Inactivation of bacterial pathogens on lettuce, sprouts, and spinach using hurdle technology

Accepted Manuscript Inactivation of bacterial pathogens on lettuce, sprouts, and spinach using hurdle technology

Paul-François Ngnitcho Kounkeu, Imran Khan, Charles Nkufi Tango, Mohammad Shakhawat Hussain, Deog Hwan Oh PII: DOI: Reference:

S1466-8564(17)30361-2 doi: 10.1016/j.ifset.2017.07.033 INNFOO 1816

To appear in:

Innovative Food Science and Emerging Technologies

Received date: Revised date: Accepted date:

28 March 2017 26 July 2017 27 July 2017

Please cite this article as: Paul-François Ngnitcho Kounkeu, Imran Khan, Charles Nkufi Tango, Mohammad Shakhawat Hussain, Deog Hwan Oh , Inactivation of bacterial pathogens on lettuce, sprouts, and spinach using hurdle technology, Innovative Food Science and Emerging Technologies (2017), doi: 10.1016/j.ifset.2017.07.033

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ACCEPTED MANUSCRIPT 1

Inactivation of Bacterial Pathogens on Lettuce, Sprouts, and Spinach Using Hurdle Technology Paul-François Ngnitcho Kounkeu¥, Imran Khan¥, Charles Nkufi Tango, Mohammad Shakhawat Hussain,

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Deog Hwan Oh*.

Department of Food Science and Biotechnology, College of Agriculture and Life Sciences, Kangwon National

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University, Chunchon 200-701, Republic of Korea.

*Author for correspondence. Tel: 82-33-250-6457; Fax: 82-33-241-0508; E-mail: [email protected] (Deog Hwan Oh) ¥

P-F, N, Kounkeu and I. Khan share the co-first-authorship

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ACCEPTED MANUSCRIPT 2 Abstract Effects of chemical treatment using slightly acidic electrolyzed water (SAEW), fumaric acid (FA), or calcium oxide (CaO) and physical treatment using ultrasonication (US), micro-bubbles (MB), or ultraviolet (UV) to inactivate bacterial pathogens Listeria monocytogenes, Escherichia

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coli O157:H7, Staphylococcus aureus, and Salmonella spp. on lettuce, spinach, and sprouts were

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determined. Fresh produce inoculated with bacterial pathogens (~9 log CFU/mL) was immersed

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in distilled water (DW), SAEW, FA (0.5%), or CaO (0.2%) alone or in combination at 23 ± 2 °C for 3 min followed by treatment with US, MB for 3 min, or UV for 10 min. Effects of combined

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treatment on shelf-life of lettuce at 4 C and 23 ± 2 C were also determined in this study.

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Results revealed that the use of a combination of CaO+SAEW+FA+US exhibited significant reduction (p < 0.05) for bacterial pathogen on fresh produce compared to individual treatment or

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other combinations. CaO+SAEW+FA+US treatment exhibited highest reduction of E. coli

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O157:H7, S. aureus, L. monocytogenes and Salmonella spp. by 4.7, 4.9, 4.84 and 5.08 log CFU/g,

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respectively on lettuce as compared to spinach and sprouts. Microbial count reducing capability for combined treatment methods were ranked in the following order: SAEW+FA <

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CaO+SAEW+FA < CaO+SAEW+FA+US. However, introduction of US to CaO+SAEW+FA treatment resulted in little detrimental effect on the overall quality of lettuce. Moreover,

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CaO+SAEW+FA treatment effectively enhanced the shelf-life of lettuce stored at 4 ºC and 23 ± 2 ºC by about 6 days and 3 days, respectively as compared to control (DW treatment), with longer lag time (23.11 h on lettuce) for naturally occurring bacteria on fresh produce. These findings suggest that significant synergistic benefit could be obtained from combined sanitizer treatment to eliminate bacterial pathogens from fresh produce. Keywords: bacterial pathogens; fresh produce; hurdle technology; inactivation; shelf-life

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ACCEPTED MANUSCRIPT 3 1. Introduction Consumption of fresh produce has continued to increase over the last decade. Such increase in consumption may be due to the higher nutritional value of fresh produce, changes in social eating habits, and accessibility to fresh produce (Garrett, et al., 2003; Khan, Ullah, & Oh, 2016). Fresh

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produce such as lettuce, spinach, cabbage, and sprouts are minimally processed. They serve as

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key vectors for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes (Alegre,

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Abadias, Anguera, Usall, & Viñas, 2010; Issa-Zacharia, Kamitani, Muhimbula, & Ndabikunze, 2010; Khan, Khan, MisKeen, Nkufi Tango, ParK, & Oh, 2016). Fresh produce has been

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increasingly implicated as a vehicle for transmission of foodborne illnesses around the world.

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These foodborne illnesses are estimated to cause 48 million illnesses, 128,000 hospitalizations, and 3,000 deaths alone in United States (CDC, 2017), resulting in economic loss of billions of

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dollars due to reduced productivity and increased medical expenses.

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Therefore, developing effective disinfectants to reduce pathogens in food and agricultural products is one of the most important steps in the food industry for ensuring product safety (Al-

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Haq, Sugiyama, & Isobe, 2005; Issa-Zacharia, Kamitani, Morita, & Iwasaki, 2010; Rahman,

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Khan, & Oh, 2016). The food industry has adopted various decontamination techniques throughout the food chain, ranging from chemical washing (such as chlorine-base components,

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acid compounds, and ozonated water) to current emerging treatments such as high hydrostatic pressure, dielectric heating, ohmic heating, ultrasonication, irradiation, and use of bacteriocins, and bacteriophages (Du, Han, & Linton, 2002; Trinetta, Vaidya, Linton, & Morgan, 2011; Khan & Oh, 2016). Although these decontamination technologies are effective in reducing microbial load, many of them are expensive and technically difficult to be applied in a field environment.

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ACCEPTED MANUSCRIPT 4 The Hurdle concept, commonly referring to application of combined preservative methods, has become a potential technology that can decrease losses of nutritional and sensory quality while enhancing food safety (Khan, Tango, Miskeen, Lee, & Oh, 2017). The goal of hurdle technology is to improve total quality of foods and reduce treatment concentrations of chemicals (Leistner,

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1985). Moreover, hurdle technology exhibits synergistic effects due to different mechanisms

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involved in the inhibition or inactivation of microorganisms in foods (Rahman, et al., 2016).

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Electrolyzed water (EW) is an oxidant sanitizer with free chlorine as its main antimicrobial agent. It is produced by electrolysis of a dilute salt solution (generally NaCl or HCl) through an

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electrolytic cell. It has been highlighted as one emerging alterative of sodium hypochlorite

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sanitizer because of its on-site and simple production, cheap and easy-to-find raw materials, low operational costs, and low trihalometanes generation (Gil, Gómez-López, Hung, & Allende,

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2015; Gómez-López, Gil, & Allende, 2017). There are two types of EW producing machines,

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those that contain membrane and produce acidic electrolyzed water (pH 2.5) and basic electrolyzed water (pH 10-13) in a 2-cell chambers and others that do not contain a membrane

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and produce neutral electrolyzed water (NEW; pH 7) and slightly acidic electrolyzed water

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(SAEW; pH 5.0-6.5) in a single-cell chamber. SAEW has many advantages over other types of EW. SAEW reduces the environmental damage and corrosive impact of the food industry (Tango,

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Khan, Kounkeu, Momna, Hussain, & Oh, 2017). In addition, the sensory quality of food products is not negatively affected by the use of SAEW (Rahman, et al., 2016). To further enhance SAEW efficacy, it can be used in combination with chemical sanitizers such as fumaric acid (FA), calcium oxide (CaO) or physical treatments such as ultrasonication, microbubble, and ultraviolet irradiation (Awad, Moharram, Shaltout, Asker, & Youssef, 2012; Tango, Wang, & Oh, 2014). Therefore, the objective of the current study was to evaluate different

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ACCEPTED MANUSCRIPT 5 decontamination treatments to establish an optimal hurdle treatment with synergistic antimicrobial effect to improve the quality and safety of fresh produce. 2. Materials and methods 2.1. Microbial culture and inoculum preparation

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The following bacteria were used in this study: L. monocytogenes (ATCC 19111, 19118, 49594),

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E. coli O157:H7 (ATCC 23150, 946, 938), S. aureus (ATCC 13150, 6538, 23235), and

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Salmonella enterica serovars (S. Typhimurium ATCC 19585 and S. enteritidis ATCC 13076). These strains were obtained from College of Agriculture and Life Sciences, Kangwon National

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University, Chuncheon, Gangwon-do, South Korea. Each strain was individually cultivated in 10

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mL of Tryptic Soy Broth (TSB, Becton Dickinson Diagnostic Systems, Sparks, MD, USA) supplied with 2% NaCl at 35 °C for 20 h to obtain a cell density of 9 log CFU/mL. Cell

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suspension was washed twice with 0.1 % sterile Buffered Peptone Water (BPW). Cell pellet was

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obtained after centrifugation at 4,000 × g for 10 min at 4 °C and resuspended in 10 mL of BPW. Four different cocktails were made by mixing bacterial suspension in same proportion (10 mL

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for each strain) according to each bacterium. Bacterial population in each inoculum was

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confirmed by spreading 100 µL of the inoculum from desired dilution onto two Tryptic Soy Agar (TSA, Difco, Becton, Dickinson and Company Sparks, USA) plates. These plates were incubated

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at 37 °C for 24 h and the number of colonies was counted. 2.2. Samples preparation and inoculation Fresh spinach (Spinacia oleracea L.), iceberg lettuce leaves (Lactuca sativa L.), and vacuum-packaged alfalfa sprouts (Medicago sativa L.) were purchased from supermarkets at Chuncheon city, South Korea. These samples were transported to the laboratory using ice bags. They were stored at 4 °C until further experimental. Debris and other particles of leaves (spinach

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ACCEPTED MANUSCRIPT 6 and lettuce) were washed with running tap water to reduce natural microbiota load, dried inside a laminar flow safety cabinet, and exposed to UV for 40 min. These samples were divided into two groups. One group was processed for decontamination treatment. The other group was processed for the shelf-life study. These leaves were inoculated with 1 mL of each bacterial cocktail on the

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abaxial-side of each leaf surface. Approximately 50 g of sprouts were separately immersed in

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each bacterial cocktail (2 mL of bacteria cocktail was individually diluted in 200 mL 0.1% BPW)

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for 45 min using a shaking incubator at room temperature to allow bacteria to equitably distribute on food surface. After bacterial inoculation, sprouts were removed using a sterile metal

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sieve, dried in a laminar flow safety cabinet for 1 h, divided into 10 g portions in a stomacher

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bag, and stored at 4 °C for 24 h. After bacterial inoculation, 10 g of each inoculated vegetable

2.3. Sanitizer solution preparation

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was set aside for microbiological analysis to determine the initial population of each bacterium.

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SAEW (30 ppm) was generated by electrolysis of a combined solution of HCl (5%) and NaCl (2M) in an electrolytic cell without membrane. This was a self-developed electrolyzed

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water generator. The flow rate of incoming tap water was adjusted to 4 L/min. The amperage and

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voltage were set at 10 A and 3.2 V, respectively. Electrolyte flow rate was set at 2 mL/min. The EW was collected at 30 min after starting the generator to allow amperage and voltage to reach

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steady state. After preparation, SAEW was either kept in polypropylene containers (BioTank, Komax, Korea) or immediately used for experimentation. Fumaric acid (FA) was directly diluted with sterile distilled water (DW) using a magnetic stirrer to obtain a final concentration of 0.5%. A solution of CaO (0.2%) was provided by Eco-Biotech Company (Hwaseong-Si, Gyeonggi-Do, Korea). The pH (5.3–5.5) and oxidation reduction potential of 818–854 mV of all SAEW were measured with a dual – scale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ,

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ACCEPTED MANUSCRIPT 7 USA). Available chlorine concentration (ACC) was determined using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan) with a colorimetric method. Combined FA+SAEW was prepared by adding FA to SAEW to obtain desired concentration. 2.4. Physical treatment

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Six liters of sanitizer (FA+SAEW) was filled into a bench-top ultrasonic cleaner (JAC-

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4020, KODO Technical Research Co., Ltd., Hwaseong, Gyeonggi-do, South Korea). The device

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was set at a frequency of 40 KHz with acoustic energy density of 400 W/L. For micro-bubbles treatment, ultrasonic cleaner was filled with 6 liters of FA+SAEW and connected to cable with

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micro-bubbles generators (Moming spa DC 40V, Moming Co., Ltd, Seoul, Korea) to produce

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FA+SAEW micro-bubbles (FA+SAEW+MB). Ultrasonication and micro-bubbles treatments were performed by immersing samples at room temperature (23 ± 2 °C) for 3 min after CaO

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washing. Ultraviolet treatment was carried after sanitizers treatment (CaO+SAEW+FA) using

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ultraviolet light (TUV 15 W; Philips Lighting, Roosendaal, Netherlands) at a distance of 30 cm in a ultraviolet cabinet (Entkeimung schrank, 220 V, Ernst Schuttjun Laborgerotebau, 3400,

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Gottingen, Germany) for 10 min.

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2.5. Decontamination procedure

For single treatments, samples (uninoculated and inoculated) were washed with 200 mL

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solutions (DW, SAEW, FA, and CaO). All treatments were performed at room temperature (23 ± 2 °C) for 3 min. For combined treatment, samples were divided into four groups (Table 1). For the first group, samples were washed with SAEW+FA for 3 min. For the second group, samples were washed with CaO solution for 3 min followed by SAEW combined with FA for 3 min (CaO+SAEW+FA). CaO treatment was applied separately to avoid mixing with SAEW+FA which might increase the pH of SAEW+FA. For the 3rd group, samples were treated with CaO

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ACCEPTED MANUSCRIPT 8 for 3 min followed by SAEW+FA in combination with ultrasonication or micro-bubbles treatment for 3 min. For the 4th group, the treatment consisted of a combination of CaO+SAEW+FA followed by ultraviolet treatment for 10 min. After each chemical treatment, samples were immediately transferred into 200 mL of neutralizing solution (0.85% NaCl + 0.5%

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Na2S2O3) in a stomacher bag for 1 min to stop the antimicrobial effect of the treatment solution.

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Chemical solution was removed from the bath. The bath was then sterilized with 70% ethanol,

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rinsed with DW twice, and left to dry prior to the next experiment. 2.6. Microbiological analysis

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Produce samples (approximately 10 g) were aseptically transferred into separate sterile

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stomacher bags (BA6041, Seward LTD, UK) containing 90 mL of 0.1 % BPW and blended with a Stomacher® 400 CIRCULATOR (Seward Laboratory Systems Inc. Port Saint Lucie, FL, USA)

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for 2 min. After stomaching, the supernatant was serially diluted with 0.1% BPW. Surviving

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bacterial populations were enumerated using culture specific media. Sorbitol MacConkey agar (SMA, Difco) supplemented with Cefixime-Tellurite (CT, Oxoid LTD, England) was used for

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enumeration of E. coli O157:H7. Brilliant green agar (BGA, Difco) was used for enumeration of

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Salmonella spp. Oxford base medium agar (OBMA, Difco) was used for enumeration of L. monocytogenes. Baird-Park Agar (BPA, Difco) was used for enumeration of S. aureus. TSA was

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used for total aerobic bacteria (TAB) enumeration. Total coliform count was enumerated using PetrifilmTM (3M Company, MN, USA). Plates and PetrifilmTM were incubated at 37 and 30 °C, respectively for 24–48 h. 2.7. Storage test Fresh vegetables were subjected to DW, CaO+SAEW+FA, and CaO+SAEW+FA+US treatments as

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ACCEPTED MANUSCRIPT 9 CaO+FA+SAEW+US) against target bacteria were chosen for shelf-life, quality, and pathogenic survival tests. After treatments, excess chemical solution on each treated sample was removed with a sterile paper towel. Treated samples were then packaged using a new stomacher bag and stored at 4 °C and room temperature (RT, 23 ± 2 °C). During storage test at 4 °C, samples were

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stored for 14 days and analyzed every 48 h. For storage test at room temperature, samples were

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kept for 7 days and analyzed every 24 h.

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During storage, growth kinetics of TAB, pathogenic bacteria and growth parameters were determined using modified Gompertz model. The end of shelf-life was considered when TAB

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population reached 7.0 log CFU/g (Ragaert et al., 2007). Overall visual qualities (OVQ,

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including color change, textural breakdown, and off odor) were used to determine the end of sensory shelf-life since these parameters reflected major deterioration in sensory quality of

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minimally processed vegetables during storage (Ragaert et al., 2007). These sensory parameters

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were scored using a numeric rating scale proposed by Gomez-Lopez et al. (2008). Sensory evaluation of treated samples was carried out by a trained panel consisting of six persons in a

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special room with individual booth. On each day of testing, panelists were presented with fresh

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produce as reference. The scores were given to each treated sample basing on the organoleptic properties (color change, textural breakdown, and off odor) as followed: 1= excellent; 5 =

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acceptable and 9 = extremely poor. For TAB analysis during storage, the mixture and serial dilution prepared as described earlier were plated onto TSA. These plates were then incubated at 37 °C for 24–48 h. 2.8. Statistical Analyses All inactivation treatment was performed in triplicates with two repetitions. Colonies were then enumerated and microbial counts were expressed as log CFU/mL for each sample.

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ACCEPTED MANUSCRIPT 10 Log CFU/mL reduction in bacterial population was also computed. The mean of each treatment including inactivation (in vitro), decontamination (in vivo), and storage test were subjected to one-way analysis of variance (ANOVA) using SPSS statistics software version 21 (SPSS Inc., IBM Company). Differences between these means were determined using Tukey’s multiple

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comparison. Statistical significance was set at p < 0.05.

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3.1.1 Sanitization effectiveness of treatments on lettuce

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Results and discussion

The mean initial densities of E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella spp.

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on inoculated lettuce were approximately 6.85, 6.43, 6.34, and 6.95 log CFU/g, respectively.

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Inoculated lettuce were dipped in sterile DW, SAEW (30 ppm), FA (0.5 %), CaO (0.2 %), SAEW+FA, and CaO+SAEW+FA for 3 min at room temperature. The 3 min dipping time for

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SAEW along with other treatments was chosen based on previous publications (Forghani & Oh,

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2013; Huang, Xu, Walker, West, Zhang, & Weese, 2006; Nan, Yongyu, Baoming, Wang, Cui, & Cao, 2010) suggesting that it would be a suitable time for experiments with similar nature.

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Effects of SAEW, FA, CaO, SAEW+FA, and CaO+SAEW+FA treatments were prominent

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against all tested microorganisms on lettuce. All sanitizing treatments significantly (p < 0.05) reduced the counts of each pathogen to the range of 1.67-4.45 log CFU/g (Figure 1) compared to

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control treatment (DW). The CaO+SAEW+FA treatment showed the highest (p < 0.05) reduction for each pathogens when compared to other sanitizing treatments. According to reduction of microbial counts on lettuce, these sanitizing treatments were ranked in the following order: CaO, SAEW, FA < SAEW+FA < CaO+SAEW+FA. The reduction abilities for E. coli O157:H7 or L. monocytogenes by these sanitizers in this study were higher when compared to the results of Forghani and Oh (2013) showing 1.23 and 1.2 log

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ACCEPTED MANUSCRIPT 11 CFU/g reduction for E. coli and L. monocytogenes on lettuce, respectively, after treatment with SAEW (pH, 5.2–5.5; ORP, 500–600mV; ACC, 21–22 ppm). The higher activity of SAEW in our study might be attributed to 30 ppm of ACC used in this study. It has been reported that the efficacy of SAEW is highly influenced by the available concentration of chlorine (HOCl, Cl 2,

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and -OCl) (Rahman, Khan, & Oh, 2016). In contrast, Yang, Swem, and Li (2003) have reported a

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reduction of 2.0 log CFU/g for L. monocytogenes, S. Typhimurium, and E. coli on the surface of

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romaine lettuce after 5 min of dip in neutral electrolyzed oxidizing water (pH, 7; ACC, 300 ppm). The difference in the efficacy of EW may be attributed to experimental conditions provided and

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ACC concentration (Rahman, et al., 2016; Tango, Mansur, Kim, & Oh, 2014). Results of FA in

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our study were similar to those of Kondo, Murata, and Isshiki (2006) reporting a reduction of ~2 log CFU/g for bacteria on lettuce after treatment with FA (0.58%). Regarding the antimicrobial

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efficacy of CaO at 0.2% against E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella

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spp. inoculated on lettuce, it showed a total reduction of 1.78-2.1 log CFU/g, similar to results of Yoon, Bae, Jung, Heu, and Lee (2013) showing a reduction of ~1.8 log CFU/g for bacterial

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pathogens on lettuce. CaO is a strongly basic disinfectant. The antimicrobial effect of CaO is due

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to calcium hydroxide which is produced when CaO is mixed with water (Na-ngam, Angkititakul, Noimay, & Thamlikitkul, 2004). However, the exact antimicrobial mechanism of CaO is

2013).

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currently unknown (Bari, Kusunoki, Furukawa, Ikeda, Isshiki, & Uemura, 1999; Yoon, et al.,

3.1.2. Sanitization effectiveness of treatment on spinach Results of microbial inactivation efficacy of different treatments on spinach are shown in Figure 2. The mean initial counts of E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella spp. were 6.90, 6.51, 6.49, and 6.91 log CFU/g, respectively. The same pattern of microbial reduction

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ACCEPTED MANUSCRIPT 12 was observed for all treatments. The highest microbial reduction was observed for CaO+SAEW+FA hurdle treatment, followed by SAEW+FA treatment. These two treatments resulted in significantly higher microbial reduction compared to other sanitizing treatments. Reduction of microbial counts ranged from 2.97 to 3.96 log CFU/g for combined treatments.

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However, the extent of reduced counts for spinach samples with combined treatments

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(SAEW+FA, CaO+SAEW+FA) were lower compared to those for lettuce samples, in agreement

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with results of Forghani and Oh (2013).

For individual treatment (SAEW, FA, or CaO), microbial reduction ranged from 1.32 to 2.36 log

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CFU/g, similar to results of Huang and Chen (2011) after dipping baby spinach leaves in

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chlorinated water (pH 10, ACC 200 ppm) for 5 min which resulted in a reduction of E. coli O157:H7 by 1.2 and 1.4 log CFU/g at 22 and 40° C, respectively. In another study, Rahman,

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Ding, and Oh (2010) have reported that SAEW treatment (pH, 6.3; ACC, 5 ppm) reduced counts

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of E. coli O157:H7 and L. monocytogenes of 1.64 to 2.80 log CFU/g on fresh cut spinach. 3.1.3. Sanitization effectiveness of treatment on sprouts

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Results of microbial inactivation efficacy of different treatments on sprouts are shown in Figure

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3. The mean initial counts of E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella spp. were 7.05, 6.43, 7.02, and 7.01 log CFU/g, respectively. Although all treatments were effective

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against bacteria on sprouts, differences in microbial reduction were observed among treatment groups. The highest reduction by individual treatment was observed for FA with reduction of L. monocytogenes and Salmonella spp. at 2.17 and 2.12 log CFU/g, respectively. Similar reduction for L. monocytogenes and Salmonella spp. (2.4 and to 2.5 log CFU/g, respectively) has been reported previously (Kim, Kim, & Song, 2009) when alfalfa sprouts are dipped in 0.5 % of FA for 3 min. However, the reduction of E. coli O157:H7 in their study is higher (2.69 log CFU/g)

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ACCEPTED MANUSCRIPT 13 as compared to our study (1.65 log CFU/g). The difference in E. coli 0157:H7 count may be attributed to the treatment conditions provided in the beginning of the experiment. Treatment with CaO+SAEW+FA significantly (p < 0.05) reduced microbial counts the most, except with S. aureus, and Salmonella spp. where there was no significant (p > 0.05) difference with

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SAEW+FA treatment compared to other sanitizing treatments. The effect of CaO+SAEW+FA

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was higher against Salmonella spp. (with a reduction of 3.55 log CFU/g). Antimicrobial

spinach.

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3.2. Hurdle enhancement of combined treatments

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efficacies of all treatments were slightly lower for sprouts compared to those for lettuce or

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Inhibitory effects of combined chemical-physical treatments against E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella spp. in sprouts, spinach and lettuce are shown in

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Figure 4. After washing with CaO in combination with SAEW+FA followed by micro-bubbles,

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ultraviolet, and ultrasonication, the count of E. coli O157:H7 on produce was efficiently reduced when compared to treatment with CaO+SAEW+FA. It caused a total reduction of 3.23 to 4.87

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log CFU/g (Figure 4a). The efficacy of CaO+SAEW+FA+US treatment was significantly (p <

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0.05) different compared to that of CaO+SAEW+FA+MB or CaO+SAEW+FA+UV. Microbial reduction remained lower for sprouts compared to that for spinach or lettuce for all the

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treatments due to complex structures of these plants (Mustar & Nazaimoon, 2010; Mustar & Noh, 2013). The increase in microbial reduction could be expected due to the additional physical treatment (micro-bubbles, ultraviolet and ultrasonication). Same pattern of microbial reduction was observed as for E. coli O157:H7 in all produce types against L. monocytogenes, S. aureus, and Salmonella spp. (Figure 4a, b, and 4c). Microbial reduction from CaO+SAEW+FA+US treatment was significantly (p < 0.05) higher compared to that from CaO+SAEW+FA+MB or

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ACCEPTED MANUSCRIPT 14 CaO+SAEW+FA+UV, except for L. monocytogenes on spinach with CaO+SAEW+FA+UV treatment (Figure 4b) and Salmonella spp. on sprouts with CaO+SAEW+FA+MB treatment (Figure 4d). However, the highest reduction was observed for S. aureus and Salmonella spp. on lettuce after treatment with CaO+SAEW+FA+US (at 4.93 and 5.09 log CFU/g, respectively,

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Figure 4c, 4d). These results suggested that ultrasonicaton combined with CaO+SAEW+FA was

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more effective in reducing bacterial pathogens on fresh produce than other treatments.

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Micro-bubbles has already been utilized in the food industry alone or in combination with other sanitizers such as NaOCl, acetic acid, and citric acid (Klintham, Tongchitpakdee, Chinsirikul, &

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Mahakarnchanakul, 2013). When coriander, march mint, asparagus, okra, lemongrass, and

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ginger samples are washed for 15 minutes with micro-bubbles water, E. coli and Salmonella are significantly (p < 0.05) reduced (by 0.8-2.2 log and 0.6-2.7 log CFU/mL, respectively) compared

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to those with control treatment (tap water) (Klintham, et al., 2013). In addition, they reported that

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a combination of micro-bubbles with NaOCl, acetic acid, and citric acid wash for 5 min can reduce E. coli on inoculated Chinese kale by 5.5 log CFU/mL, which was significantly higher

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than other treatments (Klintham, et al., 2013). Results from Klintham, et al. (2013) and results of

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this study suggest that it is possible to achieve maximum microbial inactivation while retaining food quality with a proper combination of disinfection hurdles. Other physical treatments such as

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ultraviolet and ultrasonication have also been utilized in the food industry to control microbes. The U.S. Food and Drug Administration and US Department of Agriculture have concluded that the use of ultraviolet irradiation is safe. However, a single process of ultrasound and ultraviolet treatment cannot significantly reduce high-load microbial contamination (Gayán, Serrano, Raso, Álvarez, & Condón, 2012; Luo & Oh, 2015; Sagong, et al., 2011). Ultrasonication treatment in combination with SAEW (Forghani & Oh, 2013; Luo, et al., 2015), CaO (Yoon, et al., 2013),

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ACCEPTED MANUSCRIPT 15 heat (Condon-Abanto, Arroyo, Alvarez, Condon, & Lyng, 2016), or essential oil (Millan-Sango, McElhatton, & Valdramidis, 2015) has been investigated. Ultrasonication treatment should be kept to the minimum because of its destructive nature (Forghani & Oh, 2013) which can potentially promote degradation of pectin (Zhang, Lu, Li, Shang, Zhang, & Cao, 2011), resulting

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3.3. Sanitization effectiveness of treatment on natural microflora

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in softening of vegetable texture.

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Results for the effectiveness of combined treatment (CaO+FA+SAEW, CaO+FA+SAEW+US) for natural microbiota on fresh produce are shown in Figure 5. The initial microbial counts of

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TAB on lettuce, spinach, and sprouts were 5.28, 5.49, and 5.80 log CFU/g, respectively. In

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comparison with control (DW), CaO+FA+SAEW and CaO+FA+SAEW+US treatments resulted in significantly (p < 0.05) higher reduction for microbial population of TAB, ranging from 2.85

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to 3.54 log CFU/g. For both treatments, their ability of reducing microbial counts on different

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fresh produce could be ranked in the following: lettuce > spinach > sprouts. The efficacy of these sanitizing treatments was more prominent for microbes on lettuce, in agreement with results

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reported by Forghani and Oh (2013) and Yang, et al. (2003). The initial total coliform counts on

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lettuce, spinach, and sprouts were 3.09, 2.88, and 4.35 log CFU/g, respectively. Both combined treatments (CaO+FA+SAEW and CaO+FA+SAEW+US) significantly (p < 0.05) reduced

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microbial counts (in the range of 2.85 to 3.54 log CFU/g) compared to the control. The highest reduction was observed for CaO+FA+SAEW+US treatment with reduction on lettuce and spinach of 3.54 and 3.39 log CFU/g, respectively. 3.4. Shelf-life Results of microbial population change during 14 days of storage are shown in Figure 6. The end of shelf-life of a fresh produce is considered when TAB reaches the maximum acceptable level

15

ACCEPTED MANUSCRIPT 16 of ≥ 7 log CFU/g (V. Gómez-López, Devlieghere, Ragaert, & Debevere, 2007; Luo, et al., 2015). Results of the present study demonstrated that the TAB of the untreated group (DW) during storage at 4 and 23 ± 2 ºC exceeded the unacceptable levels (≥ 7 log CFU/g) within 6 and 3 days, respectively. The microbial count on treated (CaO+SAEW+FA) lettuce and spinach stored at 4

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and 23 ± 2 ºC exceeded the unacceptable level after 12 and 6 days, respectively, while microbial

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count on sprouts during storage at 4 and 23 ± 2 ºC exceeded the unacceptable level after 10 and 6

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days, respectively. The CaO+SAEW+FA treatment effectively enhanced the shelf-life of produce stored at 4 and 23 ± 2 ºC by about 6 days and 3 days, respectively. Interestingly, results

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for CaO+SAEW+FA+US treatment revealed that TAB level on spinach and lettuce stored at 4

AN

and 23 C remained below 7 log CFU/g throughout the storage period, reflecting that this treatment effectively extended their shelf-life during storage at 4 and 23 ± 2 ºC by about 8 days

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and 4 days, respectively. However, TAB level for sprouts stored at 4 and 23 ± 2 ºC exceeded the

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unacceptable level after 10 days and 5 days, respectively, thus increasing the shelf-life by 5 and 2

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days, respectively. Based on microbial properties of these fresh produce, 3 min of washing with CaO followed by 3 min of SAEW+FA+US treatment was determined as the optimum treatment

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among all treatments. Previously, a two-step sanitizing treatment for fresh produce has been reported (Ajlouni, Sibrani, Premier, & Tomkins, 2006; Forghani, Rahman, et al., 2013; Huang, et

AC

al., 2006; Koseki, Yoshida, Kamitani, Isobe, & Itoh, 2004; Luo, et al., 2015; Scouten & Beuchat, 2002). However, to the best of our knowledge, no study has reported the sanitizing effect of 3step treatment using SAEW, FA, and CaO with ultrasonication for fresh produce. During storage, changes in quality of produce type might be due to physicochemical stress caused by each treatment (CaO+SAEW+FA, CaO+FA+SAEW+US). To investigate this risk, the effect of optimum treatment on the quality of produce type during storage at 4 and 23 ± 2 ºC for

16

ACCEPTED MANUSCRIPT 17 14 and 7 days was determined through texture analyses. Results are shown in Tables 2 and 3. The CaO+FA+SAEW+US treatment mildly affected the quality of produce stored at 23 ± 2C. Microbial count on sprouts, lettuce, and spinach reached the unacceptable level at 3, 4, and 5 days, respectively (Table 2). The same pattern of deterioration of produce stored at 23 ± 2C

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observed for the CaO+FA+SAEW+US treatment was also observed for all produce types stored

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at 4 °C. However, as expected, the shelf-life of these produce was doubled. Sprouts, lettuce, and

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spinach lasted for 8, 10, and 12 days, respectively. The detrimental effect of

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CaO+FA+SAEW+US treatment was higher than CaO+FA+SAEW treatment. The detrimental effect of CaO+FA+SAEW+US might be attributed to ultrasonication application. Its detrimental

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effect was also temperature-dependent. It was increased with increasing temperature. The shelflife of produce at 23 ± 2 C was shorted compared to that at 4 C. Nyborg (1978) has reported

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that ultrasonic cavitation events can induce biological changes in plant tissues. In another study,

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(Ter Haar, Dyson, & Smith, 1979) have reported that cavitation can induce cell damage due to

PT

several energy density. It has been suggested that the highly localized tissue damage might be partly due to increased temperatures and pressures related to unstable collapse of a bubble (Ter

CE

Haar, et al. (1979). Both thermal and non-thermal factors might be involved in the interaction

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between ultrasonication and plant tissues. Acoustic microstreams produced during oscillation of stable bubbles in ultrasonic field (Miller, 1979) can produce shear forces and lead to tissue damage and cell disruption (Zhou, 2011). To provide residual protection against pathogens growth after treatment, TAB growth on spinach, lettuce, and sprouts during storage period was evaluated. Growth curves and parameters (lag time and growth rate) are showed in Fig. 6 and Table 4. At the first stage of storage, mean TAB counts on spinach treated with CaO+FA+SAEW and CaO+FA+SAEW+US were 2.95 ± 0.49

17

ACCEPTED MANUSCRIPT 18 and 2.60 ± 0.41 log CFU/g, respectively. TAB counts on samples treated with CaO+FA+SAEW+US remained low (under shelf-life limit) throughout the storage period. However, TAB counts on samples treated with CaO+FA+SAEW reached 7.03 ± 0.07 log CFU/g after 12 days of storage. TAB counts on DW treated samples were 4.64 ± 0.17 log CFU/g at day

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0. They reached shelf-life limit after 5 days of storage at 4 °C (Fig. 6b). Mean TAB counts on

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samples treated with DW, CaO+FA+SAEW, and CaO+FA+SAEW+US were 4.39 ± 0.18, 3.05 ±

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0.68, and 2.74 ± 0.56 log CFU/g, respectively. After 6, 12, and 14 days of storage at 4 °C, they reached 7.60 ± 0.75, 4.31 ± 0.83, and 7.13±0.14 log CFU/g, respectively (Fig. 6a). For sprouts

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treated with DW, CaO+FA+SAEW, and CaO+FA+SAEW+US treatments stored at 4 °C, TAB

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counts at day 0 were 4.98 ± 0.17, 3.29 ± 0.07, and 3.27 ± 0.32 log CFU/g, respectively. They exceeded shelf-life limit after 5, 10, 12 days of storage, respectively.

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TAB populations on spinach and lettuce treated with CaO+FA+SAEW+US remained under the

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shelf-life limit during storage period at 23 ± 2 °C (Fig. 6d and 6e). TAB populations on sprouts treated with the same combined treatment reached shelf-life limit after 6 days of storage. TAB

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populations treated with CaO–FA+SAEW exceeded the shelf-life limit after 6 days for all fresh

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produce types examined in this study (Fig. 6f). However, TAB washed with DW reached the shelf-life limit after 3 days of storage at RT. These results indicated that combined treatments

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developed in the present study might have potential to reduce naturally occurring bacteria and extend the shelf-life of fresh vegetables. They extended the shelf-life of produce for 6 days or more at 4 °C (except sprouts treated with CaO+FA+SAEW which was extended for 5 days). They also extended the shelf-life of produce for 3 days for samples stored at RT. When growth data were fitted to a modified Gompertz model, results showed that growth parameters lag time and growth rate were not likely to be affected by treatments or temperatures of storage (Table 4).

18

ACCEPTED MANUSCRIPT 19 Thus, the extension of shelf-life observed in the present might be due to the great microbial reduction

obtained

after

fresh

produce

was

treated

with

CaO+FA+SAEW

or

CaO+FA+SAEW+US. Regarding the end of shelf-life of vegetable which is usually established at 7 log CFU/g, its average shelf-life is typically 10–14 days (Mukhopadhyay, Ukuku, Juneja, &

T

Fan, 2014). These results demonstrated that CaO+SAEW+FA and CaO+SAEW+FA+US

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treatments might be useful to control naturally occurring microorganisms on fresh produce.

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Conclusion

A combination of CaO, SAEW, and FA sanitizers appears to be a favorable method to ensure

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microbial safety as well as to prolong the shelf-life of fresh produce with little damaging effect

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on their quality. Using physical treatment such as ultrasonication can enhance microbial reduction. At the same time, it might impair the quality of produce compared to other treatments.

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Alternatively, CaO+FA+SAEW without physical treatment could be used to increase microbial

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reduction and improve fresh produce quality. To the best of our knowledge, the application of a combined sequential CaO, SAEW, and FA treatment for controlling microorganisms on lettuce,

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spinach, and sprouts has not been reported previously. The synergistic effect of CaO, SAEW,

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and FA might offer valuable method for the reduction of bacterial pathogens on fresh produce. In general, a combination of different sanitizers can maintain better microbial quality for food

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product. Therefore, more studies in this area need to be performed to determine the right combination of disinfection technologies for various food products. Acknowledgment This study was supported by IPET Korea No. 314059-3 (Korea institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries). It was also partly supported by a research grant of Kangwon National University.

19

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Table 1. Treatments conditions, properties and procedure used in the study. Types of treatment

Treatment conditions Treatment properties

Treatment procedure

DW

pH 7.01

3 min washing

SAEW

ACC 30 ppm, pH 5.4

3 min washing

FA

0.5 %, pH 2.5

3 min washing

CaO

0.2%, pH 12.5

3 min washing

SAEW+FA

30 ppm + 0.5%, pH 3.2

3 min washing

CaO+SAEW+FA

0.2% + 30 ppm + 0.5%

CaO+SAEW+FA+US

CaO+SAEW+FA+MB

CaO+SAEW+FA+UV

C S

A

3 min CaO washing followed by 3 min of

M

40 KHz

SAEW+FA+US

SAEW+FA washing

D E

T P

0.2% +30 ppm + 0.5% + MB

0.2% + 30 ppm + 0.5% + UV-C

C A

I R

U N

0.2% + 30 ppm + 0.5% + US

E C

T P

3 min CaO washing followed by 3 min

3 min CaO washing followed by 3 min SAEW+FA+MB 3 min CaO washing followed by 3 min of SAEW+FA washing, followed by 10 min ultraviolet

DW: distilled water; SAEW: slightly acidic electrolyzed water; FA: fumaric acid; CaO: calcium oxide; SAEW+FA: slightly acidic electrolyzed water + fumaric acid; CaO+SAEW+FA: calcium oxide + slightly acidic electrolyzed water + fumaric acid; CaO+FA+SAEW+UV: calcium oxide + slightly acidic electrolyzed water + fumaric acid + ultraviolet treatment; CaO+FA+SAEW+MB: calcium oxide + slightly acidic electrolyzed water + fumaric acid + microbubble treatment;

26

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CaO+FA+SAEW+US: calcium oxide + slightly acidic electrolyzed water + fumaric acid + ultrasonication treatment. ACC: available chlorine concentration.

T P

I R

C S

U N

A

D E

M

T P

E C

C A

27

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Table 2. Overall visual quality of treated vegetables during storage at room temperature. Vegetables Treatment

Lettuce

Spinach

Sprouts

Time (day) 0

1

DW

1.2±0.3a

CaO-FA-SAEW

3

4

1.8±0.4ab 2.4±0.5a

3.2±0.2a

3.6±0.5a

1.2±0.2a

1.5±0.2a

2.3±0.2a

3.3±0.4a

CaO-FA-SAEW+US

1.2±0.2a

1.9±0.3b

2.9±0.3b

DW

1.2±0.3a

1.4±0.5a

2.0±0.4a

CaO-FA-SAEW

1.2±0.4a

1.6±0.5a

M

CaO-FA-SAEW+US

1.2±0.5a

1.6±0.3a

DW

1.5±0.2a

6

7

4.70.6a

5.0±0.6a

5.5±0.4a

4.60.2b

4.8±0.4a

5.1±0.4a

6.7±0.3b

3.7±0.4b

5.0±0.3c

5.6±0.5b

6.4±0.3b

7.1±0.4c

2.5±0.3a

3.9±0.1a

3.3±0.4a

4.9±0.4a

5.2±0.6a

2.1±0.3a

2.8±0.4b

3.5±0.5b

4.7±0.3b

5.3±0.5b

5.5±0.5b

2.6±0.2b

3.9±0.2c

4.5±0.4c

5.6±0.5c

6.2±0.5c

7.3±0.3c

2.6±0.3a

2.8±0.6a

3.4±0.2a

4.1±0.4a

4.4±0.6a

5.5±0.5a

1.5±0.2a

2.4±0.2ab 2.8±0.3a

3.2±0.4b

3.7±0.3b

4.8±0.5b

5.4±0.5b

5.8±0.4b

1.9±0.3b

2.6±0.4b

5.1±0.2c

5.4±0.3c

6.2±0.7c

6.6±0.4c

7.2±0.5c

T P

E C

CaO-FA-SAEW

CaO-FA-SAEW+US

C A

T P 5

D E

2.2±0.2a

2

3.6±0.4b

C S

U N

A

I R

OVQ scored as, 1: excellent; 5: acceptable; 9: extremely poor. Numbers in bold indicate scores above the acceptability limit. Within the same column, values not followed by the same lowercase letter indicate significantly (p < 0.05) different between treatments. DW: distilled water; CaO+SAEW+FA: calcium oxide + slightly acidic electrolyzed water + fumaric acid treatment; CaO+FA+SAEW+US: calcium oxide + slightly acidic electrolyzed water + fumaric acid + ultrasonication treatment. 28

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Table 3. Overall visual quality of treated vegetables during storage at 4°C Vegetables Treatment

Lettuce

Spinach

Time (day) 0

2

4

6

8

DW

1.2±0.1a

1.2±0.2a

14±0.3a

1.9±0.3a

2.5±0.5a

CaO+FA+SAEW

1.2±0.1a

1.2±0.2a

1.6±0.4b

2.1±0.4b

CaO+FA+SAEW+US 1.2±0.2a

1.2±0.4a

1.9±0.3c

DW

1.2±0.2a

1.2±0.2a

1.5±0.2a

CaO+FA+SAEW

1.2±0.2a

1.2±0.3a

M

CaO+FA+SAEW+US 1.2±0.3a

1.4±0.4b

12

14

3.2±0.2a

4.4±0.6a

5.1±0.4a

2.8±0.4b

3.9±0.5b

4.7±1.3b

5.6±0.6b

2.7±0.5c

3.5±0.7c

5.1±0.6c

5.9±0.4c

6.3±0.5c

1.9±0.6a

2.4±0.9a

3.4±0.4a

4.7±0.6b

5.3±0.7a

1.6±0.5a

1.8±0.4a

2.3±0.6a

3.5±0.9a

4.3±0.8a

5.8±0.8b

1.8±0.4b

2.8±0.6b

3.6±0.4b

4.6±0.5b

5.4±1.2c

5.8±0.9b

2.1±0.1ab 2.5±0.3a

2.9±0.5a

3.6±0.4a

4.3±1.2a

5.0±0.6a

5.2±0.87a

1.5±0.1a

2.0±0.3a

2.8±0.4b

3.8±0.9b

4.7±0.6b

5.0±1.5b

5.3±0.4b

6.0±0.5b

CaO+FA+SAEW+US 1.9±0.2a

2.2±0.2a

3.5±0.2c

4.6±0.4c

5.1±0.8c

5.7±0.4c

6.4±0.5c

6.8±0.7c

DW Sprouts

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1.5±0.1a

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CaO+FA+SAEW

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OVQ scored as, 1: excellent; 5: acceptable; 9: extremely poor. Numbers in bold indicate scores above the acceptability limit. Within the same column, values not followed by the same lowercase letter indicate significantly (p < 0.05) different between treatments. DW: distilled water; CaO+SAEW+FA: calcium oxide + slightly acidic electrolyzed water + fumaric acid treatment; CaO+FA+SAEW+US: calcium oxide + slightly acidic electrolyzed water + fumaric acid + ultrasonication treatment. 29

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Table 4. Growth parameters of total aerobic bacteria on treated vegetables during storage at 4 °C and room temperature. Vegetables Treatment

Lag time (h) 4 °C

Lettuce

RT

DW

14.15±2.3a 7.20±1.6a

CaO+FA+SAEW

23.11±1.6b 5.60±1.7a

CaO+FA+SAEW+US 7.14±3.1a

Spinach

DW

3.82±2.5a

CaO+FA+SAEW

12.0±1.8b

A

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0.002±0.001a 0.004±0.002a

C S

U N

6.79±2.4a

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4 °C

0.004±0.002a 0.004±0.001a 0.002±0.012a 0.006±0.002b

17.52±3.1b 0.001±0.003a 0.003±0.002a 2.72±3.2a

0.003±0.004a 0.002±0.002a

CaO+FA+SAEW+US 11.96±1.4b 2.53±2.8a

0.002±0.002a 0.003±0.004a

D E

T P

DW Sprouts

Growth rate (log cfu/g/h)

9.60±2.5a

2.89±1.4a

0.001±0.001a 0.005±0.001a

9.15±2.7a

6.92±1.3c

0.003±0.002a 0.004±0.003a

CaO+FA+SAEW+US 9.26±1.3a

4.39±1.5b

0.002±0.013a 0.004±0.002a

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CaO+FA+SAEW

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Within the same column, values not followed by the same lowercase letter are significantly (p < 0.05) different between treatments. RT: room temperature (23 ± 2 C); DW: distilled water; CaO+FA+SAEW: calcium oxide + slightly acidic electrolyzed water + fumaric acid treatment; CaO+FA+SAEW+US: calcium oxide + slightly acidic electrolyzed water + fumaric acid + ultrasonication treatment. 30

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Figure Legends Figure 1. Effectiveness of different treatments on bacterial decontamination on lettuce. Bars labeled with different letters in the same reduction group indicate significant (p < 0.05)

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difference. DW: distilled water; SAEW: slightly acidic electrolyzed water (30 ppm); FA:

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fumaric acid (0.5%); CaO: calcium oxide (0.2%); FA+SAEW: fumaric acid combined with

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SAEW; CaO+FA+SAEW: calcium oxide followed by combined FA with SAEW treatment. EC: Escherichia coli O157:H7; LM: Listeria monocytogenes; SA: Staphylococcus aureus; ST:

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Salmonella spp. Initial populations of E. coli O157:H7, L. monocytogenes, S. aureus, and

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Salmonella spp. on lettuce used for evaluating the efficacy of sanitizing treatments either alone or in combination were approximately 6.85, 6.43, 6.34, and 6.95 log CFU/g,

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Figure 2. Effectiveness of different treatments on bacterial decontamination on spinach. Bars

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labeled with different letters in the same reduction group indicate significant (p < 0.05)

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difference. DW: distilled water; SAEW: slightly acidic electrolyzed water (SAEW); FA: fumaric acid; CaO: calcium oxide, FA+SAEW: fumaric acid combined with SAEW;

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CaO+FA+SAEW: calcium oxide followed by combined FA with SAEW treatment. EC: Escherichia coli O157:H7; LM: Listeria monocytogenes; SA: Staphylococcus aureus; ST: Salmonella spp. Initial populations of E. coli O157:H7, L. monocytogenes, S. aureus, and Salmonella spp. on spinach used for evaluating the efficacy of sanitizing treatments either alone or in combination were approximately 6.90, 6.51, 6.49, and 6.91 log CFU/g, respectively.

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Figure 3. Effectiveness of different treatments on bacterial decontamination on sprouts. Bars labeled with different letters in the same reduction group indicate significant (p < 0.05) difference. DW: distilled water; SAEW: slightly acidic electrolyzed water (SAEW); FA:

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fumaric acid; CaO: calcium oxide, SAEW+FA: fumaric acid combined with SAEW;

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CaO+SAEW+FA: calcium oxide followed by combined SAEW with FA treatment. EC:

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Escherichia coli O157:H7; LM: Listeria monocytogenes; SA: Staphylococcus aureus; ST: Salmonella spp. Initial populations of E. coli O157:H7, L. monocytogenes, S. aureus, and

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Salmonella spp. on lettuce used for evaluating the efficacy of sanitizing treatments either

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alone or in combination were approximately 7.05, 6.43, 7.02, and 7.01 log CFU/g, respectively.

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Figure 4. Bacterial reduction on produce type using different combined treatments against

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Escherichia coli O157:H7 (a), Listeria monocytogenes (b), Staphylococcus aureus (c), and Salmonella spp. (d). Values shown are mean ± standard deviation. Bars labeled with different

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letters in the same reduction group indicate significant (p < 0.05) difference.

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CaO+SAEW+FA+MB: calcium oxide washing followed by slightly acid electrolyzed water + fumaric acid + microbubble; CaO+SAEW+FA+UV: calcium oxide washing followed by

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CaO+FA+SAEW: calcium oxide washing followed by combination of FA + SAEW treatment; CaO+FA+SAEW+US: calcium oxide washing followed by combination of FA + SAEW + ultrasonication treatment; TAB: Total aerobic bacteria; COL: total coliform count.

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Figure 6. Effect of various decontamination treatments on growths of total aerobic bacteria on

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fresh produce during storage at 4 °C and room temperature. Values shown are mean ±

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standard deviation. DW: distilled water; CaO+SAEW+FA: calcium oxide washing followed by slightly acid electrolyzed water + fumaric acid treatment; CaO+SAEW+FA+US: calcium

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oxide washing followed by slightly acid electrolyzed water + fumaric acid + ultrasonication

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treatment. a: lettuce at 4 °C; b: Spinach at 4 °C; c: sprouts at 4 °C; d: Lettuce at room

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represents the end of shelf life.

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Highlights  Hurdle technology has been used in the food industry to ensure food quality and safety.  Individual preservative treatments in this study were not as effective as combinations of treatments.  Introduction of ultrasonication to combined calcium oxide, slightly acidic electrolyzed water and fumaric acid (CaO+SAEW+FA) treatment caused slight detrimental effects in food products.  CaO+SAEW+FA treatment has shown higher reduction (~4 log CFU/g) and enhanced the shelf life by 6 days at 4 ºC.

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