The annual Research Poster Session features cutting-edge research on food safety and quality aspects of fresh and fresh-cut produce, from an international range of researchers and research organizations, providing information and new technologies to the produce industry. Posters will be available for viewing during show floor hours. Researchers will be available at their posters to discuss their research on Tuesday, June 21 from 2:00-4:00 p.m.

2016 FOOD SAFETY RESEARCH POSTER SESSION

Antimicrobial Effect of Haskap Berry Juice and Extracts on Listeria innocua and E. coli by Ban, Z., L. Fan*, W. Kalt, M. Vinqvist-Tymchuk, T. Hughes, C. Doucette and J. Guan*Kentville Research and Development Centre, AAFC, Nova Scotia, Canada

Haskap berries are the one of the rising stars of berry fruits in the Canadian marketplace. They can be used in the same way as other berries including fresh eating, jams and preserves, baking and juice. Research reports have shown that haskap berries have high levels of anti-oxidants, vitamin C and A along with high fibre and potassium.

In our study, haskap berry juice, fractions including whole fruit extracts (EXT), sugars/acids (S/A), total phenolics (TP), total flavonoids (TF), and chlorogenic acid (CA) as well as a range of pure phenolic acids were investigated for their antimicrobial effects against Listeria innocua and E. coli. Antimicrobial properties of different concentration of haskap berry juice and extracts were determined based on the OD values and bacterial growth curves generated using Bioscreen C. Live/dead BacLight bacterial viability kits were used for quantitative assays of bacterial cell damage tested with fluorescence microplate reader. This assay utilized the SYTO 9 and propidium iodide (PI) stains. When used alone, the SYTO 9 labelled all bacterial cells, while PI worked only on bacteria with damaged membranes, causing a reduction in the SYTO 9 stain fluorescence when both stains were present. Haskap berry fractions on the membrane permeability and survival of L. innocua and E. coli were determined and relative fluorescence units (RFU) were used to express the degree of cell damage. Our results indicated that TP, TF and CA played an important role in damaging cell membrane of L. innocua while TP and CA also damaged cell membrane of E. coli. Phenolic acids from haskap berry including salicylic acid, caffeic acid, m-coumaric acid, and ferulic acid were found to have strong antimicrobial effects with minimum inhibitory concentrations (MICs) ranging from 2.5-5.0 mg/mL. The antimicrobial effects of haskap berry fractions such as anthocyanins (ACN), heteropolymers (HET) and proanthocyandins (PAC) on food pathogens have been testing in our lab.

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Application of Comparative Genetic Diversity Markers and Whole Genome Sequencing of Listeria monocytogenes for Spatial Mapping and Source Tracking in Produce Packing Facilities by Janneth Pinzon1, Mariya Skots1, Adrian Sbodio1, Jeremy Roland1, Trevor V Suslow1 1Department of Plant Sciences, University of California, Davis, CA

Genetic diversity and molecular typing of Listeria monocytogenes are routinely carried out by Pulse-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) has emerged as a powerful tool for the subtyping and comparison of L. monocytogenes. While WGS-methods have been primarily applied retrospectively within culture collections or to identify and characterize an emerging outbreak, these techniques are highly informative in spatial- mapping and root-cause investigations within an operation developing an Environmental Monitoring Program (EMP). To evaluate the use of routine prospective WGS-based methods compared with the conventional molecular sub-typing methods, including PFGE and PCR serogrouping for source tracking of L. monocytogenes in produce packing environments. Sterile, neutralizing agent pre-wetted sponges and swabs (n 35-58/date and facility) were collected from multiple produce packing facilities during a two year period. Samples were enriched and following incubation were subjected to Atlas (Roka Biosciences) Listeria protocols. CHROMagar Listeria incubated at 37 ºC for 48 h was used for culture isolation with qPCR using 23S rDNA and hlyQ gene targets for Listeria spp. and L. monocytogenes, respectively. Molecular serotyping was conducted using multiplex PCR. PFGE was performed according to the standardized Centers for Disease Control PulseNet protocol using restriction enzymes AscI and ApaI. PFGE patterns were analyzed using BioNumerics 5.10. Genomic DNA from overnight cultures of L. monocytogenes was extracted using the Qiagen Genomic-tip 20/G and Genomic DNA Buffer set (Qiagen, Hilden, Germany). The DNA samples were processed according to Illumina instructions generating Nextera XT paired-end libraries, which were sequenced using the Illumina HiSeq system with minimum 50X coverage and average read length 150 - 250 bp by arrangement with Noblis, Inc. Prior to alignment, trimming was conducted on raw FASTQ sequences using the UrQt tool with a probabilistic model. Noblis BioVelocity™ software was used to align the trimmed reads to a reference database and calculated Single Nucleotide Polymorphisms (SNPs) and coverage. A matrix based on the collection of SNPs was constructed in collaboration with specialists at Noblis, Inc. from each sample against the selected reference genome and a phylogenetic tree was generated by MEGA (Molecular Evolutionary Genetics Analysis). A total of 180 L. monocytogenes were isolated from diverse produce packing facilities over two years. A subset of 81 isolates was selected for further characterization by WGS. Molecular serotyping identified group IIb (66.6%), which is usually serotype 1/2b, group

IVb (17.3%) corresponding typically to serotype 4b, group IIa (11.2%) which is typically 1/2a and IVb-v1 (4.9%), a recently described variant of serotype 4b. ApaI and AscI PFGE analyses yielded 35 and 45 patterns respectively, producing a total of 67 PFGE profiles in a dual analysis. Three isolates were untypeable by PFGE. Indistinguishable and closely related pulsotypes were observed in the same packing facility on multiple sample collection dates suggesting persistence or residency of a given strain. Furthermore, some L. monocytogenes isolates recovered from different facilities (geographically far apart), in different years were found to have indistinguishable or highly related PFGE pulsotypes. WGS-SNP based comparison revealed isolates with a given PFGE type clustered with isolates representing one or two PFGE pulsotypes. Within a single PFGE group there was considerable genetic diversity. The results of this preliminary study underscore the utility of multiple approaches, such as molecular serotyping, PFGE and WGS analysis to improve source track surveillance. Although WGS tools have evolved to point that provides timely, meaningful and relatively low-cost data for source tracking surveillance, the need of standardized methodology and analysis are still a challenge in order to apply this technology for resolving resident and transient isolates and real-time surveillance within an EMP.

Development and validation of an alternative alfalfa sprouting seed disinfection strategies: effect of mild heat and non-chlorine sanitizers by Bach, Susan1, Delaquis, Pascal1, Wang, Siyun2, and Wakeling, Carmen3 1 Agriculture & Agri-Food Canada, Summerland Research and Development Centre, 4200 Highway 97, Summerland, BC, Canada, V0H 1Z0 2 Food, Nutrition and Health, Faculty of Land and Food Systems, UBC, 241-2205 East Mall, Vancouver, BC, Canada V6T 1Z4 3 Eatmore Sprouts & Greens Ltd., 2604 Grieve Road, Courtenay, BC, Canada V9J 1S7

Many outbreaks of foodborne illness caused by Salmonella enterica, toxigenic Escherichia coli and Listeria monocytogenes have been linked to sprouted vegetables. Seed disinfection is a critical factor for the reduction of risks associated with such pathogens. The current Canadian Code of Practice for the Hygienic Production of Sprouted Seeds recommends treatment of alfalfa seed with a 2000 ppm calcium hypochlorite solution for 20 minutes or 6-10% hydrogen peroxide (H2O2) solution for 10 minutes. Such treatments entail the use of concentrated chemicals in the production environment, with attendant risks to worker health and safety. Moreover, previous research has indicated that neither treatment can eliminate pathogens on alfalfa seed. The purpose of our research is to develop an effective disinfection strategy for alfalfa seed using treatments and sanitizers compatible with organic production principles. A two-step treatment consisting of a soak in water at 50˚C followed by exposure to a mixture of H2O2 and acetic acid was first evaluated for effects on the

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germination rate and weight (yield) of sprouted alfalfa seed after treatment. Alfalfa seeds soaked in water at 50˚C for 10 minutes followed 2% H2O2 + 0.1% acetic acid (diluted vinegar) or 4% H2O2 + 0.2% acetic acid for up to 10 minutes germinated at the same rate and provided the same yield as seed soaked in water at room temperature. Alfalfa seeds were then inoculated with 5 log cfu/g of S. enterica, E. coli O157:H7 and L. monocytogenes to examine the antimicrobial efficacy of the treatment. Enrichment was required to detect each species after treatment of the seed with water at 50˚C for 10 minutes, followed by exposure to a 4% H2O2 + 0.2% acetic acid solution at room temperature for 10 minutes. This exceeded reductions achieved with a 2000 ppm calcium hypochlorite for 20 minutes (4 log cfu/g) and was equivalent to that achieved by treatment of the seed with 8% H2O2 for 10 minutes.

Development of metal-organic framework for ethylene encapsulation to facilitate user-friendly ripening stimulation by Boce Zhang1, Yaguang Luo1*, Kelsey Kanyuck2, Gary Bauchan1, Joseph Mowery1, Peter Zavalij2 1 U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD, U.S. 2 University of Maryland, College Park, MD, U.S.

Controlled ripening of climacteric fruits is a critical step to provide consumers with high-quality products while reducing post-harvest losses. Prior to ripening, these fruits can be stored for an extended period of time, but are usually not suitable for consumption. However, once ripening is initiated, they undergo irreversible changes that lead to rapid quality loss and decay if not consumed within a short window of time. Therefore, technologies to stimulate ripening immediately before consumption are in high demand. In this study, we developed a copper terephthalate metal-organic framework (CuTPA-MOF) to encapsulate gaseous ethylene for subsequent release. We evaluated the feasibility of this solid porous material for on-demand stimulated ripening of bananas and avocados. The CuTPA-MOF was synthesized via a solvothermal method and loaded with ethylene gas. Its morphology and crystal structure were characterized by electron microscopy and X-ray diffraction crystallography, and the porosity was further evaluated by N2 and ethylene isotherms. The MOF-ethylene was placed inside sealed containers with pre-climacteric bananas and avocados and stored at 16 °C. The headspace gas composition, fruit color, and texture were monitored periodically. Results showed that this CuTPA MOF is highly porous with a total pore volume of 0.39 cm3/g. A 50 mg portion of MOF-ethylene can absorb and release up to 654 µL/L ethylene in a 4-L container. MOF-ethylene significantly accelerated the ripening-related color and firmness changes of treated bananas and avocados. This result suggests that MOF-ethylene technology could be used for postharvest application to stimulate ripening just before consumption.

Effects of PROSAN, a Biodegradable Vegetable Wash, on Pathogen Viability and Selected Quality Characteristics in Fresh Basil and Cilantro by Aubrey F. Mendonça1, Aura Daraba1,2, Floyd Woods3, and Angela M. Shaw1 1Iowa State University, Ames IA 50011; 2University “Dunarea de Jos” of Galati, Romania; and 3Auburn University, Auburn, Alabama

Outbreaks of foodborne illness linked to enteric pathogens on fresh basil and cilantro have raised concerns about the safety of these herbs. Fresh basil and cilantro (inoculated with Salmonella or Listeria monocytogenes) were immersed in distilled water (H2O), chlorine (CHL; 100 ppm), or two separate PRO-SAN solutions (PRO 1% or PRO 2%) for 2 minutes then analyzed for live pathogens. Log10 reductions of pathogens on basil immersed in H2O, CHL, PRO 1% and PRO 2% were 1.09, 1.70, 1.65, and 2.00 (Salmonella), and 1.18, 1.84, 1.89, and 2.20 (Listeria), respectively. Reductions on cilantro after immersion in H2O, CHL, PRO 1% and PRO 2% were 1.06, 1.54, 1.48, and 2.14 (Salmonella), and 1.22, 1.64, 1.75, and 2.38 (Listeria), respectively. PRO-SAN solutions did not significantly change the general appearance (color) or any of the quality characteristics tested for both products. Results of this study indicate that PRO-SAN (2%) has good potential for use as a sanitizer to destroy Salmonella and E. coli O157:H7 on the surface of whole cucumbers without altering the overall quality of these vegetable products.

Food quality and safety of packaged leafy greens after storage in a refrigerated display case with doors by J. Atilio de Frias, Yaguang Luo, Bin Zhou, Ellen Turner, Patricia Millner, and Xiangwu Nou. USDA-ARS, Beltsville, MD

Food safety risks are minimized in the cold chain of packaged ready-to-eat leafy greens when temperatures are kept at 41°F (5ºC) or less. The 2009 US Food Code set this requirement to minimize the potential of pathogen proliferation in the supply chain. However, infiltration of ambient air into the open-refrigerated cases subjects product to temperature fluctuations that are above 41°F (5°C). To address this problem, we evaluated several options in a state-of-the-art research supermarket facility at USDA Agricultural Research Service (ARS), which included the use of insulators inside display cases, or installation of curtains or glass doors. The latter were determined as the best solution to improve the shelf life and quality of packaged leafy greens, while providing significant energy savings when compared to open display cases. For the validation of our quality and safety studies comparing an open case and a display case with glass doors, thermostat settings were set at 33°F (0.6°C), ambient conditions were constant at 70ºF (21°C), 60-70% RH, and product packages were fitted

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with temperature data loggers for four days. In the open case, temperatures of packaged leafy greens at the open front of the case were mostly non-compliant with Food Code, with temperatures greater than 41ºF (5°C) most of the time while the non-compliance with Food Code was largely reduced in the case with doors. These uniformly lower temperatures resulted in significantly higher visual quality scores and lower decay rates for the all products in the case with doors (P<0.001). In addition, pathogenic bacteria purposely inoculated into the bags, including E. coli O157:H7, Salmonella enterica, and Listeria monocytogenes, did not grow. Furthermore, energy consumption in the case with glass doors was reduced by 69%. A foreseeable win-win situation is within reach for the industry stakeholders and retailers.

Formation of trichloromethane, a chlorine by-product, in wash water, shredded lettuce and diced onion by Xuetong Fan, Kimberly Sokorai U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA

Washing with sanitizers is one of the critical processing steps for fresh and fresh-cut produce. The most common sanitizers used by the fresh produce industry in the U.S. are chlorine-based compounds, such as sodium hypochlorite (chlorine) and chlorine dioxide. However, there is a concern about the use of chlorine due to potential health risks associated with the formation of chlorine by-products. Commercially, the pH of chlorine solutions is often adjusted and maintained at values of 6.5-7.0 with citric acid to optimize antimicrobial efficacy and to avoid off-gas. However, it is unclear if citric acid affects the formation of chlorine by-products. The objectives of the present study were to study the formation of trichloromethane in wash water, shredded lettuce, and diced onions, to compare sodium hypochlorite with chlorine dioxide in producing trichloromethane, and to evaluate the impact of citric acid on trichloromethane formation. Results demonstrated that trichloromethane levels in chlorinated water increased after repeatedly being used to wash batches of lettuce. Nevertheless, low levels (14-22 ppb) of trichloromethane were detected in shredded lettuce. After rinsing, trichloromethane in the lettuce was further reduced to levels close to detection limit (4 ppb). Additional results showed that much lower levels of trichlormethane were produced in wash water for diced onions when compared to those for cut lettuce. Furthermore, chlorine dioxide contributed little formation of trichloromethane. Results also demonstrated that citric acid reacted with chlorine and contributed to the formation of high levels (>1,000 ppb) of trichloromethane in wash water. To lower the formation of trichloromethane in water, citric acid may be replaced with other pH adjustors such as phosphate. The information may be useful to the produce industry to minimize formation of chlorine by-products.

Growth and biofilm formation of Listeria monocytogenes in cantaloupe flesh and peel extracts by P. De. A. Abeysundara, N. Dhowlaghar, R. Nannapaneni*Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762

Listeria monocytogenes (Lm) is identified as one of the most serious foodborne pathogens affecting cantaloupe industry following the multi-state outbreak in 2011 which resulted in 33 deaths. Lm is known to persist as biofilms on food contact surfaces which can act as a continuous contamination source leading to a food safety problem. The objectives of this study were to determine the growth and biofilm formation by different Lm strains on various cantaloupe processing surfaces containing high and low concentrations of cantaloupe flesh and peel extracts at 22˚C and 10˚C. The growth of Lm BUG600 was determined in 25% and 1% of cantaloupe flesh or peel extract at 22˚C or 10˚C for 72 h. A set of six strains of Lm were evaluated for their biofilm formation on stainless steel coupons containing 25% or 1% cantaloupe flesh or peel extract at 22˚C or 10˚C for 7 days. The cantaloupe outbreak strain Lm 2011-2625 was evaluated for the biofilm formation on different cantaloupe processing surfaces containing 25% and 1% cantaloupe flesh or peel extract at 22˚C or 10˚C for 7 days. The cell density of Lm BUG600 increased to 7.5-8.1 log CFU/ml in 64 h at 22˚C compared to 3.5-4.0 log CFU/ml within 72 h at 10˚C in 1% cantaloupe flesh or peel extract. In 25% cantaloupe flesh or peel extract, the cell density of Lm increased to 8.5 log CFU/ml within 24 h at 22˚C compared to 6.5 log CFU/ml in 72 h at 10˚C. All the strains tested formed biofilms on stainless steel with some differences. Lm biofilm formation was faster (~7 log CFU/coupon within 4 days) in 25% cantaloupe flesh or peel extract at 22˚C and slower (~ 4 to 5 log CFU/coupon in 7 days) in 1% cantaloupe flesh or peel extract at 10˚C. The biofilm formation by Lm 2011L-2625 was lower on buna-rubber compared to the other food processing surfaces under all environmental conditions tested with a few exceptions. These findings show that Lm strains can grow and form biofilms at a very low concentration of cantaloupe flesh or peel extract on different food processing surfaces tested.

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Moisture regulation to control microbial growth on packaged produce by Sangeeta Chopraa,b, Sangram Dhumalc,d, Patrick Abelic, Elliot Rysere, Randolph Beaudryc, Eva Almenaraa School of Packaging, Michigan State University, MI 48824, USAb IARI, PUSA, New Delhi 110012, India,.c Department of Horticulture, Michigan State University, MI 48824, USAd Mahatma Phule Agricultural University, Rahuri 416003, Maharastra, Indiae Department of Food Science and Human Nutrition, Michigan State University, MI 48824, USA.

Enhanced growth of microorganisms is commonly associated with free water in the packaged product. The goal of this study was to explore the utility of highly porous structures with water absorption capacity to control microbial growth on packaged produce. Two metal organic frameworks (MOFs), C300 and A520, found to have high water sorptive capacity, were sealed in sachets and placed in packages containing fresh-cut Romaine lettuce. Packages with no sachets and empty sachets were used as controls and positive blanks, respectively. Bacterial and fungal loads were used to assess the effect of the highly porous structures on microbial growth and consequently, on shelf-life extension. Both bacterial and fungal loads were measured after 15, 18 and 21 days of storage at 2 °C, with 15 days representing the normal commercial shelf life of packaged fresh-cut Romaine lettuce. The results show that after 15 days, C300 reduced the bacterial count by 1 log cfu/g (p < 0.05) compared to A520, the control, and positive blank treatments. However, no difference was seen between C300 and any of the treatments in respect of fungal population after 15 days. On day 18, C300 reduced the bacterial count by ~2 log cfu/g (p < 0.05) and the fungal population by ~0.7 log cfu/g (p < 0.05) compared to the control and positive blank. That same day, A520 reduced the bacterial count by ~1 log cfu/g (p < 0.05) but not the fungal population compared to the control and positive blank. At the end of storage, A520 reduced the bacterial count by ~2 log cfu/g (p < 0.05) and the fungal population by ~1 log cfu/g (p < 0.05) compared to the control and positive blank. These results suggest that the rise in bacterial activity during storage can be suppressed in packaged fresh-cut Romaine lettuce using sachets containing C300 and A520 MOF.

Rapid detection of preexisting internal Leuconostoc mesenteroides spoilage in fresh-cut carrots during storage by Kellee Britt, Janneth Pinzon, and Trevor Suslow

During an extended period of abnormally short quality retention in mixed component packaged salads, due primarily to rapid decay, a root-cause investigation was undertaken. Ultimately, it was determined that the underlying cause could be attributed to the early onset of decay and spoilage of the fresh-carrot-shred, even under industry-accepted cold storage conditions, typically between 3-8°C. From this initial investigative assessment, this study focused on identification of the primary underlying microbiological cause and validation of a rapid detection screening of raw material. Analysis of several lots of raw, unprocessed product, approx. 6 cm abrasively peeled carrot plugs, revealed the accumulation of an aqueous slime in the shipping bag void space and around the progressively softened carrot-plug surfaces. This diagnostic sign of lactic acid bacteria (LAB) spoilage, specifically Leuconostoc mesenteroides, developed in cold storage (2.5°C) after two weeks. Efforts were undertaken to determine whether the Leuconostoc populations were internalized in raw material or environmentally contaminated with a proliferating reservoir of LAB in the primary processing and packaging environment. Characteristic dextran-producing LAB were obtained from samples and pure cultures were subjected to determinative cultural and genetic identification. Polymerase chain reaction (PCR) primers specific for the amplification of a sequenced region of the Leuconostoc spp. and L. mesenteroides 16S ribosomal RNA gene confirmed taxonomic genus and species identity. Total initial LAB and Leuconostoc spp. bacterial populations isolated from symptomatic carrots ranged from log₁₀ 7.5–8.5 and log₁₀ 3.5–4.0 CFU/g carrot tissue weight respectively, and increased log₁₀ 2 CFU/g log₁₀ and 3.5 CFU/g respectively in population density on asymptomatic and symptomatic raw carrot material during a two week refrigerated storage. The developed method can rapidly associate pre-symptomatic presence and, potentially, prevalence of Leuconostoc species, especially Leuconostoc mesenteroides, which may provide shelf-keeping timeframe prediction of mixed ingredient fresh-cut products which include shredded carrot.

Shelf life of blueberries in protein-based packages in vending machines by Christopher Wilson1, Meghan Green1, Kikyung Kim1, Matthew Costigan1, Joshua Hofmann1, George Henrique Camêlo Guimarães2, Daquan Miller3, Jaime González-Buesa4, Eva Almenar1

1 School of Packaging, Michigan State University, East Lansing, MI, USA2 Department of Agronomy, Federal University of Paraiba, Areia-PB, Brazil3 Everett High School, Lansing, MI, USA4 Department of Animal Production and Food Science, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain

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There is a growing interest in the development of food packaging materials made from renewable resources and with a biodegradable end-life scenario. The goals of this study were to investigate: (1) the shelf life of blueberries in protein-based pouches, and (2) the stability of the protein-based pouch properties under vending machine conditions (7˚C and 36% RH) for 9 days. Protein-based films were produced using the compression technique and then heat sealed to form protein-based pouches, with the same dimensions as commercial pouches used to sell produce in vending machines. The weight loss, firmness, titratable acidity, soluble solids content, quality index, off-flavors, and microbial growth of the packaged blueberries were determined over time. Pouch properties of interest in food packaging (e.g., seal strength, thermal transition changes, and water permeability) were assessed over time as well. The results showed that the studied protein-based pouches are a viable packaging alternative for blueberries commercialized in vending machines.

Survival of E.coli O157:H7, non-O157 STEC, and Salmonella within hydroponic water systems over time by Angela Shaw, Kara Helterbran, Christopher Currey, and Michael M. Evans. Iowa State University and University of Arkansas

The desire for local, fresh produce year around is driving the growth of hydroponic growing systems in the United States. Many food crops grown within hydroponics systems, such as leafy greens and culinary herbs, have their root systems submerged in recirculating nutrient dense water systems from planting through harvest. If a foodborne pathogen was incorporated into this water system, the risk of contamination to the entire crop is high. Hence, this study evaluates the ability of E.coli O157:H7, E.coli non-O157 STEC, and Salmonella to survive in three different model water systems (water control, fertilizer with plant, and fertilizer without plant) to better understand the behavior of these organisms over time. All the pathogens grew three logs over a twenty four hour period in all three systems. The E.coli O157:H7 had the highest growth in the fertilizer with plant (log 3.52), while E.coli non-O157 STEC and Salmonella showed more growth in the fertilizer without plant system than in the fertilizer with plant (1-2 logs). Overall, these findings show that if foodborne pathogens were to enter a hydroponic system there is potential for survival and growth.

The effect of cold stress on gene expression of Escherichia coli O157:H7 on fresh-cut Romaine lettuce by Bach, Susan J., Harlton, Colleen, and Pylatuk. Melanie. Agriculture & Agri-Food Canada, Summerland Research and Development Centre, 4200 Highway 97, Summerland, BC, CANADA, V0H 1Z0

Escherichia coli O157:H7 is an important foodborne pathogen that causes a variety of illnesses such as hemorrhagic colitis and the haemolytic uremic syndrome. In recent years, cases of foodborne illness have been increasingly associated with fresh produce, particularly leafy greens. Leafy greens often encounter cold temperatures during production, processing and retail distribution. The objective of this study was to evaluate the effect of cold stress on the expression of stress and virulence genes in E. coli O157:H7 on fresh-cut Romaine lettuce. Logarithmic phase cells of E. coli O157:H7 (EDL 933, ATCC 700927) (108 CFU/g) were exposed to 4 °C for 5 days on 4 x 10 cm pieces of fresh-cut Romaine lettuce (CL) and gene expression was evaluated using quantitative real-time PCR. Cold stressed inoculum (no lettuce) (C) and non-cold stressed inoculum (NC) were included as controls (n=3). Three independent experiments were performed. Cold shock protein A (cspA) and starvation related phoA were significantly (P<0.0001) upregulated in CL. Minor upregulation was observed in Stx1 (Shigatoxin)(P>0.05). Genes related to general stress (uspA), starvation (dpS), cold shock (cspC) and UV radiation (mutS) were downregulated (P<0.05). Genes associated with acid resistance (gadW) and intimin (eae) exhibited minor downregulation (P>0.05). Results of the study provide a basic understanding of the impact of cold stress on the survival and virulence of E. coli O157:H7 EDL 933. Understanding the survival mechanisms used by E. coli O157:H7 is important for minimizing contamination of fresh-cut produce and the occurrence of foodborne illness.

Use of aerosolized antimicrobials to inactivate Salmonella Typhimurium and preserve quality of cherry tomatoes by Yunbin Jianga, Xihong Lia, Joshua B. Gurtlerb, Sudarsan Mukhopadhyayb, Tony Jinb, Xuetong Fana*aKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457, ChinabU.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA

Washing with aqueous sanitizers has limited effectiveness in reducing populations of pathogenic bacteria on fresh and fresh-cut produce. This study was conducted to evaluate the effects of in-package antimicrobial aerosolization on the populations of attenuated Salmonella enterica serovar Typhimurium and

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quality attributes of tomatoes. The smooth skin surfaces and stem scar areas of cherry tomatoes were inoculated with a cocktail of attenuated S. Typhimurium ATCC 53467 and 53468 strains. The inoculated fruits inside a clamshell container were then treated with aerosolized chlorine, peroxyacetic acid (PAA), chlorine dioxide (ClO2), and combinations of organic acids. Un-inoculated fruits were treated similarly for quality analyses. Initial tests demonstrated that the cocktail of attenuated S. Typhimurium ATCC 53467 and 53468 had similar sensitivity to the antimicrobials as a cocktail of four pathogenic outbreak-associated Salmonella spp. Results showed that 400 ppm PAA, 2% lactic acid + 2% acetic acid +2% levulinic acid, 3% acetic acid + 3% lactic acid, and aqueous ClO2 (100 and 400 ppm) reduced the S. Typhimurium populations on smooth tomato surfaces by more than 5 log CFU/fruit. PAA at 80 ppm and chlorine at 200 ppm achieved only 0.85 and 1.50 log CFU/fruit reductions, respectively. On the stem scar areas, ClO2 (400 ppm) was most effective in reducing S. Typhimurium populations, achieving 4.89 log CFU/fruit reduction, followed by 400 ppm PAA (2.62 log CFU/fruit). The efficacy of ClO2 and acid combination treatments increased during 3 weeks of storage at 10 °C, achieving >3 log CFU/fruit inactivation on the stem scar areas with the acid combination and ca. 6 log with for 400 ppm with ClO2. Fruit quality attributes (color, appearance, firmness, vitamin C, lycopene or antioxidant values) of tomatoes were not affected by the treatments. However, an acidic odor was detected for samples treated with the acid combinations in the earlier period of the storage. These results show that the in-package aerosolization technique may be used to enhance microbial safety of tomato fruit.

Use of UV as a food safety intervention in a model aquaponics system by Sai Deepika Elumalai, Angela Shaw, D. Allen Pattillo, Christopher Currey, Kurt A. Rosentrater, and Kue Xie. Iowa State University

Aquaponics, which is the art of growing plants in a non-soil medium using nutrient-rich water from fish culture, is a growing trend in food production as it is seen as a sustainable, space- and energy-efficient approach for production of fruits, vegetables and seafood. Within aquaponics there has been an emphasis of following good agricultural practices and best aquaculture practices, but few microbial studies have been conducted to determine the food safety status within these units. The aim of this study was to determine the food safety status and the effectiveness of ultraviolet sterilizers as a food safety intervention in sterilizing the water system, in a model aquaponic unit that is growing lettuce, basil and barramundi (Australian Sea Bass). Large Leaf basil, Buttercrunch Bibb lettuce, water and fish samples were collected throughout the 118-day

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production period and microbial analysis was conducted for the presence of E. coli O157:H7, Aeromonas and Salmonella spp. and the prevalence of aerobic plate counts (APC), coliforms, and fecal coliforms in the systems in triplicates. Absence of foodborne pathogens was confirmed using ELISA technology and enumeration through cultural methods. A significant increase was observed in aerobic plate counts (1-3 logs) over the trial period, in the presence and absence of UV for the lettuce, basil, and barramundi. Ultraviolet treatment did not significantly reduce the APC, Aeromonas, coliform, or fecal coliform counts when compared to the control systems samples. Though the UV intervention method was not effective in reducing microbial loads, future work should focus on improving the unit design and other food safety interventions that can be effective in the presence of living system while maintaining fish homeostatic environment.

Validation of a Novel Method for Resuscitating Viable But Non Culturable (VBNC) Salmonella spp. in Water and Soil Samples by Taejo Kim Mississippi State University

When exposed extreme environmental conditions such as high/low temperature and nutritional starvation, Salmonella enter into a metabolic starvation mode or a physiologically viable but non-culturable (VBNC) state. However VBNC Salmonella survive up to 231 days in the environment and the cells resuscitated from a VBNC state can retain their pathogenic capacity. Interestingly, work conducted in the PI’s laboratory from water and soil samples have shown that Pseudomonas, one of the environmentally abundant microorganisms stressed Salmonella. This caused Salmonella to be VBNC on culture media (2% buffered peptone water, BPW) and non-culturability of VBNC Salmonella cells. This led to false negative results utilizing conventional culture detection techniques as well as molecular tools such as real-time PCR which require a minimum 10-100 cells/mL. VBNC cells are characterized by a loss of culturability and separation on routine microbial media, which impairs their isolation by conventional plating techniques. Lack of proper methods of culturing VBNC Salmonella increases the risk of human exposure to final food products. Hence, for checking the level of Salmonella contamination in water and soil in agriculture, it is very important to use a reliable culture technique for the detection of VBNC Salmonella and establish their viability. The objective of this study was to evaluate the effectiveness of a modification of RV broth and compare it to the recommended secondary enrichment from regulatory agencies for the recovery of VBNC Salmonella cells in environmental samples. Soil and water samples from a sweet potato field and catfish farm were pre-enriched in BPW, transferred to RV, TT, and biochemically modified RV2 (mRV2, novel secondary culture),

incubated at 41.5°C and further analyzed using USDA/FSIS and real-time PCR methods. Presumptive colonies separated on BGA and XLD were confirmed by conventional PCR. Isolation rates were 43% on BAX® PCR, 54% on RV, 43% on TT and 61% on mRV2. mRV2 could be a good alternative enrichment broth to resuscitate VBNC cells that would go undetected with traditional methods and that could regain viability, contaminate foods, and cause foodborne outbreaks.

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