The Pennsylvania State University. The Graduate School. College of Agricultural Sciences IDENTIFICATION AND VALIDATION OF ANTIMICROBIAL

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences IDENTIFICATION AND VALIDATION OF ANTIMICROBIAL INTERVENTIONS FOR RED MEAT CARCASSES PROCESSED IN VERY SMALL MEAT ESTABLISHMENTS A Thesis in Food Science by Sally Lucile Flowers Copyright 2006 Sally Lucile Flowers Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2006

2 The thesis of Sally Lucile Flowers was reviewed and approved* by the following: Catherine N. Cutter Associate Professor of Food Science Thesis Advisor Chair of Committee Stephanie Doores Associate Professor of Food Science William R. Henning Professor of Animal Science Edward W. Mills Associate Professor of Dairy and Animal Science Nancy Ostiguy Associate Professor of Entomology John D. Floros Professor of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School.

3 ABSTRACT Carcass decontamination strategies for very small meat plants (VSMP) were developed by systematic experimentation. First, a survey of VSMP in Pennsylvania, Washington and Idaho determined that knife trimming and cold water washing were the most common antimicrobial interventions in use. Secondly, red meat carcasses (n = 866) processed in VSMP in Pennsylvania, Washington, Idaho, and Texas were swabbed to determine prevalences of Salmonella spp., Campylobacter spp., and E. coli O157:H7 and enumerate hygiene indicators (mesophilic aerobic plate count, coliforms, and generic E. coli). Thirdly, manual water washes (various temperatures, pressures, drip times, applications, and distances) and chemical rinses at various concentrations (acetic acid, citric acid, lactic acid, peroxyacetic acid, aqueous ozone, sodium hypochlorite, chlorine dioxide, and acidified sodium chlorite) were applied in inoculation challenge studies to reduce bacteria on inoculated beef surfaces. While hot water (77 C) yielded log reductions of 2.7 to 5.1 CFU/cm 2, there were quality issues with this intervention. Relative antimicrobial effectiveness was determined: organic acids > peroxyacetic acid > chlorinated compounds > ozone. Using this information, individual and combined effectiveness of water washing and/or rinsing with an antimicrobial compound was measured in a laboratory setting. The effectiveness of a portable, pressurized stainless steel spray tank to apply a 2% lactic acid rinse was compared with a garden sprayer, retrofitted garden sprayer, and motorized backpack sprayer. The findings from these studies indicated that a manual warm water wash (54 C), followed by a 5 minute drip and 2% lactic acid rinse applied with a portable, pressurized steel tank, was the most effective multi-step antimicrobial carcass intervention iii

4 (MSACI) with reductions of pathogens between 3.5 and 5.0 log CFU/cm 2 and 3.8 to 8.9 log CFU/cm 2 for hygiene indicators. Instructional materials were developed to train VSMP employees how to implement MSACI in their plants. Lastly, red meat carcasses (n = 747) were swabbed to generate a second microbiological baseline after MSACI implementation. The first and second baselines were statistically compared to validate the reduction in carcass hygiene indicators and prevalence of harmful pathogens. Overall, MSACI was deemed an effective and feasible method of improving the microbiological safety of carcasses processed in VSMP. iv

5 TABLE OF CONTENTS List of figures.xiv List of tables..xvi Acknowledgments. xxviii Chapter One: Preliminary Survey 1 Statement of the problem Research objective Experimental plan in brief Chapter Two: Literature Review Introduction Large, small, and very small plants HACCP implementation.. 8 Primary processing.. 9 Types of interventions Chemical and physical hazards.. 14 Chemical hazards Physical hazards. 16 Biological hazards Escherichia coli O157:H Salmonella spp Campylobacter spp.. 25 Bacterial attachment to meat surfaces. 28 v

6 Antimicrobial interventions Water washing Rinsing with antimicrobial compounds...34 Lactic acid Acetic acid Citric acid Peroxyacetic acid Chlorinated compounds...41 Sodium hypochlorite Acidified sodium chlorite Chlorine dioxide Aqueous ozone Steam vacuuming Steam pasteurization Chemical dehairing Knife trimming Antimicrobial interventions used in combination Physical attributes of interventions.. 52 Future/potential interventions Conclusions.. 56 References Chapter Three: Preliminary Survey 80 Introduction vi

7 Methods...83 Results..85 Discussion.. 93 References.. 97 Chapter Four: Investigation of water washes on the elimination of meat borne pathogens from inoculated beef surfaces Introduction Types of interventions Selection of antimicrobial treatments Water washing Physical attributes of interventions 131 Research Objectives Methods. 133 Preparation of fecal slurry..133 Inoculation of beef plates with fecal slurry Treatment of inoculated plates with cold water washes Treatment of inoculated plates with cold, warm or hot water washes..136 Enumeration/isolation of pathogens and hygiene indicators. 137 Detection of pathogens at low levels. 138 E. coli O157:H Salmonella spp Campylobacter spp Enumeration of hygiene indicators 140 vii

8 Statistical analysis of cold water washes Statistical analysis of cold, warm, and hot water washes Results Cold water washes Cold, warm, and hot water washes Discussion References..152 Chapter Five: Investigation of chemical rinses on the elimination of meat borne pathogens from inoculated beef surfaces Introduction 173 Rinsing with antimicrobial compounds Lactic, citric, and acetic acids Peroxyacetic acid Sodium hypochlorite Acidified sodium chlorite..177 Chlorine dioxide Aqueous ozone Types of interventions Research objectives 182 Methods..183 Preparation of fecal slurry..183 Inoculation of beef plates with fecal slurry Treatment of inoculated plates with chemical rinses viii

9 Monitoring of chemical residues Enumeration of pathogens 190 Detection of pathogens at low levels 191 E. coli O157:H7 191 Salmonella spp. 192 Campylobacter spp Enumeration of hygiene indicators 193 Statistical analysis of chemical rinses Results 195 Discussion..201 References Chapter Six: Investigation of water washes combined with chemical rinses on the elimination of meat borne pathogens from inoculated beef surfaces Introduction 233 Research objectives 235 Methods Preparation of fecal slurry..237 Inoculation of beef plates with fecal slurry Combined treatments of inoculated plates ph of combination washes using different spraying systems Enumeration of pathogens. 242 Detection of pathogens at low levels. 243 E. coli O157:H ix

10 Salmonella spp Campylobacter spp Enumeration of hygiene indicators 245 Statistical analysis of combination treatments Results Discussion..249 References..251 Chapter Seven: Establishment of microbiological baselines of red meat carcasses to validate a multi-step antimicrobial carcass intervention in very small meat plants Introduction Biological hazards Escherichia coli O157:H Salmonella spp Campylobacter spp Research Objective Methods 280 Preparation of positive controls 282 Collection of carcass swabs Preparation of homogenate Enumeration of hygiene indicators 285 Detection of E. coli O157:H7 286 Detection of Salmonella spp. 287 x

11 Detection of Campylobacter spp..288 Statistical analysis. 289 Results Pathogen prevalence. 291 Hygiene indicators 293 Discussion. 295 References. 302 Chapter Eight: Future Research 332 References..337 Appendix A: Survey Questionnaire Appendix B: Establishment of growth curves for Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter coli, and Campylobacter jejuni Introduction Methods Results and discussion References 352 Appendix C: Effect of diluent selection on ph of homogenized beef brisket treated with a 2% lactic acid rinse and on populations of Escherichia coli O157:H7, Salmonella Typhimurium, and Campylobacter spp. 361 Introduction Methods Results and discussion References..370 xi

12 Appendix D: Determination of carcass surface areas and application time for water washing and rinsing with 2% lactic acid Introduction Methods Results and discussion References Appendix E: Video brochure Background and purpose Step 1. Water wash Step 2. Five-minute drip Step 3. Antimicrobial rinse Suggestions for establishing a critical limit Suggestions for monitoring a critical limit Suggestions for corrective actions Spray equipment selection 406 Summary Disclaimer.416 Contact information..416 Appendix F: Cost comparison of chemical treatments used to decontaminate carcass surfaces in very small meat establishments..418 Introduction..419 Spraying equipment..421 Antimicrobial compounds.423 xii

13 Aqueous ozone Chlorine dioxide 425 Acidified sodium chlorite..428 Sodium hypochlorite..431 Peroxyacetic acid Citric, acetic, and lactic acids Acknowledgments. 439 References Appendix G: Questions and comments from processors related to the implementation of a multi-step carcass decontamination treatment in very small meat plants..441 Introduction Question Question Question Question Comment Comment Comment Comment References. 447 xiii

14 LIST OF FIGURES Chapter Seven Figure 1. Sampling locations for testing of cattle carcasses Figure 2. Sampling locations for testing of swine carcasses Appendix B Figure 1. Escherichia coli O157:H7 (ATCC 43889) growth curve (n = 2) and absorbance readings (n = 2) Figure 2. Escherichia coli O157:H7 (PSUGDC ) growth curve (n = 2) and absorbance readings (n = 2) Figure 3. Salmonella Typhimurium (ATCC 14028) growth curve (n = 2) and absorbance readings (n = 2)..355 Figure 4. Salmonella Typhimurium (ATCC 13311) growth curve (n = 2) and absorbance readings (n = 2) Figure 5. Campylobacter coli (ATCC 33559) growth curve (n = 2) and absorbance readings (n = 2) Figure 6. Campylobacter coli (ATCC 33560) growth curve (n = 2) and absorbance readings (n = 2) xiv

15 Appendix E Figure 1. Bacteria and water film on a carcass surface Figure 2. 2% lactic acid rinse being applied to a carcass surface that has not been allowed to drip adequately. 395 Figure 3. 2% lactic acid is diluted as it mixes with the water film on a carcass surface..395 Figure 4. Heavy-duty stainless steel tank. 406 Figure 5. Close-up view of top of heavy-duty stainless steel tank Figure 6. Garden sprayer..410 Figure 7. Retrofitted garden sprayer Figure 8. Close-up view of retrofitted garden sprayer Figure 9. Backpack sprayer..414 Figure 10. Spray tank on wheels..414 xv

16 LIST OF TABLES Chapter Three Table 1. Slaughter interventions used to reduce microbial contamination on carcass surfaces in very small meat establishments in Pennsylvania Table 2. Approximate application time of slaughter intervention to carcass surfaces in very small meat establishments in Pennsylvania Table 3. Types of equipment used to apply slaughter interventions in very small meat establishments in Pennsylvania Table 4. Timing of the application of slaughter interventions to carcass surfaces in very small meat establishments in Pennsylvania 102 Table 5. Type of nozzle used in washing cabinets to apply hot water or organic acids in very small meat establishments in Pennsylvania..103 Table 6. Application pressure of hand-held garden sprayer or spray cabinet used to apply hot water or organic acids to carcass surfaces in very small meat establishments in Pennsylvania Table 7. Distance between hand-held hose or hand-held sprayer and carcass surfaces during application of hot water or organic acids in very small meat establishments in Pennsylvania Table 8. Proportion of very small meat establishments in Pennsylvania that apply more than one antimicrobial compound to carcass surfaces as a slaughter intervention Table 9. Method for monitoring the concentration of antimicrobial compounds used for slaughter interventions in very small meat establishments in Pennsylvania 107 xvi

17 Table 10. Interest in participating in a microbial sample study for evaluation of slaughter interventions in very small meat establishments in Pennsylvania Table 11. Interest in training for new slaughter interventions in very small meat establishments in Pennsylvania. 109 Table 12. Interest in various types of instructional methods of processors in Pennsylvania..110 Table 13. Availability of Internet access to survey participants in Pennsylvania for educational purposes..111 Table 14. Availability of CD-ROM access to survey participants in Pennsylvania for educational purposes Table 15. Most suitable time of year for employees of very small meat plant establishments in Pennsylvania to attend a training program Table 16. Best times and days of the week for employees of very small meat establishments in Pennsylvania to attend training programs Table 17. Highest level of education completed by processors in Pennsylvania Table 18. Job title of survey participants in Pennsylvania.116 Table 19. Number of years worked in meat and/or poultry processing industry by survey participants in Pennsylvania Table 20. Number of employees who work in surveyed establishments in Pennsylvania..118 Table 21. Slaughter interventions used to reduce microbial contaminants on carcass surfaces in very small meat establishments in Washington and Idaho 119 xvii

18 Table 22. Approximate application time of slaughter intervention to carcass surfaces in very small meat establishments in Washington and Idaho Table 23. Types of equipment used to apply slaughter interventions in very small meat establishments in Washington and Idaho Table 24. Timing of the application of slaughter interventions to carcass surfaces in very small meat establishments in Washington and Idaho Table 25. Type of nozzle used in washing cabinets to apply hot water or organic acids in very small meat establishments in Washington and Idaho Table 26. Application pressure of hand-held garden sprayer or spray cabinet used to apply hot water or organic acids to carcass surfaces in very small meat establishments Table 27. Distance between hand-held hose or hand-held sprayer and carcass surfaces during application of hot water or organic acids in very small meat establishments in Washington and Idaho Table 28. Method for monitoring the concentration of antimicrobial compounds used for slaughter interventions in very small meat establishments in Washington and Idaho..126 Chapter Four Table 1. Mean populations and reductions of Salmonella Typhimurium (log 10 CFU/cm 2 ± SE) on inoculated beef plates treated with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times 155 Table 2. Mean populations and reductions of Campylobacter spp. (log 10 CFU/cm 2 ± SE) on inoculated beef plates treated with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times xviii

19 Table 3. Mean populations and reductions of Escherichia coli O157:H7 (log 10 CFU/cm 2 ± SE) on inoculated beef plates treated with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times Table 4. Mean mesophilic aerobic plate counts and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times 158 Table 5. Mean populations and reductions of generic E. coli (log 10 CFU/cm 2 ± SE) on inoculated beef plates treated with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times Table 6. Mean populations and reductions of coliforms (log 10 CFU/cm 2 ± SE) on inoculated beef plates treated with a cold water wash (15 C) under various pressures, spray distances, application times, and drip times Table 7. Mean populations and reductions (log 10 CFU/cm 2 ± SE) of Salmonella Typhimurium on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances Table 8. Mean populations and reductions (log 10 CFU/cm 2 ± SE) of Campylobacter spp. on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances Table 9. Mean populations and reductions (log 10 CFU/cm 2 ± SE) of Escherichia coli O157:H7 on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances.163 xix

20 Table 10. Mean mesophilic aerobic plate counts and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances Table 11. Mean populations and reductions (log 10 CFU/cm 2 ± SE) of generic E. coli on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances Table 12. Mean populations and reductions (log 10 CFU/cm 2 ± SE) of coliforms on inoculated beef plates following a cold (15 C), warm (54 C), or hot (77 C) water wash (30 psi, 20 s application time) with varying drip times and spray distances Table 13. Mean temperature of lean tissue surfaces of beef plates manually washed with warm (54 C) or hot (77 C) water in a benchtop washing cabinet Table 14. Mean log reductions of pathogens and hygiene indicators on inoculated beef plates following cold water (15 C) washing (first, flawed analysis of cold water washing data) Table 15. Mean populations (log 10 CFU/cm 2 ) of bacterial populations on inoculated beef plates following cold water (15 C) washing under a variety of pressures, spray distances, application times, and drip times (second analysis of data) Chapter Five Table 1. Mean populations and reductions of Salmonella Typhimurium (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip) xx

21 Table 2. Mean populations and reductions of Campylobacter spp. (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip) Table 3. Mean populations and reductions of E. coli O157:H7. (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip) Table 4. Mean mesophilic aerobic plate count reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip).221 Table 5. Mean populations and reductions of generic E. coli (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip) Table 6. Mean populations and reductions of coliforms (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with water or an antimicrobial rinse (40 psi, 30.5 cm, 15 s application, 5 min drip)..225 Table 7. Mean reductions of bacterial populations (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with chlorinated rinses Table 8. Mean reductions of bacterial populations (log 10 CFU/cm 2 ± SE) on inoculated beef plates following treatment with 2% organic acid rinses Table 9. ph, concentration, and pressure observations of antimicrobial compounds before and after decontamination of inoculated beef plates. 229 Table 10. Aqueous ozone concentration before and after decontamination of inoculated beef plates. 231 xxi

22 Chapter Six Table 1. Mean bacterial populations and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates before (control) and after treatment with water washing, water rinsing, or 2% lactic acid rinsing alone or in combination using a portable, stainless steel tank Table 2. Mean bacterial populations and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates before (control) and after treatment with water washing, water rinsing, or 2% lactic acid rinsing alone or in combination using a garden sprayer.255 Table 3. Mean bacterial populations and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates before (control) and after treatment with water washing, water rinsing, or 2% lactic acid rinsing alone or in combination using a motorized backpack sprayer Table 4. Mean bacterial populations and reductions (log 10 CFU/cm 2 ± SE) on inoculated beef plates before (control) and after treatment with water washing, water rinsing, or 2% lactic acid rinsing alone or in combination using a retrofitted garden sprayer..261 Table 5. Mean reductions of bacterial populations (log 10 CFU/cm 2 ± SE) on inoculated beef plates after water washing combined with 2% lactic acid rinsing using a garden sprayer (GS), retrofitted garden sprayer (RF), portable stainless steel tank (SS), or motorized backpack sprayer (BP) Table 6. ph of inoculated beef plates homogenized after treatment with a warm water wash (54 C) and/or water or 2% lactic acid rinse xxii

23 Chapter Seven Table 1. Prevalence of pathogens on red meat carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention..316 Table 2. Prevalence of pathogens on red meat carcasses processed in very small meat establishments in Pennsylvania before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 3. Prevalence of pathogens on red meat carcasses processed in very small meat establishments in Washington and Idaho before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 4. Prevalence of pathogens on red meat carcasses processed in very small meat establishments in Texas before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention.319 Table 5. Prevalence of pathogens on beef carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention..320 Table 6. Prevalence of pathogens on pork carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention..321 Table 7. Prevalence of pathogens on lamb carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention xxiii

24 Table 8. Prevalence of pathogens on bob veal carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention..323 Table 9. Median populations (log CFU/cm 2 ) of hygiene indicators on red meat carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 10. Median populations (log CFU/cm 2 ) of hygiene indicators on red meat carcasses processed in very small meat establishments in Pennsylvania before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 11. Median populations (log CFU/cm 2 ) of hygiene indicators on red meat carcasses processed in very small meat establishments in Washington and Idaho before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 12. Median populations (log CFU/cm 2 ) of hygiene indicators on red meat carcasses processed in very small meat establishments in Texas before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 13. Median populations (log CFU/cm 2 ) of hygiene indicators on beef carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention 328 xxiv

25 Table 14. Median populations (log CFU/cm 2 ) of hygiene indicators on pork carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 15. Median populations (log CFU/cm 2 ) of hygiene indicators on lamb carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Table 16. Mean (± SE) or median populations (log CFU/cm 2 ) of hygiene indicators on bob veal carcasses processed in very small meat establishments in three geographical regions before (first baseline) and after (second baseline) the implementation of a multi-step antimicrobial carcass intervention Appendix B Table 1. Pearson correlation (r) of bacterial populations to absorbance readings 359 Table 2. Regression coefficients (R 2 ) of bacterial populations versus absorbance readings..360 Appendix C Table 1. Mean ph of diluents before and after use Table 2. Populations (log CFU/ml) of pathogens before and after recovery from acid-treated beef brisket homogenized in Butterfields phosphate diluent (BPD), buffered peptone water (BPW) or 0.1% peptone water (peptone) xxv

26 Table 3. Average reductions in bacterial populations (log CFU/cm 2 ) recovered from acidtreated beef briskets homogenized in Butterfields phosphate diluent (BPD), buffered peptone water (BPW), or 0.1% peptone water (peptone) Appendix D Table 1. Average surface areas of red meat carcasses from previous studies Table 2. Surface areas of a small number of lamb pelts and pork skins determined by plastic sheeting method in a very small meat plant..384 Table 3. Average time necessary to apply a water wash or 2% lactic acid rinse to small, red meat carcasses in a very small meat establishment Appendix E Table 1. Cost comparison assumptions Table 2. Cost comparison of cold, warm and hot water on per gallon and per carcass bases Table 3. Antimicrobial effectiveness of several food-safe compounds used to eliminate meatborne pathogens from experimentally inoculated beef surfaces. 398 Table 4. Antimicrobial effectiveness of several food-safe compounds used to eliminate hygiene indicators from experimentally inoculated beef surfaces Table 5. Average log reductions of common meat pathogens and hygiene indicators on beef surfaces xxvi

27 Appendix F Table 1. Estimated flow rate and cost of spraying equipment that can be used to apply antimicrobial rinses to carcass surfaces in very small meat establishments..422 Table 2. Estimated per usage costs of aqueous ozone to decontaminate carcass surfaces in a very small meat establishment Table 3. Estimated costs of obtaining two chlorine dioxide generators Table 4. Estimated per usage costs of chlorine dioxide to decontaminate carcass surfaces in a very small meat establishment Table 5. Cost of reagents needed to prepare acidified sodium chlorite Table 6. Estimated per usage costs of acidified sodium chlorite to decontaminate carcass surfaces in a very small meat establishment Table 7. Estimated per usage costs of sodium hypochlorite (NaOCl) to decontaminate carcass surfaces in a very small meat establishment Table 8. Cost of concentrated organic acids used to prepare antimicrobial carcass rinses Table 9. Estimated per usage costs of acetic acid to decontaminate carcass surfaces in a very small meat establishment Table 10. Estimated per usage costs of citric acid to decontaminate carcass surfaces in a very small meat establishment..437 Table 11. Estimated per usage costs of lactic acid to decontaminate carcass surfaces in a very small meat establishment xxvii

28 ACKNOWLEDGMENTS A dissertation of this size simply does not happen without help from a number of people. Firstly, I want to thank the people who helped me gather or analyze information to fulfill numerous research objectives. Dr. Naana Nti from the Penn State Office of Outreach and Marketing calculated and tabulated the results of survey questionnaire that is described in Chapter One. Chapter Two was my literature review, which was adapted from a book chapter (from Handbook of Beef Safety and Quality; The Haworth Press, Inc.; Binghamton, NY) co-authored by myself, my major advisor, Dr. Catherine Cutter, Dr. William R. Henning, who is also on my doctoral committee, and Dr. Margaret Hardin, who currently works for Boars Head Provisions. Their technical prowess has helped me deepen my understanding of meat safety during slaughter. As I round out my doctoral committee, I consider myself extremely fortunate to have been guided by Dr. Edward Mills (my major advisor for my Master of Science degree), Dr. Stephanie Doores, and Dr. Nancy Ostiguy. I attribute the quality of the documents that follow directly to their thoughtful attention to detail. Additionally, I wish to acknowledge the National Integrated Food Safety Initiative, which is administered by the Cooperative State Research, Education, and Extension Service under the United States Department of Agriculture, for funding this project. There are numerous individuals who were absolutely essential to my success. I wish to thank each of them for their willingness to work with me and for providing me with a wide range of resources from technical knowledge to workspace. Of the Penn State Meats Lab, I am grateful to Glenn Myers, Barrie Moser, Jason Monn and Katie Logan. Furthermore, I appreciate Jon Cofer and Lucas Parker for filming and producing a 17-minute training video and to Bill Houser for narrating it. Another large component of my project was the collection xxviii

29 of hundreds of carcass samples from very small meat establishments in Pennsylvania, Idaho, Washington, and Texas with the assistance of Washington State University and Texas Tech University. I received a mountain of data from Peter Gray at Washington State University under the direction of Dr. Dong-Hyun Kang, a co-investigator of this project. I genuinely appreciate everything that Peter has done for me. He has approached each aspect of this project with an admirable sense of integrity and professionalism. Alejandro Echeverry, Jason Mann, and other students from Texas Tech University also provided me with the data that we needed to answer our research questions. Dr. Mindy Brashears, also a co-investigator, provided much guidance to the Texas Tech crew and was quick to answer all of my questions. In order to develop recommendations to very small meat processors, I spent countless hours in the laboratory studying the antimicrobial efficacies of eight chemicals. As these experiments were costly to perform, Dr. Cutter and I were fortunate to benefit from the generosity of the following individuals and their companies for donating and loaning supplies and/or equipment: Rick Hess of Hess Machines, Ephrata, Pennsylvania; Joy Herdt and Keith Johnson of ECOLAB, St. Paul, Minnesota; Rich O Reskie of Halox Technologies, Bridgeport, Connecticut; and Dr. Neeraj Khana of Bio-Cide International, Norman, Oklahoma. These professionals have donated their time and industrial expertise and some of them traveled to University Park to set up equipment and provide one-on-one training. Additionally, I called upon several experts to help me answer tough questions. I am thankful for lactic acid experts at Birko Corporation in Henderson, Colorado and PURAC America in Lincolnshire, Illinois. Also, I want to thank several individuals from the Food Safety and Inspection Service of the United States Department of Agriculture: Ms. Patricia White, a xxix

30 meat product labeling expert; Jan Behney, District Manager of District 60; and Dr. Ronald Jones, District Manager of District 15. Although I am unable to reveal the identities of the very small meat establishments that allowed us to gather our carcass samples, I do wish it to be known that their participation in this project is tremendously appreciated. Personally, I am very grateful to all of them for the privileges that they extended to me so that I could earn this degree. Several of these companies have had long-term partnerships with the meat research programs at Penn State, Washington State, and Texas Tech and I certainly hope that these special relationships continue to thrive. Next, I thank my wonderful labmates and research assistants who have given me advice, helped me prepare media and reagents, washed my dirty labware, and performed various unpleasant laboratory tasks so that I could do my job better. Since January 2002, I have had the pleasure of working with Donna Miller, Naveen Chikthimmah, Kerry Fabrizio, Peyman Fatemi, Mary Nguyen, Niraja Ramesh, Renee Britton, Amie Geiger, Tim Strittmatter, Ben Joyce, Ginger Fenton, Matt Fenton, Nathan Angstadt, Donna Abdullah, Kristin Wernosky, Rich Sweich, Alison Bell-St. John, Steve Adoff, Kate Lamm, Aubri Carmichael, Drew Aurand and Kim Norton in the food microbiology facilities of Borland Laboratory. Many of these individuals were also volunteers for Borland Day Care, a wonderful group of students, faculty, and staff who watched Hazel, my daughter, for a few minutes or for several hours while I carried out my research tasks under hazardous conditions. I would be remiss not to recognize the administrative support that I have received from the Penn State Department of Food Science. Dr. John Floros, the head of the xxx

31 department, has been especially supportive of my academic success. Furthermore, there is a long list of wonderful ladies, who have always been willing to point me in the right direction regarding matters of the Graduate School and use of budget funds. Juanita Wolfe, Amy Larimer, Donna Merrill, Martha Neiheisel, Melissa Strouse, Ellie Chapman, and Carole Donald, thank you for all of your help. I wish everyone knew how much your efforts impact the success of each graduate student. On a personal note, I want to recognize my family for their unconditional support of me throughout this degree program. I began this program of study in January 2002 and I graduated four and half years later. My parents, Rose and Dale, always supported my decision to move forward with my research through some of the most difficult phases of my life. My sister, Susan, has also been steadfast in her support of my academic objectives. She and her husband, Nate, willingly cared for Hazel for six months of her infancy so that I could complete the most labor-intensive segments of my research. It is amazing how much love this precious, little gem has brought into my life. Finally, I want to acknowledge Tim Yoder, the love of my life. Since we have been together, Tim has shown me more patience, thoughtfulness, and genuine kindness than I feel I deserve, especially during the final stretches of research and writing. Without my family, my failsafe support network, I would still be in the laboratory conducting washing experiments or collecting samples from meat establishments. I devotedly recognize the unconditional support that Dr. Cutter has given to me during my time in the Food Science Department. As her first doctoral student, I provided her with an abundance of mentoring challenges. I can think of several people who would have shown me to the door. Instead, Dr. Cutter advised me to make the right choices and for that I xxxi

32 am forever grateful to her. It is my hope to pattern my professional and personal life after hers one distinguished by integrity, goodwill, and resolve. Thank you, Cathy. xxxii

33 CHAPTER ONE INTRODUCTION 1

34 STATEMENT OF THE PROBLEM While meat and poultry establishments in the United States were in the midst of developing and implementing HACCP plans as required by the Pathogen Reduction Act of 1996, the Food Safety and Inspection Service openly admitted that more research efforts would be needed to provide documentation for the validation of critical control points in very small plants (USDA-FSIS, 1996b). As the decisions were made to establish acceptable levels for generic E. coli and Salmonella spp. on raw meat and poultry, FSIS depended mostly on data that had been collected from large or small establishments. While these performance standards may certainly apply to processing in the large or small plant, it is not clear that these same standards are suitable objectives for very small meat and poultry plants. Though the data do not exist, the levels of pathogenic microorganisms on carcasses processed in very small meat and poultry plants may be as low as those detected in large plants. Even in the absence of adequate data, very small establishments are required to cite reliable sources, typically from refereed journals, in their HACCP plans to demonstrate that antimicrobial interventions to reduce microbiological hazards are, indeed, effective. 2

35 RESEARCH OBJECTIVE Given the labor constraints to implement and monitor elaborate decontamination methods and limited financial resources for capital investment, it is necessary to identify and validate alternative methods of reducing bacterial populations on carcass surfaces to meet the unique needs of very small plants in the United States. Very small meat plants do not have the available resources to dedicate to a third-party for the generation of sufficient and valid data. When valid techniques for carcass decontamination and supporting documentation become available, then there will be a logical basis to assess the prevalence of pathogenic microorganisms on carcass surfaces in very small meat plants. With such a basis for process control, appropriate decontamination techniques can be implemented in very small meat plants and the meat industry should come one step closer to preventing food borne illness in humans. 3

36 EXPERIMENTAL PLAN IN BRIEF Described below is a series of five research objectives that achieve the overall goal of providing scientific documentation for the validation of HACCP plans of very small meat plants. While the study of a subset of very small plants may not precisely represent all very small meat and poultry establishments (approximately 3,400), the selection of three geographical regions in the United States represented by Pennsylvania, Texas, Washington, and Idaho should illustrate the general preferences for and characteristics of carcass decontamination techniques practiced in the United States. To represent all very small meat plants in the United States, three state universities (The Pennsylvania State University, Texas Tech University, and Washington State University) collaborated to survey the decontamination practices and sample red meat carcasses in very small establishments in the states listed above. In the first phase of this project, very small meat plants in three states were surveyed to ascertain which antimicrobial interventions were being used to eliminate bacteria from carcass surfaces. The next objective involved the sampling of red meat carcasses processed in very small meat plants to determine a baseline for Salmonella spp., Campylobacter spp., E. coli O157:H7, aerobic plate counts, and counts of generic E. coli and coliforms. For the third research objective, a variety of antimicrobial compounds to reduce bacteria on meat surfaces was selected for laboratory study based on survey results, peer-reviewed literature, and personal communication. Next, the individual and combined effectiveness of water washing and/or rinsing with an antimicrobial compound was measured in a laboratory setting. The results of this fourth research objective were used to produce a training video and 4

37 instructional booklet to transfer technical knowledge to employees of very small plants. The fifth objective served as a real-world validation of the decontamination strategies that were investigated under laboratory conditions. After implementation of a multi-step antimicrobial intervention in selected very small plants, carcasses were sampled again to generate a second microbiological baseline to determine whether the implementation of this intervention reduced the prevalence of target bacterial populations. The entire project will be summarized into a report and distributed to all very small meat plants. In the near future, the very small plants that were sampled during the secondary baseline are to be surveyed again to assess the adoption rate of the new technology and gather employee opinions of the feasibility and success of the multi-step antimicrobial intervention. 5

38 CHAPTER TWO LITERATURE REVIEW 6

39 INTRODUCTION Meat processors have an ethical responsibility to provide wholesome products to consumers. From the moment the live animal sets foot on the slaughter floor until the beef carcass is fabricated into primals and throughout processing, there are hazards that must be controlled. To fulfill this responsibility, plant employees must be aware that the procedures performed on the slaughter floor and in the fabrication and processing rooms can have a tremendous impact on the safety of the meat products that reach the end user. Currently, every meat plant under federal inspection conducts daily operations under Hazard Analysis and Critical Control Point (HACCP) systems. Each plant develops its own HACCP plan focused on the prevention of problems to assure the safety of meat products through control of chemical, physical, and biological hazards. Large, small, and very small plants The United States meat and poultry industry comprises approximately 6,000 establishments dedicated to harvest, fabrication, processing, and/or purveying. The USDA classifies plants into one of three categories based on the number of employees and annual revenue. About 5% or 300 of these plants are classified as large plants as they employ more This literature review was adapted from: S. Flowers Yoder, M. D. Hardin, W. R. Henning, C. N. Cutter, IN PRESS. Beef safety during slaughter, fabrication, and further processing. Ch. 3 in Handbook of Beef Safety and Quality. D. (Roeber) Van Overbeke (Ed.), The Haworth Press, Inc., Binghamton, NY. 7

40 than 500 workers, whereas small plants (38%, 2,300 establishments) employ between 10 and 500 people. Plants considered very small employ 10 or fewer employees and generate average annual revenue of $2.5 million or less (USDA-FSIS, 1996). Given these definitions, it is obvious that very small plants have limited labor and finances for the purchase, operation and maintenance of elaborate decontamination methods such as carcass cabinet washers and steam vacuum sanitizers. HACCP implementation In the interest of public health, it has become unacceptable for contamination with pathogens to persist in muscle foods. The Pathogen Reduction: Hazard Analysis and Critical Control Point Systems final rule of 1996 mandated the implementation of HACCP plans for all meat and poultry establishments, established performance standards for the incidence of Salmonella in raw and ground meat products, required periodic testing by the company for generic E. coli, and required the development and implementation of Sanitation Standard Operating Procedures (SSOPs) in each plant (USDA-FSIS, 1996c). Employees also should follow the good manufacturing practices (GMPs) that are prescribed within individual meat plants (Katsuyama and Humm, 1995). By doing these practices and adhering closely to the plant s HACCP plan, the chemical, physical, and biological hazards associated with meat should be under control at all times. Generally, biological hazards require the most consideration because of the potential for widespread outbreaks of illness in humans. 8

41 Primary processing Several steps must take place in the slaughter facility to convert a live animal to a meat carcass. These steps are described in a general manner, although there is some variation among plants due to existing workspace, available equipment, religious practices and other factors. During slaughter, exsanguination takes place after stunning and it is the first step in the conversion of muscle to meat. Exsanguination also is the first opportunity during slaughter for microbes to contaminate the carcass, unless captive bolt stunning or some other invasive procedure is used to render the animal unconscious (Aberle et al., 2001). Moreover, microbes can gain access to the bloodstream through the opening made by severing the major blood vessels with a contaminated knife. Next, the integument is removed. In some plants, chemical dehairing or hide washing may take place prior to exsanguination; then the hide is removed. Hide or pelt removal can be achieved by manual skinning or an automated puller. Pork carcasses may be scalded, dehaired, and singed in place of skinning. When performed manually, the employee should minimize cross-contamination and be careful to keep the skinning knife clean and avoid letting the skin touch the carcass surface as it is being cut away. Automated removal of hides can release dust and debris into the ambient air as the skin is pulled away from the carcass by a hydraulic arm. Some of these particles could be desiccated manure that have the potential to carry viable cells of Escherichia coli O157:H7, which could be deposited on an otherwise clean carcass surface or processing equipment (Delazari et al., 1998; Elder et al., 2000). In fact, the bovine hide is known to be a major source for carcass contamination with E. coli O157:H7 (Elder et al., 2000 and Reid et al., 2002). 9

42 Typically, the head is removed after the carcass has been skinned. At some point, the tail, if present, also is removed. Removal of viscera (stomach, spleen, intestines, and anus) and pluck (tongue, esophagus, heart, diaphragm, lungs, and liver) from the body cavity, or evisceration, follows. Fecal matter can be released onto carcass surfaces if the intestinal tract is punctured or the bung is not properly tied. In some plants, carcasses are steam vacuumed for removal of fecal matter and other debris. The beef carcass is then sawed into halves while other red meat species may be left intact until fabrication. As a preventive measure for controlling bovine spongiform encephalopathy (BSE), specified risk materials (SRM), such as the spinal cord, brain and skull, must be removed from cattle that are 30 months of age or older (USDA-FSIS, 2004). At the end of primary processing, the carcass is visually inspected for contamination by employees, undergoes knife trimming as necessary and is then washed thoroughly with water. In most plants, an intervention step follows the water wash. Interventions may include a sanitizing step with an antimicrobial substance or surface pasteurization. After the final wash, carcasses are placed in a chill cooler or hot box to control the lowering of carcass temperature. In a very small meat plant, the final washing step prior to chilling provides the best opportunity to decontaminate carcass surfaces. While an intervention (washing, knife trimming) may take place during slaughter and provide an effective means of removing bacteria, carcass surfaces could become recontaminated before the final wash. The debris that may have accumulated on meat surfaces during the slaughter sequence is most effectively removed by a worker who is profoundly familiar with the effort that is necessary for each carcass to pass federal inspection. Given the relatively small labor force in these plants, one 10

43 or two workers are usually dedicated to the quality assurance tasks of visual inspection, knife trimming, washing, and application of interventions to each carcass before it enters the chill cooler. Types of interventions Currently, there are several types of antimicrobial treatments or interventions shown to substantially reduce bacteria levels on red meat carcasses and are approved for use to meet HACCP requirements. The most common carcass decontamination strategies include water washing, knife trimming, chemical treatments, and moist heat under pressure or vacuum. When considering which interventions to implement, plants of all sizes should consider the expenses of purchase, maintenance, and daily operation (fixed costs); available space and manpower; and documented antimicrobial effectiveness. Some interventions, which are wellsuited for large plants, are difficult or impossible to implement in small or very small plants. As part of HACCP, FSIS requires the removal of all visible fecal contamination, ingesta, or milk from carcasses, by knife trimming or vacuuming with hot water or steam (USDA-FSIS, 1996b). Carcasses are then subject to re-inspection and must meet finished product standards applied by plant employees and verified by FSIS inspectors before the carcasses can enter the chiller. Despite the use of sanitary dressing procedures to reduce or eliminate contamination during slaughter and processing, pathogenic bacteria still may be present on red meat carcasses (USDA-FSIS, 1996a). Therefore, antimicrobial treatments or interventions are recommended by USDA to reduce the prevalence of pathogens in raw products. 11