For Abstracts

Elliot T. Ryser (Michigan State University), Haiqiang Wang (Michigan State University (Andrew Scollon) (Michigan State University), Chelsea Kaminski (Michigan State University), Rocky Patil, (Michigan State University), Bradley P. Marks (Michigan State University), Sanghyup Jeong (Michigan State University), Eva Almenar (Michigan State University) and Naalie Page (Michigan State University)

Public health concerns surrounding the safety of fresh-cut fruits and vegetables have escalated over the last five years in response to a series of nationwide foodborne outbreaks traced to leafy greens, tomatoes, celery, onions, and cantaloupe. The overall goal of this 4-year multi-disciplinary, multi-institutional, $1.8 million USDA National Integrated Food Safety grant is to enhance the microbial safety and quality of ready-to-eat, fresh-cut fruits and vegetables via integrated research and outreach/training targeted at processing, packaging, distribution, risk modeling/economics and education/training. Once products such as tomatoes, onions, celery and cantaloupe become contaminated in the field or during harvest, washing in various sanitizer solutions typically removes only 90 to 99% of the bacterial population, including pathogens, with the efficacy of chlorine-based sanitizers negatively impacted by the organic load of the wash water. Any remaining bacterial contaminants can be spread to subsequent product during mechanical shredding, dicing, slicing, conveying and fluming as well as shaker table and centrifugual dewatering. Various packaging strategies employing different gaseous atmospheres specific to the type of produce in question have been successfully used to optimize both end-product quality and safety. However, bacterial pathogens such as Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes can still remain viable in fresh-cut and in some cases grow, particularly if the product is temperature abused during shipment, retail storage, retail display and subsequent home storage. Careful attention to sanitizer washing of fresh produce, minimizing cross-contamination during slicing/dicing and maintaining the cold chain throughout distribution is essential for maximizing the safety of fresh produce.

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Andrea Cossu (Food Science and Technology Department, University of California - Davis, Davis, CA 95616 USA)Rohan V. Tikekar (Department of Nutrition and Food Science, University of Maryland - College Park, College Park, MD 20742 USA)Nitin Nitin (Food Science and Technology Department, University of California - Davis, Davis, CA 95616 USA) and (Department of Biological and Agricultural Engineering, University of California-Davis, Davis,CA 95616 USA)

Peroxides and chlorite based sanitizers are commonly used for washing procedures. Efficacy of these sanitizers is significantly reduced in the presence of organic content and may result in formation of harmful by-products. Therefore, new food grade sanitizers with enhanced antimicrobial efficacy are needed. In this work, we tested a new treatment for the sanitation of the wash-water based on the use of photosensitizers (PS) that continuously produce oxidative stress upon illumination. Antimicrobial effect of the food-grade PS dye Rose Bengal (RB) photosensitized with a visible light source on E. coli and bacteriophage T7 was evaluated. Effect of factors such as duration of illumination, growth phase of the microorganism and presence of organic matter on efficacy of inactivation was also studied. Data from exponential phase bacteria with high organic content (2,000 mg/L LB) showed that 10 µM and 100 µM RB achieved -1.83 ± 0.16 and -2.85 ± 0.05 logs reductions after 30 minutes and -5 and -6 logs reduction after 60 minute of illumination respectively. Stationary phase bacteria were more resistant than exponential phase bacteria since 10 µM RB achieved only a -3.96 ± 0.26 logs reduction after 60 minute of exposure. Experiments with T7 bacteriophage showed -3.58 ± 0.29 and -5 ± 0 logs reductions achieved in 30 minutes with 10 µM and 100 µM of RB respectively at 2,000 mg/L of LB. These results highlight the efficacy of RB as an antimicrobial against bacteria and viruses and demonstrate its potential as an alternative sanitizer for the food industry.

Todd Baggett (CEO of RedLine Solutions, co-chair of the PTI Technology working group and author of Produce Traceability For Dummies®)

Today consumers expect that the produce industry has the capability to track any fresh produce back to its source. However is this really the case? In this session we will answer that question and explore the state of produce traceability past, present, and future.

The E. coli outbreak in 2006 was the 9/11 of the produce industry. Consumers were getting sick, three died, and the FDA identified spinach as the culprit. However, the FDA was unable to quickly trace the contamination back to the source farm, so they issued an industry-wide spinach warning leading retailers and restaurants to remove all spinach from sale. Ten years later, spinach consumption in the US has still not returned to the volumes sold prior to the outbreak. This wakeup call for the produce industry led to action on multiple fronts:

• In 2008 leading retailers, growers, and three of the largest industry trade associations introduced the Produce Traceability Initiative (PTI)
• Many forms of legislation, including 2011’s Food Safety Modernization Act, were introduced to improve food safety and protect consumers.

Produce traceability is essential to expedited recall of contaminated food, keeping the consumer and the industry healthy. However for produce traceability to function effectively, retailers, grower- shippers, and the FDA all need to be on the same page. So is our produce supply chain safer in 2015? Come hear PTI Technology Working Group Co-Chair, Todd Baggett, provide an insider view into produce traceability.

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Hetong Lin (College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China); Yifen Lin (College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China); Yen-Con Hung (Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA); Yihui Chen (College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China); Meirong Fan (College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China)

Nisin has been widely used in the food industry as a safe and natural preservative to increase the shelf-life of many foods. In order to evaluate the possibility of nisin for anti-microorganism development and maintaining quality of fresh-cut Chinese yam, fresh tubers of harvested Chinese yam (Dioscorea opposita Thunb.) cv. Shangehuaishan No.1 were washed, peeled and cut into approximate 30-mm blocks. The cut tubers were dipped with 0% (control), 0.01%, 0.02%, or 0.03% nisin solution for 10 min, respectively, then air-dried and packed into sealed polyethylene bags (0.015 mm thickness) and stored at 4 degrees C for 10 days. At 2-day intervals during storage, aerobic plate count (APC), CIE (Commission Internationale de L'Eclairage) parameter of L, browning index, value of browning degree, polyphenol oxidase (PPO) activity and weight loss were determined. The results showed that compared to the control yam samples, different doses (0.01%, 0.02%, or 0.03%) of nisin treated-fresh-cut Chinese yam exhibited lower APC, higher value of CIE L, lower browning index, lower value of browning degree and lower PPO activity, as well as lower weight loss. Among the nisin-treated yam samples, the yam treated with 0.03% nisin for 10 min was the most effective in antimicrobial activity, reducing the development of browning and weight loss. From the results it could be concluded that the postharvest treatment with .03% nisin for 10 min might be a feasible and safe technique for suppressing the growth of microorganism and maintaining quality of fresh-cut Chinese yam during storage at 4 degrees C.

Ann Charles (Rutgers University), Elliot Ryser (Michigan State University) and Donald Schaffner (Rutgers University)

Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes have been implicated in a number of outbreaks linked to fresh-cut produce. This presentation presents models for bacterial transfer during slicing and bacterial growth during transport for inclusion in a fresh-cut quantitative microbial risk assessment. Three separate scenarios are considered: (1) Transfer of Salmonella during tomato slicing considering four variables: slicing method, slicing temperature, blade thickness and moisture on the tomato before slicing. (2) Transfer of L. monocytogenes during onion slicing considering three different inoculation concentrations between 8.5 and 5.5 log CFU/onion. (3) Temperature data collected from sixteen tractor-trailer leafy green shipments over a 1-year period to model E. coli O157:H7 and Listeria monocytogenes growth. Temperature loggers were located in pallets at the front, middle and rear and along the sidewalls of each trailer. About 17% of the sensors used in the study recorded temperatures greater than 5 degrees C. Our results showed that the concentration of Salmonella and Listeria generally decreased logarithmically slicing. Because such a model would be difficult to incorporate into a QMRA, an alternative modeling approach considering transfer as a function the number of microorganisms remaining on the slicer was also investigated, and found suitable. Growth modeling results showed that L. monocytogenes growth was more likely than E. coli O157:H7 growth, but that no significant growth was the most likely occurrence. Prediction of Salmonella and Listeria transfer during onions and tomato slicing and E. coli O157:H7 and Listeria monocytogenes growth during transport of leafy greens appears complex but feasible.

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Karin Söderqvist (PhD student, Department of Biomedical Sciences andVeterinary Public Health, SLU, Uppsala, Sweden), Ivar Vågsholm (Professor in Microbial Food Safety, Department of Biomedical Sciences andVeterinary Public Health, SLU, Uppsala, Sweden), Susanne Thisted Lambertz (Microbiologist, The National Food Agency, Microbiology division, Uppsala, Sweden) and Sofia Boqvist (Associate Professor in Infectious Disease Epidemiology, Department of Biomedical Sciences andVeterinary Public Health, SLU, Uppsala, Sweden)

There is a trend towards healthier eating habits with increased consumption of fresh vegetables, often sold prepared and ready-to-eat (RTE). Consumers consider these products safe and healthy. However an increasing number of foodborne outbreaks have been associated with consumption of RTE salad. Foodborne pathogens may contaminate at several steps during the production of RTE salad, e.g. via contaminated water or cross-contamination from human handling, and there is no process in the production chain that inactivates pathogens. Consequently, the safety of RTE salad depends on good hygiene practices and a well-functioning cold chain. The aim of the study was to investigate the growth of different pathogens in RTE salad, mimicking recommended fridge temperature (8 degrees C) and temperature abuse (15 degrees C). Two matrices were studied; salad (baby spinach) and mixed salad (baby spinach and grilled chicken). Rifampicin-resistant strains of Salmonella typhimurium, Listeria monocytogenes and pathogenic Yersinia enterocolitica together with a green fluorescent protein (GFP)–labeled strain of Escherichia coli O157:H7 were inoculated in low numbers and enumerated after 3 and 7 days. The results demonstrated that storage of mixed salad at 15 degrees C strongly supported growth of E. coli, L. monocytogenes and Y. enterocolitica. For L. monocytogenes and Y. enterocolitica growth was also supported in mixed salad at 8 degrees C. Cold storage of RTE salad is essential to reduce the risk of foodborne disease and for mixed RTE salad the recommended storage temperature should be lower than 8 degrees C –the current recommended temperature in Sweden.

Deirdre M. Holcroft (Holcroft Postharvest Consulting) Stephen T. Lacasse (Fresh Appeal)

Fresh Appeal has designed and patented a fresh-cut processing system that incorporates innovative cutting technology, a multi-hurdle approach to wash water sanitation, and unique drying systems. The sanitation treatment uses a chemical sanitizer, ultraviolet light (UV-C) and mild heat in sequential steps. The exposure to UV-C has optimized by delivering the UV-C to produce through a column of circulating, turbulated, pasteurized water. In addition to providing good exposure to the produce, the turbulence of the water helps to physically remove microbes from the product surfaces which helps improve the efficacy of the UV-step. Microorganisms are more vulnerable in water than on produce. This system is designed to maintain the UV-C dose within strict parameters. The process water is continuously monitored for UV transmissivity. The water is filtered and replenished to maintain optimal performance. The dwell time in the ‘turbulator’ is controlled by flow rate. The heat treatment is usually applied after the UV-C step and is followed by rapid and effective cooling and, where necessary, antioxidant infusion. The processing line was initially developed for apples and has a unique means of aligning fruit to optimize coring and slicing, as well as an antioxidant recovery step. Several modifications have been made so that other produce items can processed. For example, blueberry and strawberry shelf life has been extended as a result of the berry drier. This system will be discussed along with relevant research and commercial data on sanitation efficacy, product quality and shelf life.

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Debanjana Roy (Department of Plant Sciences, UC Davis) and Maeli Melotto (Department of Plant Sciences, UC Davis)

Plants have small pores on their leaf surfaces known as stomata. These pores are formed by two kidney-shaped guard cells that serve as ports for gas exchange with the environment and plant transpiration. Pathogenic bacteria can use these pores to get inside the plant interior (intercellular space) and often scaping sanitation procedures. Plants have evolved mechanism to quickly perceive the presence of bacteria and close the stomatal pore, thus preventing leaf contamination. This phenomenon is recognized as stomatal immunity. In this study, we assessed the ability of several fresh leafy vegetables in mounting stomatal immunity against Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 and determined the influence of environmental variables on the effectiveness of the stomatal response. Butter lettuce, romaine, basil, and cilantro showed strong stomatal closure in response to O157:H7 as compared to stomatal response to the water control. Salmonella, however, induced a transient stomatal closure in butter lettuce, romaine, and basil. Unlike the other plants tested, cilantro stomata remained closed in response to Salmonella. Furthermore, air relative humidity and temperature had variable effects on the plant immunity depending on the plant-bacterium combination tested. These results indicate that the extent and the kinetics of stomatal closure and re-opening may vary among plants providing opportunities to discovering the genetic basis for stomatal immunity in plants and to proposing plant-specific control measure to reduce pathogen load on/in leafy greens.

Brooke Schwartz (Brooke Schwartz Consulting)

Reliable pathogen detection methods play a key role in an effective food safety program. A variety of pathogen detection platforms and methods are commercially available, and test kit manufacturers are striving to develop increasingly rapid methods to address the specific needs of the fresh cut industry. In developing new methods, test kit companies must take into account the diverse set of needs and requirements that span fresh cut growers, testing labs, retail customers, and the regulatory and public health agencies that monitor the safety of these products. This presentation addresses some of the critical questions and challenges facing both the test kit developers and the fresh cut industry, including: The determination of whether and where to test; the need for speed; the challenges of sampling; validated and verified methods; and interpretation of results.

Irwin R. Donis-González (Department of Biosystems and Agricultural Engineering, 524 S. Shaw Ln., Michigan State UniversityEast Lansing, MI, 48824. USA)Daniel E. Guyer (Department of Biosystems and Agricultural Engineering, 524 S. Shaw Ln., Michigan State UniversityEast Lansing, MI, 48824. USA)

This research was designed to develop and test an automatic image analysis algorithm to detect the presence of undesirable fibrous tissue before dicing carrots (Daucus carota L.). Fibrous carrot dices are difficult to detect, and are highly problematic when found in ready-to-eat infant food, where they might represent a choking-hazard (safety-concern). A visible/near-infrared (VIS/NIR) hyperspectral imaging (400-1000 nm) system was used to obtain a set of 520-images per sample, from 1233 sections (samples). Samples were collected during the 2013 and 2014 harvesting seasons. Classification accuracy was evaluated by comparing the classes obtained using VIS/NIR hyperspectral images obtained per carrot section to their undesirable fibrous tissue class, based on the industry-simulated invasive quality assessment (% of fiber). Class-0 represents fibrous-free samples, and class-1 denotes samples containing fibrous tissue. After VIS/NIR image preprocessing, cropping, selection, and segmentation, 1045 grayscale intensity and textural features were extracted per sample from five selected VIS/NIR images. A 4-fold cross-validation neural-network classifier with a performance accuracy of 82.4 ± 2.5 % was developed using 122 relevant features, which were selected using a sequential forward selection algorithm with the Fisher discriminant objective function. Findings showed that this methodology is an objective, accurate, and reliable tool to determine the presence of undesirable fibrous tissue in processing carrots, and would be applicable to an automated noninvasive inline sorting system.

Trevor Suslow, Cooperative Extension Specialist, University of California, Davis

The final FDA rule-making under the Food Safety Modernization Act (FSMA) is very close to becoming a reality and moving towards ‘Implementation’ timelines. The Current Good Manufacturing Practice and Hazard Analysis and Risk-Based Preventive Controls for Human Food (aka Preventive Controls) rule are anticipated to be released in final form by the convening of the International Conference on Fresh Cut Produce. This major FSMA rule will cover both domestic and foreign fresh-cut processing facilities exporting to the U.S. The standards may also apply to other establishments that hold fresh-cut products, such as distribution centers. Two key provisions of the anticipated Preventive Controls final rule is that each facility develop and implement a written Food Safety Plan which covers their suppliers (for example, farms under the Produce Rule which do not have to have  a written plan) and includes documented validation and verification of all preventive and process controls. The elements of the Food Safety Plan must include: (i) identification of all hazards (biological, chemical, physical, and radiological) specific to each food at the facility, (ii) documented evaluation of which hazards are “reasonably likely to occur,” (iii) implementation of validated preventive controls for each “reasonably likely to occur” hazard, and (iv) specific monitoring, corrective actions, and verification activities for each preventive control. This presentation will summarize the key provisions of the final Preventive Controls and use the challenges of validation of wash water process controls and Environmental Monitoring Programs as examples of essential actions needed by supply-chain stakeholders for compliance expectations.

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Maria Grudén (SLU, Swedish University of Agricultural Sciences, Dep. of Biosystems and Technology, Microbial horticulture)Lars Mogren (SLU, Swedish University of Agricultural Sciences, Dep. of Biosystems and Technology, Microbial horticulture)Beatrix W. Alsanius (SLU, Swedish University of Agricultural Sciences, Dep. of Biosystems and Technology, Microbial horticulture)

The bacterial colonization of rocket leaves was studied before processing and after bagging. At the same time the raw and process water quality at different stages during processing under commercial conditions was studied. Both leave and water was analyzed with respect to microorganisms at 22 degrees C, slow growing bacteria, total coliform bacteria, E. coli, intestinal enterococci, as well as Listeria monocytogenes, Salmonella spp., and Campylobacter spp.. Samples were collected at four occasions with 3 replicates per event. The bacterial load in the process water increased substantially after produce came in contact with the raw water. Microorganisms at 22 degrees C and slow growing bacteria exceeded 300 000 CFU ml-1 and 50 000 ml-1. Also intestinal enterococci were very frequent, in contrast to total coliform bacteria and E. coli. Microbial reduction rate before and after washing was log 0.5. Microbial communities inhabiting the produce before and after washing as well as the raw and process water were collected and analyzed using DGGE. From these specimens, microorganisms were grown on semi-selective media (0.1 TSA, King Agar B, Enterococci agar, and VRBD). Five randomly selected colonies were identified using Biolog GenIII panels. Various Pseudomonas species and Pantoea agglomerans were frequent in the process water. Also the phyllosphere flora before washing was dominated by Pantoea agglomerans. After washing, Pantoea agglomerans, Rahnella aquatilis and Pseudomonas were abundant. Pseudomonas fluorescens as well as Enterobacter cloaceae resisted washing procedures in the washing line and under laboratory conditions.

Adrian Sbodio (Department of Plant Sciences; University of California, Davis)Jeremy Roland (Department of Plant Sciences; University of California, Davis)Janneth Pinzon (Department of Plant Sciences; University of California, Davis)Chelsea Kaminski (Department of Plant Sciences; University of California, Davis)Renee Leong (Department of Plant Sciences; University of California, Davis)Trevor Suslow (Department of Plant Sciences; University of California, Davis)

Contamination by soil amendments, flooding, contaminated irrigation water, or other sources has resulted in substantial losses of abandoned tender leafy greens for packaged salads.

We hypothesize that short-duration, low-residue cover crops, solarization, or a combination will be effective in the practical elimination of Salmonella enterica contamination in soil.

Replicated 2 x 5 m plots were inoculated with chicken manure/litter containing an attenuated Salmonella at 10^3 or 4 CFU/g. Plots were treated using solarization or by cover crops. For solarization, plots were covered with clear polymer for 36 days. Cover crops (Buckwheat, Mustard and Canola) were grown up to 50 days. Following incorporation, soil was sampled before plots were replanted with baby spinach.

Trials in clay loam soil revealed limited difference between the fallow controls and cover cropped plots. Unlike our preliminary trials in a high organic matter soil, the die-off of inoculated S. enterica occurred within 30 days. This represented greater than 3-log reduction from applied inoculum levels, intentionally low to reflect a ‘real-world’ natural contamination incident. For solarization, Salmonella enterica was not detected in any covered plot at 39 days whereas 100% of the non-solarized plots were positive for the applied Salmonella. Temperatures at 6 cm under the polymer row covers reached 42-45C during daily cycles while non-covered plots did not exceed 33C. In plots where baby spinach was re-planted, no contamination was detected at harvest.

To determine the practical strategy that will enhance remediation of Salmonella contaminated soil and prevent subsequent product contamination following replant of leafy greens.

De la Riva-Álvarez, S. L. (Colegio de Postgraduados–Montecillo, México) Hernández-Anguiano A. M.* (Colegio de Postgraduados–Montecillo, México) Corrales -García J.E. (Universidad Autónoma Chapingo-México) Soto-Hernández M. (Colegio de Postgraduados–Montecillo, México) Patel J. (USDA-Beltsville, Maryland, USA) Reyes Trejo B.(Universidad Autónoma Chapingo-México) Zavaleta-Mancera H. A. (Colegio de Postgraduados-Montecillo, México)

Tender cactus pads or nopalitos (Opuntia ficus-indica L) are an important vegetable in México. They are often pre-trimmed, cut and packaged, and while usually consumed cooked, they may also be eaten raw in salads. Salmonella is an enteropathogenic bacterium that can adapt to adverse environmental conditions. Although it has been reported that Salmonella infected plants are asymptomatic, wilting, yellowing, biomass loss and a hypersensitive response (HR) have recently been demonstrated in plant tissues. The aim of this study was to investigate tissue response to different strains of S. enterica in two varieties of cactus as well as the persistence of Salmonella strains in the tissue. The top of pads or cladodes (20-25 cm) of greenhouse-grown (at 17.3 degree C and 39.8% RH, average) “Milpa Alta” and Atlixco varieties were inoculated with water or 1 mL 8.0 log CFU of strains S. Typhimurium (N4), S. javiana (N7) which were both isolated from Opuntia ficus-indica, and S. Typhimurium ATCC23564 (Sal 4). There were 20 cladodes per cactus variety with evaluations every 24 h for 5 days starting 4 h after inoculation for visual symptoms and persistence in the tissue determined according to SSA1-1994-NOM-109 and Madigan et al. (2001). Data was subjected to analysis of variance with mean separation by Tukey’s (? = 0.05) test. Color and appearance changed in longitudinal sections of pads of both varieties only in the area where the bacteria were infiltrated and this could be considered a hypersensitive response. All three Salmonella strains persisted 48 h or longer.

Vanessa Lieberman (Staff Researcher), Linda J. Harris (Principal Investigator)

Fresh-cut onions were recalled in 2012 after Listeria monocytogenes was isolated from finished product. The purpose of this study was to evaluate the survival or growth of L. monocytogenes in both whole and diced onion. Whole and diced yellow onions (Allium cepa) were inoculated with a six-strain cocktail of rifampin-resistant L. monocytogenes and stored under conditions to simulate food service or consumer handling. The inoculum was prepared on agar plates (for both whole onion experiments) or in broth (for diced onion experiments). Marked circles (3.3 cm in diameter) on the outer papery skin of whole onions were spot inoculated (10 µl) at 7 log CFU per circle, and after drying for 30 min onions were stored at 4 or 23 degrees C, 30 to 50% relative humidity, for up to 56 days. Diced onions were inoculated at 3 log CFU/g and then stored in closed containers at ambient conditions. Populations were determined by plating onto tryptic soy agar and Modified Oxford or Chromagar Listeria with added rifampicin (for diced onion). Populations of L. monocytogenes declined to below the limit of detection by day 42 at 23 degrees C; at 4 degrees C populations fell to 4.04 log CFU/disk at day 56. Both agar and broth cultured inoculum increased by 3 log CFU/g over 38 h of storage at 23 degrees C in diced onion; growth rates were not significantly different (P < 0.05; 0.15 and 0.12 log/CFU/g/h, respectively). A longer lag phase was observed for broth-prepared inoculum (9.8 h) compared to agar-prepared inoculum (7.0 h). L. monocytogenes can survive for long periods on onion skin. Multiplication on diced onion is evident at times that exceed food service or consumer ambient hold times (4 and 2 h, respectively).