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Photographs included in the original manuscript have been reproduced xerographically In this copy. Higher quality 6" x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Leaming 300 North Zeeb Road, Ann Artwr, Ml 48106-1346 USA 800-521-0600 UMI CONTROL OF CLOSTRIDIUM BOTULINUM BY BACTERIOCINS AND CHARACTERIZATION OF NISIN ACTION DURING SPORE-TO-CELL TRANSFORMATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yoon-Kyimg Chimg, M. S. ***** The Ohio State University 2oei Dissertation Committee: Approved By Dr. Ahmed E. Yousef, Advisor Dr. Grady W. Chism m Dr. David B. Min Food Science and Nutrition Dr. Polly D. Courtney UMI Number; 3031190 UMI' UMI Microform 3031190 Copyright 2002 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Titie 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Bacteriocins from lactic acid bacteria (LAB) can serve as a natural preservative to control foodbome pathogens in minimally processed foods. For effective biocontrol, type of bacteriocin, level of application, and stage of application against target pathogen, such as C. botulinum in this study, are the some of the factors need to be optimized. The purposes of this study were (1) to determine the most effective bacteriocin against C. botulinum, (2) to verify the effect of bacteriocin in a food system, (3) to monitor the germination of C. botulinum spores after heat treatment and in the presence of bacteriocin, and (4) to define the function of bacteriocin at different stages of spore-to-cell transformation Clostridium botulinum was more effectively inactivated by the culture supernatant of a nisin-producing bacterium than that of pediocin P02 or lacidin A. Combination of nisin and its-producer effectively delayed the growth of C. botidinum in mashed potatoes stored at 25°C for 5 days. In addition, commercial nisin (50-250 pg/g potatoes), was a very effective bactericidal agent against C. botulinum in roasted potatoes packaged under modified atmosphere (100 % N% or 20% CO 2 :80% Nz). The effect of nisin and pH (2.8 and 3.4) on the activation and subsequent germination of C. botulinum spores was investigated. Spores of C. botulinum were not ii inactivated by heat treatments ug to 92“C for 2 hours. Heat treatment at 85°C for 90 min was selected as the optimal activation condition based on monitoring subsequent germination. L-alanine alone or combined with L-cysteine was not sufficient to germinate the spores of this strain. Additional germinant(s) in Tryptone-Peptone- Glucose-Yeast extract (TPGY) broth were required for germination. Lower pH and presence of nisin during heat activation increased the degree of phase darkening, i.e., germination. However, extended heating in the presence of nisin at pH 2.8 decreased the spore culturability on recovery medium (TPGY agar). The action of nisin against vegetative cells and spores of C. botulinum was investigated. Nisin was added at various stages of spore-to-vegetative cell transition and changes in sensitivity to the bacteriocin were observed. Nisin was bactericidal against vegetative cells, but was sporostatic against spores of C. botulinum under the same concentration. The degree of bactericidal and sporostatic action of nisin was dependent on the concentration of nisin used. Under combined treatment of heat and nisin, the bacteriocin enhanced spore activation, and subsequently triggered the germination. Nisin delayed germination of activated spores, when it was added after heat activation. In addition, nisin prevented the late event of germination stage (after loss of heat resistance and before phase darkening) when added after heat activation. Finally, nisin inhibited the outgrowth of germinated spores for 20 days when added after germination stage. Findings firom this study indicate that LAB bacteriocins may be effective biocontrol agents against C. botulinum or other pathogenic/spoilage microorganisms in ready-to-eat refrigerated foods such as roasted potatoes. In addition, it is suggested that iii the bacteriocin application time i& very important for the effective control of spores during the heating process of foods. It may be possible to apply nisin at the stage of processing that coincides with the most sensitive stage of spore transformation. Finally, the action of nisin is influenced by other factors such as pH and NaCl in food systems. IV Dedicated to my mother and late father ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. Ahmed Yousef for his constant guidance, encouragement, advice, and patience throughout my study. I gratefully acknowledge that this fruition comes largely from his support. I greatly thank him for being a mentor, not just for serving as my advisor. I also would like to express thanks to Dr. Grady Chism, Dr. David Min, and Dr. Polly Courtney, my dissertation committee members, for their valuable advices, discussions and time. I wish to deliver special thanks to Dr. Mohammed BChadre, Dr. Jin-Gab Kim, and Dr. Xia Liu for their helpful suggestions and discussions. Many thanks to our laboratory group. Dr. Robert Gilmore, Dr. Abdullatif Tay, Luis Rodriguez-Romo, Hyun-Jung Chung, and Beatrice Lado, for their friendship and help in many ways. 1 wish to thank the Department of Food Science and Technology, Hillshire Farm and Kahns Company (Cinciimati, Ohio), and The Center for Advanced Food Processing and Packaging Studies (Raleigh, North Carolina) for supporting my research. Most of all, I wish to share the joy of this accomplishment with my family. I want to give special thanks to my daughter, Mee-Ree, who was bom during my first year of PhT). study. She has shared all difSculties with me throughout the study. My deepest thank goes to my mother, who has not hesitated to bear all kinds of sacrifices for me; to whom I owe everything. VI VITA December 2, 1964................................. Bom, Seoul, Korea 1987 .......................................................B. S., Seoul National University, Seoul, Korea 1987-198 8 ............................................. Graduate school, Seoul National University, Seoul, Korea 1988-199 1 ............................................. M. S., Iowa State University, Ames, Iowa 1992-1994............................................. M. S., University of Maryland, College Park, Maryland 1995-Present..........................................Graduate Research Associate, The Ohio State University, Columbus, Ohio FIELDS OF STUDY Major Field: Food Science and Nutrition vu TABLE OF CONTENTS Page Abstract......................................................................................................................... ü Dedication ..................................................................................................................... v Acknowledgments ........................................................................................................ vi Vita..............................................................................................................................vii List of Tables ................................................................................................................ x List of Figures .............................................................................................................. xi Introduction ....................................................................................................................1 Chapters: l . Literature Review....................................................................................................4 Clostridium botulinum............................................................................................ 4 Clostridium botulinum and botulism ....................................................................4 Spore structure and resistance ...............................................................................5 Spore-to-cell transition ..........................................................................................6 Bacteriocins of lâche acid bacteria .................... Il Definition of bacteriocins ...................................................................................11 Chemical composition