Doctor of Philosophy in Engineering, Cleveland State University, 2021, Washkewicz College of Engineering

In this study, we developed a comprehensive mathematical model to predict the free chlorine (FC) concentration and bacterial cross-contamination during produce wash processes. A second-order chemical reaction model for FC decay which utilizes a proportion of chemical oxygen demand (COD) as an indicator of organic content in the wash water was employed, yielding an apparent reaction rate of 9.45 ± 0.22 × 10-4 µM-1.min-1. Using a proportion of successive changes in COD in the wash water due to produce washing, typically ranging from 6 to 11% across produce types, the model was able to consistently predict experimental FC levels, however, we note that while the FC level drops, the COD level stays constant. Therefore, we established the total amino acids concentration as an alternative indicator of organic load, and modified our model based on modeling the reaction kinetics of chlorine and amino acids. Apparent reaction rate between FC and amino acids was in the range of 15.3 – 16.6 M-1.s-1 and an amplification factor in the range of 11.52 - 11.94. This study also presents a modified disinfection kinetics model to evaluate the potential effect of organic content on the chlorine inactivation coefficient of Escherichia coli O157:H7 in produce wash process. While the chlorine inactivation coefficient of E. coli was 70.39 ± 3.19 L.mg-1.min-1 in the absence of organic content, it dropped by 73% for a COD level of 600 - 800 mg.L-1. Finally, the mechanisms by which FC inactivates E. coli was studied. Results showed that at low levels of FC and shorter exposure times, cell surface became rough and plicate; however, holes and wrinkles formed on the cell surface at higher FC concentrations or at longer exposure times, causing significant damage to the cell membrane. The cellular permeability changed due to chlorination, resulting in a significant decrease in the number of viable cells. Besides, around 3.45% ± 0.62 of cells lost their culturability and transform to viable but (open full item for complete abstract)

Committee: Chandrasekhar Kothapalli (Committee Chair); Jorge Gatica (Committee Member); Joanne Belovich (Committee Member); Daniel Munther (Committee Member); Srinivasan Parthasarathy (Committee Member) Subjects: Chemical Engineering; Microbiology

Doctor of Philosophy, The Ohio State University, 2018, Plant Pathology

Salmonellosis cases caused by Salmonella enterica through pre-harvest contamination of fresh produce represent a risk to human health worldwide; however, little is known about the interactions between Salmonella, phytopathogens, environment, and the plant host contributing to this food safety issue. Furthermore, the control of Salmonella from “farm to fork” is challenging due to the development of resistance mechanisms towards current control methods and restrictions on use of antimicrobials imposed by regulatory agencies. We investigated the effects of specific environmental conditions on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) following artificial contamination of `Tiny Tim' tomato plants. We found that higher temperatures (30°C day/25°C night) reduced the persistence of S. Typhimurium in the phyllosphere compared to lower temperatures (20°C day/15°C night) when plants were sprayed on the leaves with a S. Typhimurium -contaminated solution. Wounding cotyledons with contaminated tools increased S. Typhimurium persistence and internalization in planta compared to spray inoculation. Low relative humidity enhanced the dissemination of Salmonella into non-inoculated plant tissues. S. Typhimurium was detected in the root systems for at least 98 days-post inoculation. Further, we showed that splice-grafting (`Celebrity' with 'MaxiFort') is a major risk for the internalization and long-term survival of S. Typhimurium inside the tomato plant. S. Typhimurium was detected in the root system for over 137 days if at least 5 x 10^3 colony-forming units were introduced during grafting. The survival of S. Typhimurium in tomato foliage was also affected by the presence of phytopathogens, the genotype of S. Typhimurium and tomato variety used. We found that rfbV, involved in O antigen synthesis, might be essential for S. Typhimurium persistence in inoculated tomato plants and especially in `Tiny Tim' plants (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Sally Miller (Advisor); Laurence Madden (Committee Member); Christopher Taylor (Committee Member); Corey Nislow (Committee Member) Subjects: Agriculture; Bioinformatics; Biology; Environmental Health; Molecular Biology; Plant Pathology; Public Health

Master of Science in Chemical Engineering, Cleveland State University, 2018, Washkewicz College of Engineering

Fresh produce continues to be a key part of a healthy diet, with more and more people opting for ready-to-eat freshly cut produce. Bacterial outbreaks have been linked to the consumption of freshly-cut produce. The sources of bacterial contamination include farm conditions and cross-contamination in the sanitization stage of processing. Produce is chopped and sanitized at a processing facility which typically handles produce from multiple farms. The efficacy of free chlorine (anti-microbial agent in wash water) depends on the organic load in the wash water system, the efficacy is reduced if the organic load is too high, thus promoting cross-contamination. To prevent cross-contamination in the washing phase, an adequate understanding of the relationship between free chlorine and organic load is required. This study investigates the dynamics between free chlorine and organic load, and presents a mathematical model that can predict such parameters in wash water systems.

Committee: Chandrasekhar Kothapalli (Advisor); Daniel Munther (Committee Member); Parthasarathy Srinivasan (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2017, Food Science and Technology

Fresh produce is low in calories and high in the fibers, essential vitamins and minerals. Consumption of fresh fruits and vegetables helps prevention of cardiovascular diseases, cancer, diabetes and improves gastrointestinal health. Thus, fruit and vegetables have been gaining popularity among consumers and the increased consumption has been endorsed by many health organizations. On the contrary, fresh produce has continuously been associated with foodborne diseases outbreaks. It is technically challenging to decontaminate fresh produce due to the lack of microbial kill-step that effectively eliminate pathogens without causing product quality deterioration. Currently, chlorine wash is the most-used antimicrobial treatment of postharvest fresh produce. Gaseous ozone and bacteriophages have become popular as natural and environmental-friendly alternative decontamination technologies. Bacteriophages can be applied as alternative to chlorine spray or used as a final rinse before packing. Gaseous ozone was previously found very effective when combined with vacuum cooling to inactivate Escherichia coli O157:H7 on baby spinach. The objectives of this research were: (i) to evaluate the suitability of a single lytic bacteriophage, Escherichia phage OSYSP, as a fresh produce decontaminant; (ii) to assess the efficacy of bacteriophage OSYSP and gaseous ozone against E. coli O157:H7 on spinach leaves; and (iii) to develop a combination treatment involving bacteriophage and gaseous ozone to eliminate E. coli O157:H7 on spinach leaves. To justify using Escherichia phage OSYSP in fresh produce decontamination and other applications, the phage characteristics and genomic makeup were investigated (Chapters 2-4). The method to determine phage titer was optimized for maximum phage recovery and plaque clarity during enumeration. Stability of the phage at different incubation temperatures was investigated. The titer of a phage preparation did not change considerably during storage (open full item for complete abstract)

Committee: Ahmed E. Yousef (Advisor); V.M. Balasubramaniam (Committee Member); Farnaz Maleky (Committee Member); Jiyoung Lee (Committee Member) Subjects: Food Science

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Human norovirus (NoV) is responsible for more than 95% of nonbacterial gastroenteritis and over 60% of foodborne illness each year in the US. However, it has been a challenge to conduct research on human NoV because it cannot be grown in cell culture and lacks a small animal model. Despite major efforts, there is no antiviral therapeutic or vaccine for human NoV. In addition, the survival of human NoV is poorly understood. Fresh produce is major high risk food for human NoV-related outbreaks of gastroenteritis because it can easily become contaminated at any point during pre-harvest and post-harvest stages of production. However the ecology, persistence, and interaction of human NoV in produce systems is poorly understood. Determining the mechanisms underlying human NoV fresh produce contamination can facilitate the development of effective preventative and control measures to limit human NoV outbreaks. The objectives of this research are to determine whether human NoV and its surrogates become internalized via the root of growing produce and disseminated to the edible portion of the plants grown in soil; to determine whether biotic and abiotic stress can affect the rate of internalization and dissemination of human NoV in fresh produce; and to determine whether non-thermal food processing technologies can effectively inactivate internalized viruses while maintaining the fresh quality of produce. Romaine lettuce grown in soil was inoculated with 2 × 108 plaque forming unit (PFU) of human NoV surrogates [Murine norovirus-1 (MNV-1); Tulane virus (TV)] via the roots of plants. Plants were grown for 14 days and leaves, shoots, and roots of each plant were harvested and homogenized and viral titer was determined by plaque assay. It was found that both MNV-1 and TV can efficiently be internalized via plant roots and disseminated to shoots and leaves of lettuce. At day 14 post-inoculation, the titer of MNV-1 and TV in leaves of lettuce plants reached a level of (open full item for complete abstract)

Committee: Jianrong Li PhD (Advisor); Melvin Pascall PhD (Committee Member); Mark Peeples PhD (Committee Member); Wondwossen Gebreyes (Committee Member) Subjects: Food Science; Virology

Master of Science, The Ohio State University, 2014, Food Science and Technology

The safety and quality of fresh produce sold at grocery stores is a point of competition between many grocery store chains. Companies that can offer the most fresh, safest and least-expensive fresh produce are well-equipped to increase their customer base. Grocery stores may use antimicrobial agents to improve the quality of fresh produce and one of these agents is electrolyzed water. Solutions of antimicrobial agents may be used during washing or misting of fresh produce while it is on the produce rack shelves. Washing the produce with electrolyzed water is also referred to as conditioning and is typically done daily while the produce is held on the produce rack. Misting is performed periodically with diluted electrolyzed water sprayed onto the produce on the produce rack. This research first analyzed the effectiveness of using conditioning and misting during simulated produce rack storage. The effectiveness of using electrolyzed water as the conditioning agent versus tap water, along with the impact of trimming the stem of the produce during conditioning was then analyzed. Results were collected for both sensory scores for crispness and important microbiological counts. Produce from a nationwide grocery store's regional distribution center was shipped to Ohio State University and subjected to simulated grocery store procedures. Asparagus, red leaf lettuce and romaine lettuce were the produce chosen for testing. The produce was stored for 72 hours simulating grocery store conditioning and misting procedures on an industrial produce rack. For the analysis of the effectiveness of electrolyzed water vs. tap water, simulation of consumer storage after purchasing the produce was added. The produce was stored for an additional seven days in a refrigerator following the 72 hour produce rack storage. Results from the analysis of the effectiveness of conditioning and misting showed that conditioning had a positive impact on both the microbial counts and sensory scor (open full item for complete abstract)

Committee: Ahmed Yousef Ph.D. (Advisor); Hua Wang Ph.D. (Committee Member); Farnaz Maleky Ph.D. (Committee Member) Subjects: Food Science; Microbiology

Doctor of Philosophy, The Ohio State University, 2012, Food Science and Technology

Internalization of human pathogens is common in fresh produce.This threatens human health because the pathogens cannot be fully inactivated by conventional washing. This study examined the impact of water stress (abiotic factor) on the internalization of Salmonella Typhimurium in iceberg lettuce and green onion through soil and plant surface contamination during the pre-harvest stage. In addition, lettuce mosaic virus (LMV) infection (biotic factor) was introduced to determine whether the internalization via surface inoculation was affected in lettuce. Finally, different doses of ultraviolet-C radiation and their combination with chlorine/peracetic acid (PAA) were applied in an attempted to inactivate the internalized Salmonella. Salmonella was labeled with green fluorescence protein (GFP) and different concentrations of GFP-labeled Salmonella were inoculated into the rhizosphere-soil or sprayed on the leaf surface of the 4-week-old plants. LMV infection was only conducted in the surface-contaminated lettuce. The plants were irrigated with different volumes of water (drought, storm), collected after 2 days and cut into two parts (leaf, root/bottom) after surface disinfection to remove the attaching bacteria. Plate count and qPCR were used to determine the viable and total internalized Salmonella. The internalized Salmonella (via surface contamination) was also visualized using a laser scanning confocal microscope. This study discovered S. Typhimurium can disseminate throuought the entire plant after penetrating into the tissues through the roots or leaves. The viable Salmonella was detected in both parts of green onion, however, was only found in the lettuce leaves regardless of contamination method. However, once the lettuce was infected with LMV, the viable Salmonella showed up in the both parts. The water stress alone did not influence the internalization level in the lettuce when contaminated on the surface, or in the green onion using either contamination me (open full item for complete abstract)

Committee: Ahmed Yousef PhD (Committee Chair); Jiyoung Lee PhD (Advisor); Luis Rodriguez-Saona PhD (Committee Member); V.M. Balasubramaniam PhD (Committee Member) Subjects: Food Science

Master of Science, The Ohio State University, 2012, Food Science and Technology

Fresh produce is a high risk food for human norovirus (NoV) contamination, because it can easily become contaminated at both the pre- and post-harvest stages of cultivation. Disease surveillance has shown that human NoV is attributed to 40% of all fresh produce related outbreaks reported each year in the U.S. However, the ecology, persistence, and interaction of human NoV and fresh produce are all poorly understood. Increasing outbreaks of viruses in fresh and fresh-cut vegetables and fruits give high urgency to understanding the interaction of human NoV with fresh produce in order to develop effective preventive measures. In this research, the attachment, uptake, internalization, and dissemination of human NoV and its surrogates (murine norovirus, MNV-1; and Tulane virus, TV) were evaluated. First, the attachment of human NoV surrogates to fresh produce was visualized using confocal microscopy. Purified human NoV virus-like particles (VLPs), TV, and MNV-1 were conjugated with biotin, and subsequently applied to either Romaine lettuce or green onion. The biotinylated virus particles were visualized by incubation with streptavidin coated Quantum Dots (Q-Dots 655), which emit fluorescence that can be viewed using a confocal microscope. It was found that all three surrogates attached to the surface of Romaine lettuce leaves and were found aggregating in and around the stomata. Similarly, human NoV VLPs, TV, and MNV-1 were found to attach to the surface of Romaine lettuce roots. In the case of green onions, human NoV VLPs were found between the cells of the epidermis of both the shoots and roots. However, TV and MNV-1 were found to be covering the surface of the epidermal cells in both the shoots and roots of green onions. The results indicate that different viruses vary in their attachment patterns to different varieties of fresh produce. A quantitative assessment of the level of attachment of a human NoV GII.4 strain, TV, and MNV-1 was executed using Romaine lettuce a (open full item for complete abstract)

Committee: Jianrong Li PhD (Advisor); Ken Lee PhD (Committee Member); Yael Vodovotz PhD (Committee Member) Subjects: Food Science; Virology

Doctor of Philosophy, The Ohio State University, 2012, Veterinary Preventive Medicine

Water related disease outbreaks threaten public health and safety worldwide. In the United Sates, notwithstanding public drinking water systems strictly regulated, acute gastrointestinal illnesses (AGI) are continuously reported to health agencies . In agricultural intensive areas, surface and ground water resources are more likely to be exposed to be contaminated with zoonotic bacteria, given the close proximity to sources of feces from livestock, dairy farms and wildlife. The aim of this dissertation was to determine a role of drinking and irrigation water as a vehicle for the transmission of zoonotic bacteria of fecal origin and the need of risk management in rural areas. First, we investigated the microbial quality of private well drinking water system located in six Townships in northeastern Ohio, regions with high concentration of dairy farms. Water samples were collected in 180 households (summer, 2009) and processed to detect fecal indicative organisms, E. coli O157 and Campylobacter jejuni by using commercial MPN methods and quantitative PCR analysis. Around 46%, 9 % and 4% of wells were contaminated with coliforms, E. coli and E. coli O157 respectively. There were no positives for C. jejuni. Second, current guidelines for microbial irrigation water quality recommended by relevant agencies were evaluated in the regard with their practicality and feasibility to detect water quality deterioration in practical applications. Water samples (n=227) were collected in six surface water sources providing irrigation water to each six farm located in Northeastern Ohio over one irrigation season (Apr to Nov ,2010). Bootstrap analysis was applied to estimate optimal water testing frequency compared to those in current guidelines based on the value of fecal indicators detected in the water samples. Current guidelines for microbial quality of irrigation water imprecisely reflected the quality of water over one irrigation season in the context of sampling frequency recom (open full item for complete abstract)

Committee: Jeffrey LeJeune PhD (Advisor); Gireesh Rajashekara PhD (Committee Member); Rebecca Garabed PhD (Committee Member); Song Liang PhD (Committee Member) Subjects: Agriculture; Environmental Health; Environmental Management; Environmental Science; Epidemiology; Microbiology; Molecular Biology; Public Health

Master of Science, The Ohio State University, 2012, Food Science and Technology

The relationship between the consumption of fresh produce and reduced risk of cardiovascular diseases makes these products a necessary component of healthy diet. However, concerns have been raised because of increased disease outbreaks associated with the consumption of contaminated produce. The development of new and promising decontamination technologies is necessary to provide safe produce to the public and to prevent economic losses. The objectives of this study were (i) to assess the efficacy of gaseous ozone in combination with vacuum cooling for the inactivation of Escherichia coli K12 and E. coli O157:H7 spot inoculated on leaves of baby spinach and Romaine lettuce, and (ii) to evaluate the efficacy of a pre-washing step, integrated into the gaseous ozone treatment, to enhance the inactivation of E. coli K12 and E. coli O157:H7 on baby spinach and cut Romaine lettuce leaves. The efficacy of gaseous ozone in combination with vacuum cooling was evaluated for inactivation of Escherichia coli on ready-to-eat baby spinach and Romaine lettuce leaves. In a preliminary experiment, baby spinach leaves were spot inoculated with E. coli K12. Inoculated leaves were vacuum cooled (-28.5 in. Hg) and then treated with gaseous ozone at the following conditions: 1.5 g/kg ozone in gas mix at 10 psig holding pressure for 30 min holding time. Compared to inoculated non-treated samples, oxygen only (control) and gaseous ozone treatment decreased the microbial populations 0.27 and 1.29 log CFU/g, respectively. Similarly, baby spinach leaves were spot inoculated with E. coli O157:H7 and treated at the same conditions described in the preliminary study. Efficacy of the treatment varied with the crop season. Reductions on baby spinach were 1.86 and 1.59 log CFU/g of E. coli 0157:H7 in January, 2011 and February, 2012 respectively, and these reductions were significantly higher than the average 0.97 log reduction obtained between May and September, 2011. The color of baby spinach l (open full item for complete abstract)

Committee: Ahmed Yousef Dr. (Advisor); Sheryl Barringer Dr. (Committee Member); Sudhir Sastry Dr. (Committee Member) Subjects: Food Science

Master of Science, The Ohio State University, 2011, Food Science and Technology

Viruses are the leading cause of foodborne illness worldwide (67%). Specifically, human norovirus (HuNoV) is the major foodborne virus. Fresh produce is often at high risk for norovirus contamination because it can be easily contaminated at both pre-harvest and post-harvest stages and it undergoes minimal or no processing. There is an urgent need to develop novel interventions to eliminate foodborne enteric viruses in fresh produce. High pressure processing (HPP), a non-thermal processing technology may provide a new approach to reduce the virus load in fresh produce and related products. In the present study, we systematically investigated the effectiveness of HPP on inactivating norovirus in aqueous medium, lettuce, strawberry, and fruit puree using murine norovirus (MNV-1) as a surrogate for noncultivable HuNoV. Approximately 5 log virus reduction was observed in all food items upon treatment at 400 MPa for 2 min at 4°C, demonstrating that HPP is effective in reducing the MNV-1 load in fresh produce. Moreover, our results showed that pressure, pH, temperature, and the food matrix affected the virus survival. MNV-1 was more effectively inactivated at 4°C than at 20°C. MNV-1 was also found to be more sensitive to high pressure at neutral conditions (pH 7.0) than at acidic conditions (pH 4.0). Taken together, these findings support the notion that HPP is a promising intervention to eliminate the norovirus risk in fresh produce and related products while the organoleptic and nutritional properties of these foods are affected to the minimal extent. To further evaluate the potential of HPP in inactivating viruses, we continued to investigate the effectiveness of HPP on the inactivation of human rotavirus (HRV), vesicular stomatitis virus (VSV), and avian metapneumovirus (aMPV). HRV represents a major foodborne virus other than HuNoV and is non-enveloped; VSV and aMPV are enveloped and their virion structures are strikingly different from those of MNV-1, HuNoV, and HRV (open full item for complete abstract)

Committee: Jianrong Li (Advisor); Sheryl Barringer (Committee Member); Steven Schwartz (Committee Member) Subjects: Food Science

Doctor of Philosophy, The Ohio State University, 2009, Food Science and Nutrition

Although good agricultural practices (GAP) and good handling practices (GHP) may reduce the incidence of pathogens in fresh produce, there is no known defense against contamination due to wildlife or surface water. Additionally, current strategies designed to minimize the risk of contamination during post-harvest operations of fresh leafy greens are not completely effective. Contaminations of fresh fruits with spoilage microorganisms also create economical losses by decreasing products shelflife. Therefore, it is necessary to develop sound mitigation strategies to minimize the health hazards associated with fresh produce. The main objectives of this study are to enhance the safety of fresh produce in general, and baby spinach in particular, by integrating ozone-based sanitization steps into existing processing practices, while targeting Escherichia coli O157:H7 as the pathogen of concern, and to assess the feasibility of using ozone, carbon dioxide or their combinations, for reducing natural microbiota, and extending the shelf-life of strawberries. A pilot-scale system has been successfully assembled that allows vacuum cooling and ozone treatment of fresh produce simultaneously. Combinations of vacuum cooling and ozonation (SanVac) successfully inactivated up to 2.4 log E. coli O157:H7 CFU/g spinach. Contribution of important treatment variables (ozone concentration, pressure and treatment time) to process lethality was also investigated using response-surface methodology. Parameters for the optimized SanVac process are of 1.5 g O3/kg gas-mix (935 ppm, vol/vol), 10 psig holding pressure, and 30 min holding time; these conditions achieve 1.8 log inactivation against E. coli O157:H7 with no apparent damage to quality of baby spinach. A long-term, low-ozone process was also developed to treat fresh produce during transportation or temporary refrigerated storage. This treatment, termed “SanTrans”, involves sparging moist gaseous ozone at 16 mg/kg (10 ppm, vol/vol) (open full item for complete abstract)

Committee: Ahmed E. Yousef PhD (Advisor); Sheryl Barringer PhD (Committee Member); V.M. Balasubramaniam PhD (Committee Member); Luis E. Rodriguez-Saona PhD (Committee Member) Subjects: Food Science