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Validation of Thermal Destruction of Pathogenic Bacteria in Rendered Animal Products Mary Hayes Clemson University, [email protected]

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Validation of Thermal Destruction of Pathogenic Bacteria in Rendered Animal Products Mary Hayes Clemson University, Mmh@G.Clemson.Edu Clemson University TigerPrints All Dissertations Dissertations 12-2013 Validation of Thermal Destruction of Pathogenic Bacteria in Rendered Animal Products Mary Hayes Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Microbiology Commons Recommended Citation Hayes, Mary, "Validation of Thermal Destruction of Pathogenic Bacteria in Rendered Animal Products" (2013). All Dissertations. 1255. https://tigerprints.clemson.edu/all_dissertations/1255 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. VALIDATION OF THERMAL DESTRUCTION OF PATHOGENIC BACTERIA IN RENDERED ANIMAL PRODUCTS A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Microbiology by Mary Melissa Hayes December 2013 Accepted by: Annel K. Greene, Committee Chair Thomas A. Hughes Xuiping Jiang Paul L. Dawson ABSTRACT This study tested enumeration techniques for high fat food matrices and determined thermal death times in rendering animal products. Standard Class O phosphate/magnesium chloride dilution buffer series (Dilution Series A) and a modified (pre-warmed to 32ºC) lecithin phosphate dilution buffer series (Dilution Series B) were used to enumerate a Salmonella cocktail from both poultry and beef rendering materials. Results of this study indicate use of a modified lecithin buffer did not improve Salmonella enumeration accuracy from rendering materials. Instead, the results suggested use of xylose lysine deoxycholate agar (XLD) with either buffer system accurately enumerated Salmonella from rendering materials. The thermal death of four pathogenic strains of Salmonella recognized by the FDA as hazardous in animal feeds (Salmonella Choleraesuis (SC), Salmonella Enteriditis (SE), Salmonella Newport (SN), and Salmonella Dublin (SD)) was not a straight line decrease. After periods of appearing to be destroyed, some cultures reappeared at later treatment times. In thermal treatments up to 420 s at 240ºF (115.6ºC), SC was last detected at 120 s, SE at 120 s, SN at 300 s and SD at 360 s in inoculated beef rendering materials. In thermal treatments up to 420 s at 240ºF (115.6ºC), SC, SE, SN, and SD were last detected at 360 s, respectively, in inoculated poultry rendering materials. Controls indicated thermally resistant strains in the background of both beef and poultry rendering materials which when tested using standard FDA Bacteriological Analytical Manual (BAM) techniques indicated Salmonella. Hypotheses to explain the results of this study include: 1) thermally resistant sub-particles such as bone or tissue protected bacteria from ii thermal treatment; 2) presence of thermally resistant species in the background of rendering samples caused false positive results on BAM procedures; or 3) presence of thermally resistant Salmonella. Further research will need to be conducted at 240ºF (115.6ºC) for longer time intervals to ensure that SC, SE, SN and SD are destroyed and to identify the impact of particles on thermal conductivity through the rendering matrices. Additionally, future experimentation will be needed to verify that the microorganisms identified are indeed Salmonella or other another microorganism(s) cross-reacting as Salmonella. iii DEDICATION This dissertation is dedicated to my family and many friends. A special dedication in gratitude goes to my grandmother, Ellen Davis, whose words of encouragement helped me to achieve my goals. I dedicate this work to my parents, Ann and Michael Hayes, and my brother, Michael Hayes. Without their love and support, this could not have been possible. I also dedicate this work and give special thanks to my fiancé, Brian Gaines, for being there for me on the tough days. Finally, I would like to dedicate my dissertation to Neko, Bella, Vixen, Hitch, Nokie, Kitty, and Willow, for their hugs and unconditional love. iv ACKNOWLEDGMENTS I acknowledge, with much gratitude, my debt of thanks to Dr. Annel K. Greene for her advice, support, and foresight. I also would like to thank my committee members, Dr. Thomas A. Hughes, Dr. Xiuping Jiang, and Dr. Paul L. Dawson for their constant guidance during my research project. I am eternally grateful to all the undergraduate assistants for helping with all the tedious grunt work in the laboratory. Finally, I would like to thank Steven D. Chambers and Yubo Zhang for being a surrogate family offering continued moral support in and outside of the laboratory. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ......................................................................................................... x CHAPTER 1. LITERATURE REVIEW .............................................................................. 1 Introduction .............................................................................................. 1 The Rendering Process ............................................................................ 2 Thermal Processing Principles ................................................................. 6 Salmonella................................................................................................ 8 Salmonella and Rendering Co-Products ................................................ 10 Emulsifiers ............................................................................................. 13 Conclusion ............................................................................................. 14 References .............................................................................................. 15 2. METHOD VALIDATION FOR ENUMERATING BACTERIA IN HIGH FAT MATRICES ................................................................................... 21 Abstract .................................................................................................. 21 Introduction ............................................................................................ 22 Materials and Methods ........................................................................... 23 Rendering Sample Preparation .................................................. 23 Salmonella Preparation .............................................................. 24 Salmonella Enumeration in Rendering Materials ...................... 25 Statistical Analysis ..................................................................... 27 Results .................................................................................................... 27 Discussion .............................................................................................. 29 vi Table of Contents (Continued) Page Figures and Tables ................................................................................. 32 References .............................................................................................. 34 3. VALIDATION OF THERMAL DESTRUCTION OF SALMONELLA IN RENDERED BEEF PRODUCTS.......................................................... 35 Abstract .................................................................................................. 35 Introduction ............................................................................................ 37 Materials and Methods ........................................................................... 38 Rendering Sample Preparation .................................................. 38 Salmonella Preparation .............................................................. 39 Thermal Death Time Trials ........................................................ 41 Bone Particle Size Determination .............................................. 44 Determination of Estimated D Values ....................................... 44 Results .................................................................................................... 45 Discussion .............................................................................................. 49 Figures and Tables ................................................................................. 54 References .............................................................................................. 71 4. VALIDATION OF THERMAL DESTRUCTION OF SALMONELLA IN RENDERED POULTRY PRODUCTS ................................................. 73 Abstract .................................................................................................. 73 Introduction ............................................................................................ 74 Materials and Methods ........................................................................... 76 Rendering Sample Preparation .................................................
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