DEVELOPMENT OF OZONE-BASED PROCESSES FOR DECONTAMINATION OF FRESH PRODUCE TO ENHANCE SAFETY AND EXTEND SHELFLIFE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mustafa Vurma, M. S. ***** The Ohio State University 2009 Dissertation Committee: Dr. Ahmed E. Yousef, Adviser Approved by Dr. Sheryl Barringer Dr. V. M. Balasubramaniam _____________________________ Adviser Dr. Luis Rodriguez-Saona Food Science and Technology Graduate Program ABSTRACT 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 the current study are to (i) 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 (ii) 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, ii 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) for up to 3 days. The process inactivated up to 1.4 log E. coli CFU/g spinach, and the optimum process resulted in 1.0 log inactivation with minimal effect on product quality. In order to maximize inactivation on E. coli O157:H7, contaminated spinach was sequentially subjected to optimized SanVac (at 1.5 g/kg for 30 min and 10 psig) and SanTrans (at 16 mg/kg) processes, using freshly-harvested, unprocessed spinach that was shipped directly from California fields. These sequential treatments inactivated 4.1 to ≥5.0 log E. coli O157:H7, depending on the treatment time. Inoculated and vacuum-cooled spinach was analyzed by scanning electron microscopy. It was apparent that vacuum-cooled leaves often contained bacterial cells throughout the intercellular spaces, compared with the non-vacuum cooled leaves. An ozone-based treatment system was designed and constructed to enhance the storage life of fresh strawberries (Fragaria x ananassa). The O3/CO2 combination treatments showed synergistic effect in delaying mold growth and quality deterioration of the strawberries. When samples were treated with O3/CO2 for 4 h and stored at 4°C, the initiation of visual mold appearance was delayed until the 16th day of storage; an 8-day iii shelflife extension, compared to untreated samples. These novel technologies are promising alternatives to conventional processes and should enhance the safety and extend the shelflife of fresh fruits and vegetables. Additionally, the new treatments should be relatively easy to integrate into existing fresh produce processes and practices. iv ACKNOWLEDGMENTS I would like to express my sincere thanks to Dr. Ahmed E. Yousef for his outstanding guidance, support, encouragement, and enthusiasm during my graduate studies. As a scientist and a great person, he has taught me many lessons. I am so grateful for the wonderful opportunity to work with him. I would also like to thank Dr. Sudhir Sastry for providing excellent collaboration and guidance. I would like to thank Dr. Sheryl Barringer, Dr. V.M. Balasubramaniam, and Dr. Luis Rodriguez-Saona for serving on my committee and for their helpful guidance. Throughout the study, I received exceptional collaboration from a number of people. I would like to thank all the current and past members of the Food Safety Research Laboratory; Dr. Abdullatif Tay, Dr. Yoon-Kyung Chung, Dr. Kim Jin Gab, Dr. Mohammed Khadre, Dr. Luis Rodriguez-Romo, Dr. Joy Waite, Joe Jones, Jenifer Perry, Amrish Chawla, and Yuan Yan for their friendship, cooperation, advice, criticism, and suggestions. I would especially like to thank my wife Nurdan, for her intellectual advice, assistance, and endless patience; my son Arın Ozan for his love, and never-ending energy, and my parents for all their love and support. I also would like to thank DuPont Enterprise and Fresh Express Inc. for their financial support and the Food Science and Technology Department for providing me the opportunity to pursue my Ph.D. degree at The Ohio State University. v VITA March 30, 1972……………………………………….. Born, Tarsus, Turkey 1995…………………….........................................……B.Eng. Food Engineering, Istanbul Technical University 2004……………………………………………….……M.S. Food Science and Nutrition, The Ohio State University 2005…………………………………………………….Optional Practical Trainee, Kraft Foods R&D 2005-2009………………………………………………Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Rodriguez-Romo, L.A., Vurma, M., Lee, K., and Yousef, A.E. 2007. Penetration of ozone gas across the shell of hen eggs. Ozone: Science and Engineering 29: 1-4. 2. Vurma, M.., Chung, Y.K., Shellhammer, T.H., Turek, E.J., and Yousef, A.E. 2006. Use of phenolic compounds for sensitizing Listeria monocytogenes to high-pressure processing. International Journal of Food Microbiology 106: 263-269. 3. Chung, Y.K., Vurma, M., Turek, E.J., Chism, G.W., and Yousef, A.E. 2005. Inactivation of barotolerant Listeria monocytogenes in sausage by combinations of high-pressure processing and food-grade additives. Journal of Food Protection 68: 744-750. FIELDS OF STUDY Major Field: Food Science and Technology vi TABLE OF CONTENTS Page Abstract......................................................................................................................ii Acknowledgments......................................................................................................v Vita.............................................................................................................................vi List of Tables .............................................................................................................ix List of Figures............................................................................................................xi Chapters: 1. Literature Review.................................................................................................1 Introduction ...................................................................................................1 Ozone chemistry and physics.........................................................................2 Ozone production...........................................................................................8 Ozone measurement.......................................................................................9 Antimicrobial properties of ozone .................................................................11 Ozone and food applications..........................................................................18 Selected food applications .............................................................................21 Combination treatments.................................................................................29 Limitations, safety considerations, and regulatory status ..............................33 Fresh produce and microbial food safety.......................................................35 Conclusions....................................................................................................42 References......................................................................................................43 2. Development of sanitization technologies to integrate gaseous ozone treatments into fresh produce vacuum cooling operations ..................................71 Abstract..........................................................................................................71 Introduction....................................................................................................72 vii Materials and methods ...................................................................................75 Results and discussion...................................................................................82 References......................................................................................................90 3. Inactivation of Escherichia coli O157:H7 and natural microbiota on spinach leaves using gaseous ozone