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Ph.D. Dissertation Efficacy of Ultraviolet Light and Antimicrobials to Reduce Listeria monocytogenes in Chill Brines By Priti P. Parikh Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Science and Technology Approved: Robert C. Williams, Chair Kumar Mallikarjunan Joseph D. Eifert Joseph E. Marcy November 1, 2007 Blacksburg, Virginia Keywords: Listeria monocytogenes , ultraviolet light, brine, citric acid, dimethyl dicarbonate, hydrogen peroxide Copyright 2007, Priti Parikh Efficacy of Ultraviolet Light and Antimicrobials to Reduce Listeria monocytogenes in Chill Brines by Priti P. Parikh ABSTRACT Chill brines used in ready-to-eat meat processing may be an important source of post- processing contamination by Listeria monocytogenes . The purpose of this study was to determine the efficacy of ultraviolet light (UV) in combination with antimicrobials to reduce L. monocytogenes in fresh and used chill brines. Three different antimicrobials were used in combination with UV; citric acid (CA, 0.2 and 0.5%), dimethyl dicarbonate (DMDC, 250 and 500 ppm), and hydrogen peroxide (HP, 2000 and 4000 ppm). For fresh brine studies, brine (8.0% w/v NaCl) was prepared and inoculated with a cocktail of three L. monocytogenes strains (approximately 6 log CFU/mL). Brine was treated with UV alone, antimicrobials alone, and combination of UV and antimicrobials. Moreover, to observe the effect of treatment temperature and brine circulation through the UV system on survival of listeriae cells, inoculated brine was circulated through the system without any treatment that served as control for all the treatments. For UV treatment, inoculated brine solution was exposed to UV in an Ultraviolet Water Treatment Unit (Model: AMD 150B/1/2T D; Aquionics Inc., Peak output: 254 nm) fitted with an inline chiller to maintain brine temperature of -1°C. Samples were withdrawn at regular intervals for 120 minutes. When L. monocytogenes population was no longer detectable via direct plating on MOX, enrichment was performed and suspect colonies were confirmed using API-Listeria . For antimicrobial-only (i.e., no UV) treatments, a specific concentration of antimicrobial was added in inoculated brine and samples were taken for 120 minutes. For the brine that received combination of UV and antimicrobial treatments, UV was turned on once a specific concentration of antimicrobial was added in inoculated brine and samples were withdrawn at regular intervals for 120 minutes. When treated with UV alone, L. monocytogenes population decreased from approximately 6 log CFU/mL to below the detection limit (i.e., 1 log CFU/mL) in 15 minutes with the reduction rate of 0.87 log CFU/mL per minute. However, cells were detectable by enrichment through 120 minutes. The highest rate of decline (0.90 log CFU/mL per minute) was achieved by the combination of UV and 500 ppm DMDC (UV+500 ppm DMDC), which was not significantly different from the reduction rates of UV and UV+0.5% CA. UV+500 ppm DMDC reduced L. monocytogenes to the detection limit in 15 minutes and the organism was not detected by enrichment after 60 minutes. Though the reduction rate of UV+0.5% CA was not significantly lower than the rate of UV+500 ppm DMDC (P>0.05), the former treatment resulted in non-detectable levels more quickly (45 minutes) than the latter (60 minutes). Thus, based on enrichment studies UV+0.5% CA was the most effective treatment in reducing the population of L. monocytogenes in fresh brine. Moreover, when brine was treated with 0.5% CA alone the population decreased to below detection limit in 15 minutes with the rate significantly lower than UV+500 ppm DMDC and UV+0.5% CA (P<0.05). However, L. monocytogenes was not detectable by enrichment from 60 minutes. To summarize, through enrichment studies we observed that UV+0.5% CA, UV+500 DMDC, and 0.5% CA Control were more effective than other treatments in reducing the listeriae population to a non-detectable level. Spent brine is recycled brine that was obtained from a frankfurter processor after its maximum usage. Results of spent brine studies showed that when brine was treated with UV+4000 ppm HP and UV+2000 ppm HP, L. monocytogenes population decreased to the detection limit in 45 minutes and was not detected by enrichment from 120 minutes. These iii treatments were observed to be the most effective treatments with a reduction rate of 0.12 log CFU/mL per minute. The reduction rate of some other treatments such as, UV+250 and 500 ppm DMDC, UV+0.2% and 0.5% CA, and UV alone was not significantly different from UV+4000 and 2000 ppm HP. However, the population was detected through enrichment up to 120 minutes in all other treatments. The results of these studies indicate that combinations of UV and antimicrobial may be more effective than either treatment alone (except 0.5% CA treatment) to process fresh chill brines. However, the antimicrobials and UV were less effective for controlling L. monocytgoenes in spent brine; presumably due to the presence of organic matter. iv ATTRIBUTION Author Priti Parikh is the major contributor and writer of the manuscripts in chapter four and chapter five of this dissertation. Co-authors Dr. Robert Williams, Ph.D., Food Science, University of Tennesse, 2001, Committee Chair, and Prof. Joseph Marcy, Ph.D., Food Science and Technology, North Carolina State University, 1980; Joseph Eifert, Ph.D., Food Science, Virginia Tech, 1994; Kumar Mallikarjunan, Ph.D., Biological Engineering, University of Guelph, 1993, Committee members, provided advice, supervision, funding, and laboratory support. Parikh, Williams, Marcy and Eifert are with Dept. of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061 and Mallikarjunan is with Dept. of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061. v DEDICATION I dedicate this dissertation to my parents Drs. Kishor and Ranjan Shastri , and my wonderful husband, Dr. Pratik Parikh vi ACKNOWLEDGEMENTS I joined the Department of Food Science and Technology (FST) at Virginia Tech in Spring 2005. It might not be a long time for me being in this wonderful department, but a lot of things have happened in this short time. I initially was enrolled in the Masters program at FST and then changed my status from M.S. to Ph.D. after a year. There are many people who helped me take that decision and I would always be grateful to all of them. So let me take this opportunity to thank everyone who directly or indirectly helped me fulfill my dream. First, I would like to express my deepest gratitude to my major professor Dr. Robert Williams without whom I would have never thought of pursuing a Ph.D. degree. He showed more confidence in me than I had when I was given an option of joining a Ph.D. program. He was always there to help me when I was confused whether it was in research or in life. I truly enjoyed the times when he would try to learn my language (Gujarati/Hindi) and then experiment it with Joell. He has been a great mentor who would not only give you a freedom or flexibility in terms of work, but also would support you and make you laugh whenever needed. I also thank my committee members, Dr. Eifert, Dr. Marcy, and Dr. Mallikarjunan who supported me and guided me whenever needed. I specially thank Dr. O’Keefe who was always willing to help me with some random chemistry questions and let me use his laboratory whenever I needed. I truly thank Joell and Walter for everything they did for me. Had Joell not helped me, I would have not received my spent brine along with other lab supplies and without Walter I could have not run the UV machine. So without their support I could have not finished my degree on time. I also thank Govind and Leslie for all their help with media preparation and cleaning the UV system that I used. Many thanks to all the graduate students at FST who made my tough journey so pleasant. Very special thanks to my close friend and roommate, Supriya vii Ratnaparkhe, who took care of me as an elder sister during the last (but crucial) year of my journey. I remember that during my prelims she did all the household work and cooking so that I get enough time to study. I will never forget this wonderful person who used to make me laugh like anything, especially when I was stressed with my work. I also thank my parents (Drs. Kishor and Ranjan Shastri), parents-in-laws (Dr. Jitesh Parikh and Smita Parikh), brother, sister, brother-in-laws, uncle, and ant for their moral support and all the prayers. Their blessings and prayers have helped me thrive in many discouraging moments. I truly realized the value of my “family” when I left India, my country. But I am glad that my beloved husband, Dr. Pratik Parikh, was always with me whenever I needed him. He is one of the most important persons in my life. He not only showed me the other side of the world, but also different face of life! He gave me new, but exciting life. I am glad that up on graduation I am going to join him again in Atlanta (after a year of separation due to education) and continue enjoying this exciting life with him forever! viii TABLE OF CONTENTS ABSTRACT.................................................................................................................................... ii ATTRIBUTION...............................................................................................................................v ACKNOWLEDGEMENTS.........................................................................................................
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