatius, F., Abadias, M., 2010. Microbiological quality of fresh lettuce from organic and conventional production. Food Microbiol. 27, 679e684. Park, W.P., Lee, D.,S., 1995. Effect of chlorine treatment on cut water cress and onion. J. Food Qual. 18, 415e424. Pirovani, M.E., Guemes, D.R., Piagnetini, A.M., 2001. Predictive models for available chlorine depletion and total microbial count reduction during washing of freshcut spinach. J. Food Sci. 66, 860e864. Prakash, A., Inthajak, P., Huibregtse, H., Caporaso, F., Foley, D.M., 2000. Effects of Low-dose gamma irradiation and conventional treatments on shelf life and quality characteristics of diced celery. J. Food Sci. 65, 1070e1075. lix, E., Díaz-Cinco, M., Islas-Osuna, M.A., Gonz Ruiz-Cruz, S., Acedo-Fe alezAguilar, G.A., 2007. Efficacy of sanitizers in reducing Escherichia coli O157: H7, Salmonella spp. and Listeria monocytogenes populations on fresh-cut carrots. Food Contr. 18, 1383e1390. Seo, K.H., Frank, J.F., 1999. Attachment of Escherichia coli O157: H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy. J. Food Protect. 62, 3e9. Takeuchi, K., Frank, J.F., 2000. Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. J. Food Protect. 63, 434e440. Taormina, P.J., Beuchat, L.R., 1999. Behavior of enterohemorrhagic Escherichia coli O157:H7 on alfalfa sprouts during the sprouting process as influenced by treatments with various chemicals. J. Food Protect. 62, 850e856. Ukuku, D.O., Fett, W.F., 2002. Relationship of cell surface charge and hydrophobicity to strength of attachment of bacteria to cantaloupe rind. J. Food Protect. 65, 1093e1099. Vandekinderen, I., Devlieghere, F., De Meulenaer, B., Ragaert, P., Van Camp, J., 2009. Optimization and evaluation of a decontamination step with peroxyacetic acid for fresh-cut produce. Food Microbiol. 26, 882e888. Wachtel, M.R., Whitehand, L.C., Mandrell, R.E., 2002. Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water. J. Food Protect. 65, 18e25. Willson, V.A., 1935. Determination of available chlorine in hypochlorite solutions by direct titration with sodium thiosulfate. Ind. Eng. Chem. Anal. Ed. 7, 44e45.
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Decontamination and survival of Enterobacteriaceae on shredded iceberg lettuce during storage Tareq M. Osaili a, *, Akram R. Alaboudi b, Heba N. Al-Quran a, Anas A. Al-Nabulsi a a b
Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan Department of Veterinary Pathology and Public Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, 22110, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 3 February 2018
Enterobacteriaceae family can contaminate fresh produce at any stage of production either at pre-harvest or post-harvest stages. The objectives of the current study were to i) identify Enterobacteriaceae species on iceberg lettuce, ii) compare the decontamination efficiency of water, sodium hypochlorite (free chlorine 200 ppm), peroxyacetic acid (PA 80 ppm; Kenocid 2100®) or their combinations and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce and iii) determine the survival of Enterobacteriaceae post-treatment storage of shredded iceberg lettuce at 4, 10 and 25 C, for up to 7 days. Klebsiella pneumonia spp. pneumonia, Enterobacter cloacae, Klebsiella oxytoca, Pantoea spp., Leclercia adecarboxylata and Kluyvera ascorbate were identified on iceberg lettuce. No significant difference (P 0.05) among Enterobacteriaceae survival after washing with water or sanitizing with sodium hypochlorite or Kenocid 2100® (reduction 0.6 log CFU/g) were found. Combined sanitizer treatments were more effective against Enterobacteriaceae than single washing/sanitizing treatments. Sanitization of iceberg lettuce with combined washing/sanitizing treatments reduced Enterobacteriaceae by 0.85e2.24 CFU/g. Post-treatment growth of Enterobacteriaceae during storage on samples sanitized with sodium hypochlorite and Kenocid 2100® was more than on samples washed with water. The D10-value of Enterobacteriaceae on shredded iceberg lettuce was 0.21 KGy. The reduction of Enterobacteriaceae populations on iceberg after gamma radiation (0.6 KGy) was 3 log CFU/g, however, Enterobacteriaceae counts increased post-irradiation storage by 4e5 log CFU/g. Therefore, washing shredded iceberg lettuce with combined sanitizing treatment (sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, or Kenocid 2100®/Kenocid 2100®) for total time of 6 min or exposing it to gamma irradiation (0.6 KGy) can decrease the risk of Enterobacteriaceae (reduction 2 log). Post-washing storage of sliced iceberg lettuce (4, 10, 25 C) could increase the risk of Enterobacteriaceae as their counts increased during storage even at low temperatures. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Enterobacteriaceae Decontamination Sodium hypochlorite Peroxyacetic acid Ionizing radiation
1. Introduction Consumption of fresh leafy green vegetables that are eaten raw or with minimal processing has increased because of healthy lifestyle patterns. Contamination of these products with microorganisms may be a health hazard to consumers and cause foodborne illness outbreaks. Leafy green vegetables may be contaminated with pathogenic and non-pathogenic bacteria at several points throughout the pre-harvest and post-harvest systems from irrigation water, feces, dust, soil, human handlings, storage facilities,
* Corresponding author. Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan. E-mail address: [email protected] (T.M. Osaili). https://doi.org/10.1016/j.fm.2018.01.022 0740-0020/© 2018 Elsevier Ltd. All rights reserved.
distribution systems, processing equipment and poor hygienic conditions (Beuchat, 1996). These bacteria can attach to the surface of leafy green vegetables and survive for extended periods time (Brandl and Amundson, 2008; Delaquis et al., 2007). Iceberg lettuce is one of the produce commodities most susceptible to contamination. Doyle and Erickson (2008) noted that part of the plant closest to the soil contained higher concentration of the microbes. Enterobacteriaceae is classified as large family of bacteria with common characteristics comprise of Gram-negative, rod-shaped, facultative anaerobic, non-spore forming, either capsulated or noncapsulated in addition of being motile or non-motile. Presence of pathogenic and antimicrobial resistant members of Enterobacteriaceae on fresh produce at retail constitute severe threats to consumers. Pathogenic or antimicrobial resistant members of
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Enterobacteriaceae have been isolated from green leafy vegetables (Al-Kharousi et al., 2016; Al-Holy et al., 2013; Hassan et al., 2011). Al-Holy et al. (2013) and Hassan et al. (2011) isolated Klebsiella pneumoniae, Enterobacter sp., Escherichia coli, Citrobacter sp., Acinetobacter, Shigella flexneri and other Enterobacteriaceae members that are resistant to antibiotics from green leafy vegetables sold in Saudi Arabia. Many methods have been used to decontaminate bacteria on fresh produce. Sodium hypochlorite (form of chlorine) has been widely used to reduce microbial contamination on fresh produce including lettuce. Escudero et al. (1999) reported that exposure of shredded lettuce to 100 and 300 ppm chlorine for 10 min reduced the populations of Yersinia enterocolitica 2 to 3 log CFU/g. However, other studies have found that chlorine (<200 ppm) are not effective at reducing microbial populations on lettuce (Li et al., 2001a; Taormina and Beuchat, 1999). Peroxyacetic acid (PA) has also been widely used to reduce microorganisms on fresh produce. Masson (1990) found that 90 ppm PA reduced total counts and fecal coliforms on cut salad mixtures by 2 log CFU/g. However, Davidson et al. (2013) showed that 50 ppm PA (Tsunami® 100), and 50 ppm of mixed peracid (Tsunami® 200) were not significantly more effective than water at reducing of E. coli O157:H7 on iceberg lettuce. Ionizing radiation maybe considered one of the most promising technology to improve the safety of fresh produce. In 2008, U.S. Food and Drug Administration (FDA) (2008) conferred its acceptance to use irradiation for killing pathogens on iceberg lettuce. Niemira (2007) compared between the effect of sodium hypochlorite wash and irradiation against E. coli O157:H7 internalized in romaine lettuce leaves. The author reported that the D10-value of E. coli O157:H7 in lettuce leaves was 0.39 KGy, while sodium hypochlorite (chlorine 300 and 600 ppm) resulted in <1 log reduction and water wash was ineffective. No studies were found in the literature on the effect of washing/ sanitizing solutions (water, sodium hypochlorite, Kenocid 2100® (PA) or their combination) and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce during post treatment storage. Thus, the objectives of the current study were to i) identify Enterobacteriaceae species found on iceberg lettuce, ii) compare the decontamination efficiency of washing/sanitizing solutions (water, sodium hypochlorite, Kenocid 2100® (PA) or their combination) and ionizing radiation against Enterobacteriaceae on shredded iceberg lettuce and iii) determine post-treatment survival of Enterobacteriaceae on shredded iceberg lettuce during storage at 4, 10 and 25 C for up to 7 days. 2. Materials and methods 2.1. Preparation of iceberg lettuce Iceberg heads samples were purchased from a local market, at the day of each experiment. Damaged leaves were removed and only intact leaves were used in the experiments. Iceberg leaves samples of each experiment were prepared from five iceberg heads. The leaves were shredded manually (ca 1 3 cm) by sanitized knife and cutting board. The shredded leaves were mixed manually in a sterile bag to be used in the experiments (25 g quantities). 2.2. Determination of aerobic plate count and Enterobacteriaceae count on iceberg samples Each sample (25g) was placed into sterile Stomacher bags (Seward, UK) under aseptic condition and combined with 225 ml quantities of peptone water (0.1%; Oxoid, UK), and then was homogenized in the Stomacher (AES-Chemunex, France) for 2 min.
The samples were serially diluted in 9 ml of sterile peptone water (0.1%) and volumes of 0.1 ml of suitable dilutions were plated in duplicate on Nutrient agar (Oxoid) to enumerate total plate counts and in Violet Red Bile Glucose agar (VRBGA, Oxoid) by pour plate method to enumerate Enterobacteriaceae. The plates were incubated aerobically at 37 C for 24 h. The typical Enterobacteriaceae colonies were counted expressed as log (CFU/g). 2.3. Identification of Enterobacteriaceae on the iceberg samples To reveal Enterobacteriaceae members on iceberg lettuce, thirty morphological different Enterobacteriaceae colonies grown on/in Violet Red Bile Glucose agar (VRBGA) from different shredded iceberg samples were identified using the VITEK 2 automated rieux S.A., Marcy L'Etoile, France). The ID-GNB card system (bioMe was used for Enterobacteriaceae identification. 2.4. Decontamination of iceberg lettuce 2.4.1. Washing/sanitation-single treatment In this method, water washing (sterile), sodium hypochlorite and Kenocid 2100® were used separately to sanitize the shredded iceberg samples. Commercial sodium hypochlorite (NaOCl) was purchased from the local market. Free chlorine concentration was determined using the method described by Willson (1935). Sodium hypochlorite solution was freshly prepared prior to each experiment by diluting 5 ml NaOCl (free chlorine 4%) with 1000 ml of 0.05M potassium phosphate buffer (pH 6.8 at 25 C) to achieve free chlorine concentration of 200 ppm (Lang et al., 2004). Kenocid 2100® (Ieper, Belgium), a peroxyacetic acid-based sanitizer, was obtained from the local market. It contains hydrogen peroxide (H2O2 20%), peracetic acid (PA 5%) and acetic acid (AA 10%). The solution was freshly prepared prior to each experiment to obtain PA concertation of 80 ppm. The solution was prepared by adding 1.6 ml of the Kenocid 2100® to 1000 ml sterile distilled water. Shredded iceberg leaves (25g) were placed into sterile Stomacher bag and 100 ml of distilled water (at room temperature, 21±1 C), sodium hypochlorite (chlorine 200 ppm) or Kenocid 2100® (PA 80 ppm) was added to the bag. After mixing thoroughly for 3 min, the solution was drained out from the bag and the leaves were dried by tissue paper to remove the remaining solution on the samples. 2.4.2. Washing/sanitation-combined treatment In this part of the study, combinations of treatments were used [(washing with water followed by washing with water), (washing with water followed by sanitizing with sodium hypochlorite), (washing with water followed by sanitizing with Kenocid 2100®), (sanitizing with sodium hypochlorite followed by sanitizing with sodium hypochlorite), (sanitizing with sodium hypochlorite followed by sanitizing with Kenocid 2100®) and (sanitizing with Kenocid 2100® followed by sanitizing with Kenocid 2100®)]. Shredded lettuce sample (25g) was firstly treated with the first solution (distilled water, sodium hypochlorite or Kenocid 2100®) for 3 min. Then, the solution was drained out from the bag and the shredded lettuce was kept at room temperature for 5 min. Consequently, the second solution was added to the bag and mixed with the shredded lettuce thoroughly for 3 min. After that, the solution was drained out from the bag and the shredded lettuce samples were dried by tissue paper without water rinse to shorten the washing/sanitizing process. 2.4.3. Ionizing radiation Quantities of 25 g of shredded lettuce was placed in sterile Stomacher bags, pressed by hand to remove air and sealed. The
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samples were transmitted to the research gamma unit (Gammafacility PX-g -30, Issledovatelj, Techsnabexport, Moskivia) at Jordan Atomic Energy Commission, Amman, Jordan by ice box. The instrument is spherically shaped and has a sample capacity of 4400 ml. The irradiation source is Co60; the current activity and dose rate of the source are 768.27Ci and 480.19 Gy/h, respectively. Dosimetry was performed using 2 ml ethylene chlorobenzene (ECB) dosimeters (Institute of Isotopes and Chemical Research Centre, Hungarian Academy of Sciences, Budapest, Hungary), and the dosimeter response was measured with oscillotitrator OK-302/ 1 (Radelkis, Budapest, Hungary). These dosimeters were calibrated against an international standard (RISØ, Roskilde, Denmark). The samples were exposed to irradiation doses of 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 KGy in order calculate D10-value of Enterobacteriaceae. Other samples prepared for storage study were exposed to irradiation dose of 0.6 KGy. 2.5. Storage of iceberg lettuce To determine the effect of storage on the survivals of Enterobacteriaceae on shredded iceberg lettuce post treatment (washing/ sanitation and ionizing radiation), the samples were kept in sterile bags at 4 and 10 C for 7 days and at 25 C for 4 days. Preliminary study showed that shredded iceberg lettuce deteriorated after 7 days of storage at 10 C and 4 days of storage at 25 C. 2.6. Microbiological analysis Shredded iceberg samples were analyzed to enumerate Enterobacteriaceae on the samples at 0, 1, 2, 3, 4, 5, 6 and 7 days of storage at 4 and 10 C and at 0, 1, 2, 3 and 4 days of storage at 25 C. The samples were placed into sterile Stomacher bags (Seward) under aseptic condition and combined with 225 ml quantities of peptone water (0.1%), and then were mixed in the Stomacher for 2 min. The samples were serially diluted by adding 1 mle9 ml of sterile peptone water (0.1%) and volumes of 0.1 ml of suitable dilutions were plated in duplicate in VRBGA by pour plate method. The plates were incubated aerobically at 37 C for 24 h. The typical colonies counted and the numbers were expressed as log (CFU/g). 2.7. Statistical analysis The statistical analysis was performed using the Statistical Package for Social Sciences software (SPSS, version 19, Chicago. Inc). The survivals of Enterobacteriaceae on shredded iceberg lettuce after treatments and during storage were compared. A P-value of < 0.05 was considered the level for statistical significance. One way ANOVA was used to examine the effect of treatments and storage time on the survivals of Enterobacteriaceae on shredded iceberg samples. Duncan post-hoc was conducted to determine the difference among variables groups. Data are expressed as mean and standard deviation. Each experiment was replicated three times. 3. Results 3.1. Total plate count and identification of Enterobacteriaceae on iceberg lettuce The total plate count and Enterobacteriaceae count on fresh iceberg lettuce samples were 7.03 ± 0.30 log CFU/g and 6.73 ± 0.26 log CFU/g, respectively. Enterobacteriaceae isolates (n ¼ 30) from iceberg lettuce samples were identified as Klebsiella pneumonia spp. pneumonia (13 isolates) that was mostly isolated from our samples followed by Enterobacter cloacae (11 isolates) and Klebsiella oxytoca (3 isolates), while Pantoea spp, Leclercia adecarboxylata and
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Kluyvera ascorbata were identified from only three samples. 3.2. Effect of washing/sanitation single treatments and storage on the populations of Enterobacteriaceae on shredded iceberg lettuce Fig. 1 shows the survival of Enterobacteriaceae on shredded iceberg lettuce after treatment with water, sodium hypochlorite or Kenocid 2100® for 3 min followed by storage for up to 7 days at 4, 10 and 4 days at 25 C. Water, sodium hypochlorite and Kenocid 2100® reduced the initial level of Enterobacteriaceae on the samples (6.73 log CFU/g) immediately (time 0) by 0.3, 0.6 and 0.5 log CFU/g, respectively. The differences among the survivals of Enterobacteriaceae on shredded iceberg lettuce after treatment immediately or during storage at 4 C were < 0.7 log CFU/g. Post-treatment storage (4 C) of iceberg lettuce did not change Enterobacteriaceae populations on the samples significantly regardless of the washing/ sanitizing solution. Although, there was a significant (P < 0.05) drop in the cell counts on the second day of storage, the populations increased significantly during the subsequent storage period to reach levels no significantly (P 0.05) different than the original levels. At storage temperature of 10 C, there were no significant differences (P 0.05) among the survivals of Enterobacteriaceae on the lettuce regardless of storage times. Enterobacteriaceae populations on samples washed with water did not change significantly during storage, while the populations on samples sanitized with Kenocid 2100® and sodium hypochlorite increased significantly (P < 0.05) after the third and fourth day of storage, respectively. At storage temperature of 25 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on the lettuce after 2, 3, and 4 days of storage; however, the differences were <0.7 log CFU/g. Enterobacteriaceae populations on the samples increased significantly (P < 0.05) after the first day of storage at 25 C regardless of the washing/sanitizing solutions. At the fourth day of storage, the populations on samples treated with water, sodium hypochlorite and Kenocid 2100® increased by 1.5, 2.2 and 2.4 log CFU/g, respectively. 3.3. Effect of washing/sanitation combined treatments and storage on the populations of Enterobacteriaceae on iceberg lettuce Combinations of the treatments reduced the initial level of Enterobacteriaceae on the shredded iceberg lettuce (6.73 log CFU/g) by 0.85, 1.65, 1.30, 2.24, 2.09 or 1.99 log CFU/g immediately after treatment with water/water, water/sodium hypochlorite, water/ Kenocid 2100®, sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, or Kenocid 2100®/Kenocid 2100® respectively, (Fig. 2). There were significant differences (P < 0.05) among the survivals on the samples after treatment immediately and after 1, 2 and 3 days of storage at 4 C. The survivals on the samples washed with water-included treatments were higher than the survivals on the samples sanitized with non-water-included solution. In addition, the survivals on samples sanitized with sodium hypochlorite-included treatment were lower than the survivals on samples sanitized with Kenocid 2100®-included treatment. On the fourth day until the end of the storage period, there were no significant differences (P 0.05) among the survivals on the samples regardless of treatments. Post-treatment storage of lettuce reduced Enterobacteriaceae populations on the samples non-significantly in the first two days of storage regardless of the treatment, but after that, the populations increased significantly (P < 0.05) during storage. At a storage temperature of 10 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on lettuce after treatments on day 1, 2, 3 and 5 of storage (Fig. 3). Similar to the results obtained from the samples stored at 4 C, during the
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ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
A 9 8 7 6 5 4 3 0
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Fig. 1. Survival of Enterobacteriaceae on iceberg lettuce after washing with water, sodium hypochlorite or Kenocid 2100® and storage at 4 C (a), 10 C (b) and 25 C (c) for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
ENTEROBACTERIACEAE POPULATION (LOG CFU/G)
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9 8 7 6 5 4 3 0
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Fig. 2. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 4 C for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
9 8 7 6 5 4 3 0
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Fig. 3. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 10 C for up to 7 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
first 3 days of storage, the survivals on the samples treated with water-included solutions were higher than the survivals on the samples treated with non-water-included solutions. Furthermore, the survivals on the samples sanitized with sodium hypochloriteincluded treatments were non-significantly lower than the survivals on the samples sanitized with Kenocid 2100®-included treatments. After the fifth day of storage, there were no significant differences (P 0.05) among counts on samples. Enterobacteriaceae on lettuce increased significantly (P < 0.05) at the third day of storage. At the end of storage period, the populations on samples increased by 0.9e2.5 log CFU/g. At storage temperature of 25 C, there were significant differences (P < 0.05) among the survivals of Enterobacteriaceae on the lettuce after day 1 and 2 of storage (Fig. 4). The survivals on the samples washed with water alone were lower than the survivals on the samples treated with sanitizer-included solutions. The
populations on the samples increased significantly after 1 day of storage; the populations on the samples increased by 2.0e3.8 log CFU/g. 3.4. Effect of gamma radiation and storage on the populations of Enterobacteriaceae on shredded iceberg lettuce Irradiation reduced Enterobacteriaceae on lettuce (6.65 ± 0.06 log CFU/g) by 0.44, 1.10, 1.53, 2.14, 2.53 or 2.84 log CFU/g at 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 KGy, respectively. The D10-value of Enterobacteriaceae on shredded iceberg lettuce was 0.21 KGy (r ¼ 0.996). Fig. 5 shows that the level of Enterobacteriaceae on lettuce after irradiation was 3.70 ± 0.35 log CFU/g (3 log reduction). Enterobacteriaceae populations on the samples increased on the first day of storage and during the subsequent storage periods (P < 0.05). The survivals on samples stored at 25 C were higher than those on
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9 8 7 6 5 4 3 0
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Fig. 4. Survival of Enterobacteriaceae on iceberg lettuce after washing with W/W (water/water), W/S (water/sodium hypochlorite), W/K (water/Kenocid 2100®), S/S (sodium hypochlorite/sodium hypochlorite), S/K (sodium hypochlorite/Kenocid 2100®), and K/K (Kenocid 2100®/Kenocid 2100®) and storage at 25 C for up to 4 days. The average Enterobacteriaceae count on iceberg lettuce samples before treatment was 6.73 ± 0.26 log CFU/g.
9 8 7 6 5 4 3 0
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Fig. 5. Growth of Enterobacteriaceae on iceberg lettuce after exposure to 0.6 KGy irradiation followed by storage for 7 days at 4 C and 10 C, and 4 days at 25 C.
samples stored at 4 or 10 C (P < 0.05). At the end of storage period, the populations on samples stored at 4, 10 and 25 C increased by 3.67, 4.29 and 5.17 log CFU/g, respectively. 4. Discussion The current study showed that iceberg samples were highly contaminated with Enterobacteriaceae family members, they were found at high levels on the surface of the samples. Our results showed that Enterobacteriaceae count of 6.73 log CFU/g on fresh iceberg lettuce samples was in the range of that reported in other studies for lettuce and other green vegetables (Al-Holy et al., 2013; €der et al., 2007). The attachment of Oliveira et al., 2010; Fro Enterobacteriaceae on the plant surfaces may occur by several
mechanisms including extracellular polymeric substances, cell surface hydrophobicity, bacterial surface charge, divalent cationic bridges and may depend on presence or absence of fimbriae (Hassan et al., 2011; Ukuku and Fett, 2002; Fratamico et al., 1996; Junkins and Doyle, 1992). In the current study, the identified Enterobacteriaceae genera and species on iceberg lettuce were similar to those isolated from iceberg lettuce and other green vegetables in other studies (Al-Holy et al., 2013; Johannessen et al., 2002). Predominantly, Klebsiella spp. and Enterobacter spp. have been isolated from soil, water, vegetation and intestinal contents of animals and humans. The link between these two bacterium and fresh produce was considered as an indicator for the contact between soil and iceberg (Doyle and Erickson, 2008; De Roever, 1998). E. coli and other enteric
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foodborne pathogens were not isolated from our samples; this suggests that iceberg lettuce was not contaminated by feces. Washing iceberg lettuce with sodium hypochlorite (free chlorine 200 ppm) for 3 min reduced Enterobacteriaceae by 0.6 log CFU/ g. Similar observation was reported by Takeuchi and Frank (2000) who found that washing lettuce with 200 ppm chlorine for 5 min reduced the E. coli O157:H7 populations between 0.7 and 1 log CFU/ g. Similarly, Garg et al. (1990) reported that dipping lettuce in chorine solution (100 ppm) for 5 min reduced E. coli population by l log CFU/g. However, Nascimento et al. (2003) reported that washing lettuce with chlorine (200 ppm) for 15 min reduced aerobic mesophilic and total coliform by 2.63 and 1.91 log CFU/g, respectively. These reductions might be explained by the metabolic inhibition of chlorine as it attaches the amino groups of the cell protein, prevents cell metabolism and then leads to cell death (Denyer and Stewart, 1998). Washing iceberg lettuce with Kenocid 2100® (80 ppm PA) for 3 min reduced Enterobacteriaceae by 0.5 log CFU/g. Other studies have reported higher effect of PA against bacteria than numbers reported in the present study. Hilgren and Sailverda (2000) showed that washing cabbage and celery with 80 ppm PA for 30 s reduced aerobic count by 0.84 and 1.07 log CFU/g, respectively. Ruiz-Cruz et al. (2007) showed that washing shredded carrots with 40 ppm PA for 2 min reduced Salmonella and E. coli O157:H7 populations by 2.1 and 1.24 log CFU/g, respectively. Furthermore, Nascimento et al. (2003) reported that 80 ppm AA (Tsunami 100®) for 15 min reduced aerobic mesophilic and total coliform on lettuce by 1.85 and 1.44 log CFU/g, respectively. PA is a very strong oxidizing agent, which can lead to bacterial cell membrane destabilization. The damage of bacterial membrane lipid and protein can cause loss of its solidity and its ability of attachment to the thio group of proteins which inhibits several essential enzymes necessary for protein synthesis (Denyer and Stewart, 1998). Sodium hypochlorite was more effective than water and slightly more effective than Kenocid 2100® in reducing the populations of Enterobacteriaceae from the surface of iceberg lettuce. Although Kenocid 2100® solution used in the current study contained H2O2, it was not more effective against Enterobacteriaceae than sodium hypochlorite. Unlike our findings, Alasri et al. (1993) found that addition of H2O2 to PA caused a synergistic antimicrobial effect with strong correlation between increases the concentration of H2O2 and microbial reduction. Strain variation, sanitizer concentration, exposure time, presence of soil and foreign materials on the surface of the vegetables, and type of fresh produce may explain the variation among the studies. Our results showed that combined treatments were more effective than single treatments. Washing iceberg lettuce with sodium hypochlorite two times (3 min each) was the most effective in Enterobacteriaceae decontamination compared to the other washing treatments; it reduced the population of Enterobacteriaceae up to 2.2 log CFU/g. This reduction could be explained by the increases in contact time between washing solution and attached bacteria on the surface of iceberg lettuce. Similar finding was reported by Pirovani et al. (2001) who found that increasing washing time of fresh-cut spinach with chlorine from 2 to 8 min increased the reduction in total microbial count from 2.8 to 3.5 log CFU/g. Vandekinderen et al. (2009) also reported that increasing washing time with PA from 1 to 10 min increased the reduction of microflora on of fresh-cut produce from 0.5 to 1.5 log CFU/g. Washing with water and sanitizers did not eliminate the entire bacteria on the surface of iceberg lettuce. This could be explained by the presence of Enterobacteriaceae cells in inaccessible sites in lettuce leaf such as pores, punctures and damage fraction that may prevent the direct contact between the washing solutions and attached bacteria (Burnett et al., 2000; Takeuchi and Frank, 2000;
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Seo and Frank, 1999). Seo and Frank (1999) found that E. coli O157:H7 can attach to the surface, trichomes, stomata, and cut edges of lettuce leaves. The pathogen gathered inside the stomata and attached preferentially to cut edges. Strong attachment of the bacteria to the tissue and formation of biofilms can also reduce the efficacy of the washing solution against the microorganisms (Doyle and Erickson, 2008). Enterobacteriaceae populations on sliced iceberg lettuce increased during post-washing storage at 4, 10 and 25 C. Slicing of iceberg lettuce released intracellular liquids, which may also enhanced growth of inaccessible bacteria that have been protected in small niches on the leaves. According to Wachtel et al. (2002) survival of E. coli O157:H7 on lettuce tissue from heads dropped 6 feet was higher than in undamaged tissue after storage at room temperature for 4 h and then stored at 4 C for 48 h. Similar to our findings, previous researchers reported that Enterobacteriaceae are able to grow at refrigeration temperatures. Li et al. (2001b) found that Enterobacteriaceae counts increased on chlorine-sanitized lettuce during storage at 5 or 15 C for 18 or 7 days, respectively. Prakash et al. (2000) reported that the populations of aerobic bacteria on diced celery increased through storage for 22 days at 5 C after washing with acidified solution (vinegar 5% AA), chlorinated solution (200 ppm) for 5 min and water. The increase was 6.2, 2.9 and 1.9 log CFU/g, respectively. Growth of Enterobacteriaceae during storage at 25 C was higher than 10 and 4 C. Decreasing the temperature (refrigerate temperature) minimizes microbial growth, though, psychrotrophic species are able to grow. Samples that were stored at 25 C deteriorated more rapidly than at 10 and 4 C. Qualitative changes occurred at high temperature may cause deleterious effect on fresh produce; this may enhance microbial growth (Allwood et al., 2004). The comparison among the washing solutions in our study showed that Enterobacteriaceae growth during storage on samples washed with Kenocid 2100® and with sodium hypochlorite was more than on samples washed with water. This could be explained by reduction of competitive background microflora (other than Enterobacteriaceae) that had been reduced by washing treatment. This inturn allows Enterobacteriaceae to grow under the specified storage condition (Carlin et al., 1996). Also, presence of inactive sanitizer residues on the leaves could be utilized by bacteria to support their growth. In addition, sanitizers may have caused damage to vegetable tissues; this may enhance microbial proliferation on vegetable too (Park and Lee, 1995). This study found that low irradiation dose (0.21 KGy) achieved 90% (1 log) reduction in the populations of Enterobacteriaceae on shredded iceberg lettuce. Similar findings were previously reported for members in the Enterobacteriaceae family. Niemira (2007) found that the D10-values of E. coli O157:H7 internalized in romaine lettuce leaves was 0.39 KGy. Martins et al. (2004) found that D10-values of Salmonella spp. on watercress ranged from 0.29 to 0.43 KGy. Treating iceberg lettuce with 0.6 KGy ionizing radiation reduced Enterobacteriaceae by ca 3 log CFU/g. Similar values were reported by Niemira et al. (2002) who found that irradiation dose of 0.5 KGy reduced E. coli O157:H7 populations by 3.6e3.8 log CFU/g on different types of lettuce. Irradiation sensitivity of microorganisms is affected by type of fresh produce, type of bacteria, and microbial attachment on the surface of the produce (Niemira et al., 2002). The increase in Enterobacteriaceae populations on sliced iceberg lettuce post-irradiation storage could be explained by the released of intracellular liquids which enhanced growth of survival bacteria on the leaves and by the ability of injured cells to recovery during storage. According to Prakash et al. (2000) the populations of aerobic bacteria on celery increased during storage at 5 C for 22 days after irradiation with 1.0 or 0.5 KGy. The use of 0.6 KGy in the current study is safe and did not change the texture and quality of
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iceberg lettuce samples. Niemira et al. (2002) found that irradiation dose up to 0.5 KGy did not change the quality of different types of lettuce. 4.1. Conclusion Enterobacteriaceae members are found on the surfaces of iceberg lettuce leaves at high levels. Klebsiella pneumonia and Enterobacter cloacae were mostly isolated from our samples. Enterobacteriaceae populations on sliced iceberg lettuce decreased < 1 log after washing with water, sodium hypochlorite, or Kenocid 2100® for 3 min but combined sanitizing treatments (sodium hypochlorite/sodium hypochlorite, sodium hypochlorite/Kenocid 2100®, and Kenocid 2100®/Kenocid 2100®) for total time of 6 min decreased the populations by 2 log. During post-washing storage of sliced iceberg lettuce, Enterobacteriaceae counts increased (4 < 10 < 25 C), the populations were higher on samples treated with sanitizers. Gamma radiation improved the microbial safety and enhanced quality of leaves, however, Enterobacteriaceae counts increased at post-irradiation storage. Acknowledgements The authors thank the Deanship of Scientific Research at Jordan University of Science and Technology for funding this project (231/ 2015). References Alasri, A., Valverde, M., Roques, C., Michel, G., Cabassud, C., Aptel, P., 1993. Sporocidal properties of peracetic acid and hydrogen peroxide, alone and in combination, in comparison with chlorine and formaldehyde for ultrafiltration membrane disinfection. Can. J. Microbiol. 39, 52e60. Al-Holy, M., Osaili, T., El-Sayed, S., Alshammari, E., Ashankyty, I., 2013. Microbiological quality of leafy green vegetables sold in the local market of Saudi Arabia. Ital. J. Food Sci. 25, 446e452. Al-Kharousi, Z.S., Guizani, N., Al-Sadi, A.M., Al-Bulushi, I.M., Shaharoona, B., 2016. Hiding in fresh fruits and vegetables: opportunistic pathogens may cross geographical barriers. Int. J. Microbiol., 4292417 https://doi.org/10.1155/2016/ 4292417, 14 pages. Allwood, P.B., Malik, Y.S., Hedberg, C.W., Goyal, S.M., 2004. Effect of temperature and sanitizers on the survival of feline calicivirus, Escherichia coli, and F-specific coliphage MS2 on leafy salad vegetables. J. Food Protect. 67, 1451e1456. Beuchat, L.R., 1996. Pathogenic microorganisms associated with fresh produce. J. Food Protect. 59, 204e216. Brandl, M.T., Amundson, R., 2008. Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157: H7 and Salmonella enterica. Appl. Environ. Microbiol. 74, 2298e2306. Burnett, S.L., Chen, J., Beuchat, L.R., 2000. Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy. Appl. Environ. Microbiol. 66, 4679e4687. Carlin, F., Nguyen-the, C., Da Silva, A.A., Cochet, C., 1996. Effects of carbon dioxide on the fate of Listeria monocytogenes, of aerobic bacteria and on the development of spoilage in minimally processed fresh endive. Int. J. Food Microbiol. 32, 159e172. Davidson, G.R., Buchholz, A.L., Ryser, E.T., 2013. Efficacy of commercial produce sanitizers against nontoxigenic Escherichia coli O157: H7 during processing of iceberg lettuce in a pilot-scale leafy green processing line. J. Food Protect. 76, 1838e1845. De Roever, C., 1998. Microbiological safety evaluations and recommendations on fresh produce. Food Contr. 9, 321e347. Delaquis, P., Bach, S., Dinu, L.,D., 2007. Behavior of Escherichia coli O157: H7 in leafy vegetables. J. Food Protect. 70, 1966e1974. Denyer, S.,P., Stewart, G.S.A.B., 1998. Mechanisms of action of disinfectants. Int. Biodeterior. Biodegrad. 41, 261e268. Doyle, M.P., Erickson, M.C., 2008. Summer meeting 2007ethe problems with fresh produce: an overview. J. Appl. Microbiol. 105, 317e330. n, A.M.S., 1999. EffectiveEscudero, M.E., Vel azquez, L., Di Genaro, M.S., De Guzma ness of various disinfectants in the elimination of Yersinia enterocolitica on fresh lettuce. J. Food Protect. 62, 665e669. Food and Drug Administration (FDA), 2008. Code of Federal Regulations 21 CFR Part 179: irradiation in the production, processing and handling of food FDA-1999-F-
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