POST-HARVEST INTERVENTIONS AND FOOD SAFETY OF LEAFY GREEN VEGETABLES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sanja Ilic, M.Sc. Graduate Program in Food Science and Technology The Ohio State University 2011 Dissertation Committee: Dr. Jeffrey T. LeJeune, Adviser Dr. Brian McSpadden Gardener Dr. Douglas Doohan Dr. Ken Lee Copyright by Sanja Ilic 2011 Abstract Foodborne illness outbreaks associated with the consumption of leafy green vegetables are a growing public health concern worldwide. While there has been great progress in using practical and cost-effective interventions to reduce the risk of contaminations and prevalence of pathogens on farm, effective post-harvest interventions to remove field acquired contamination are still lacking. The body of literature related to microbial hazards in leafy green vegetables has accumulated since 1990, offering often contradictory information on the efficacy of food safety interventions. In this work, I identified, characterized, and assessed the quality of available research on prevalence, risk factors, and interventions for 16 microbial hazards in leafy green vegetables. Systematic literature review, a replicable two-level relevance screening, and a two-phase quality assessment and data extraction procedure were performed by two independent reviewers following general principles of systematic review methodology. A lack of well designed, executed, and reported prevalence studies investigating the efficacy of intervention(s) under real-life conditions was observed. Additional identified knowledge gaps and research areas included equipment sanitation and cross-contamination potential, survival of pathogens in organic leafy greens, and the lack post-harvest intervention studies applicable to the developing regions. ii Several identified knowledge gaps were further addressed. The concentration of coliforms and generic Escherichia coli and prevalence of E. coli O157:H7, Salmonella spp., Shigella spp. and Listeria monocytogenes on two spinach types before and after minimal processing under commercial conditions was investigated. A total of 1356 spinach samples were collected daily in two processing plants over a period of 14 months. Average total coliform counts increased significantly after processing. The proportion of coliform positive samples increased from 53% before to 79% after minimal processing. Generic E. coli prevalence was 8.9% (mean 1.81 ± 0.14 log CFU/g) with no difference after processing. Salmonella spp. and L. monocytogenes were isolated from 0.4% and 0.7% of samples, respectively. The contribution of each commercial processing step to generic E. coli, coliform, and total aerobic bacterial load on fresh-cut lettuce and food contact surfaces in a processing plant were further quantified. Plausible bacterial populations on fresh-cut, minimally processed lettuce using T-RFLP analysis were also identified. Washing initially decreased coliforms on lettuce but microbial populations increased to the pre-washed levels during subsequent steps. Peak contamination on food contact surfaces was detected after two hours of processing, and then declined after 4-6h of processing without intermediate sanitation interventions. No association between coliform increase on lettuce and contact-surfaces was found. Removal of outer leaves was the single most effective step of large-scale minimal processing. T-RFLP analysis identified representatives of 12 phyla/classes with the bacterial population profile from minimally processed lettuce dominated by Gammaproteobacteria and Bacteroidetes. iii The ability of E. coli O157:H7 to survive in processed packed organic and conventional baby spinach under household storage conditions for the duration of shelf life was further compared. Epiphytic bacteria present and the effect of leaf size on survival of E. coli O157:H7 were assessed. E. coli O157:H7 survived equally well on both spinach types. Organic spinach had overall smaller leaves that supported higher counts of E. coli O157. Finally, existing data pertaining to post-harvest interventions was compiled and synthesized. The options for post-harvest risk reduction were summarized and the existing information was translated to vegetable production conditions in developing countries. The applicability of multiple-barrier approach in developing regions was discussed. This work fills several critical gaps in knowledge. The scoping study provides a model for similar studies that can identify gaps in knowledge and thus, target limited resources and guide policy development. Information gained in studies of minimal processing can be used to develop more effective measures appropriate to control food safety risks in commercial settings. This work has a direct impact on pre-harvest food safety enhancement for leafy green vegetables. iv To Alex, Daniel & Anna v Acknowledgments I would like to sincerely thank my advisor, Dr. Jeffrey LeJeune for his support and guidance in my work, for his confidence in me and the encouragement to persist in this process. Thank you for nurturing the independence in thought and research and for giving me many opportunities to be productive and advance as a scientist. I especially thank you for your patience and support with my growing family situation. It is my pleasure to thank my advisory committee. I thank Dr. Brian McSpadden Gardener for hosting me in his laboratory and providing valuable guidance in work and data analysis. Thanks to Dr. Douglas Doohan for giving me the opportunity to be involved in the extension efforts and Dr. Ken Lee for adopting me during my course work in Columbus. This work would not be possible without support and guidance of Dr. Andrijana Rajic. A big thank you to Andrijana and her team in Public Health Agency of Canada for passing on their knowledge and for the endless hours spent in reviewing papers. Sincere thanks to Dr. Michele Williams for her help in formating this thesis. I am grateful to all who I shared the lab with over the past four years: Mike Kauffman, Pam Schlegel, Jennifer Van Pelt, Dan Aruscavage, Ann Kersitng, Terry Kline, Gayeon Won, Gaochan Wong, Katie Dodson, Rose Schelapi, Laura Harpster, Ryan Chapman, and Mark Morasch. Thanks to Food Animal Health Research Program, all faculty, staff, student and scholars for making this a second home, and thanks to Food Science Department for accepting me into this program. A very special thank you to all with whom I shared nice moments at The Ohio State University, especially Gayeon and Anastasia for being good friends. vi Finally, my gratitude to my partner Alex Rodriguez, who talked me into coming to Ohio, his help with my work and for being the greatest manny to our two little kids. I thank Daniel and Anna for their love, hugs and smiles. Thanks to my Dad for never questioning my dreams, and to my Mum for always questioning my dreams. vii Vita 1989-1995 ......................................................B. Sc. University of Belgrade 2004-2006 ......................................................M.Sc., University of Guelph 2007-2011 ......................................................Graduate Research Associate, The Ohio State University Publications Ilic S, Drechsel P, Amoah P, and LeJeune J. (2010) Applying the Multiple-barrier Approach for Microbial Risk Reduction in the Post-harvest Sector of Wastewater- irrigated Vegetables, In Wastewater Irrigation and Health Edited by Drechse P, Scott C, Raschid-Sally L, Redwood M, and Bahri A. EarthScan London Sterling, VA, p 239-261. Zhang J, Shi J, Ilic, S; et al. (2009) Biological Properties and Characterization of Lectin from Red Kidney Bean (Phaseolus vulgaris). Food Reviews International, 25: 12-27. Ilic S, Odomeru J, and LeJeune, J (2008) Coliforms and Prevalence of Escherichia coli and Foodborne Pathogens on Minimally Processed Spinach in Two Packing Plants. Journal of Food Protection.71: 2398-2403. Shi, J; Xue, SJ; Kakuda, Y; Ilic, S; et al. (2007) Isolation and characterization of lectins from kidney beans (Phaseolus vulgaris) Process Biochemistry, 42: 1436-1442 Obradovic D., Ilic S. (1997) Concentrated starter cultures for production of cheese; advantages and applications. In Cheese Making, editor Pudja P.D. Ilic S., Ivanovic M., Obradovic D. (1996) Selection of LAB strains for the production of Kashkaval cheese. Current Trends in Dairy Science. Novi Sad. Fields of Study Major Field: Food Science and Technology viii Table of Contents Post-Harvest interventions and food safety of leafy green vegetables ............................... 1 DISSERTATION ................................................................................................................ 1 Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables .................................................................................................................... xv List of Figures ................................................................................................................