Echeverry_Alejandro_Diss
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Echeverry_Alejandro_Diss

Course Number: ETD 11082007, Fall 2009

College/University: Texas Tech

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DEVELOPMENT AND VALIDATION OF INTERVENTION STRATEGIES TO CONTROL ESCHERICHIA COLI O157:H7 AND SALMONELLA TYPHIMURIUM DT 104 IN NEEDLE TENDERIZED AND INJECTED BEEF (USDA CHOICE STRIP LOINS - LONGISSIMUS LUMBORUM) UNDER TWO SIMULATED INDUSTRIAL CONDITIONS by ALEJANDRO ECHEVERRY, BS., MS. A Dissertation In Animal Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of The...

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AND DEVELOPMENT VALIDATION OF INTERVENTION STRATEGIES TO CONTROL ESCHERICHIA COLI O157:H7 AND SALMONELLA TYPHIMURIUM DT 104 IN NEEDLE TENDERIZED AND INJECTED BEEF (USDA CHOICE STRIP LOINS - LONGISSIMUS LUMBORUM) UNDER TWO SIMULATED INDUSTRIAL CONDITIONS by ALEJANDRO ECHEVERRY, BS., MS. A Dissertation In Animal Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Dr. Mindy M. Brashears Chairperson of the Committee Dr. Chance Brooks Dr. Guy H. Loneragan Dr. Mark Miller Dr. Christine Alvarado Dr. Ronald D. Warner Accepted Fred Hartmeister Dean of the Graduate School December, 2007 Copyright 2007, Alejandro Echeverry Texas Tech University, Alejandro Echeverry, December 2007 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Mindy Brashears. I really appreciate your valuable and continuous support. I want to thank you for the opportunity to work in such a great place as is your laboratory at Texas Tech University. Throughout these years I have found not only an advisor and mentor, but a friend. I also would like to thank my committee members for their guidance and support: Dr. Chance Brooks, Dr. Guy H. Loneragan, Dr. Christine Alvarado, Dr. Ronald Warner, and Dr. Markus Miller. I really appreciate your valuable time and all the input and assistance that have allowed me to complete this program. I wish to thank all colleagues and friends at TTU, especially Loree Branham, Angela Laury, Evan Chaney, Sara Gragg, Melissa Hughes and Tanya Jackson: I really appreciate all your help, collaboration and time that allowed me to complete this project. To the other members of the Brashears Lab Rats: Thank you for your hard work, dedication and service. Finally, I would like to thank my family for their support and patience. I miss you so much and wish I was able to have you closer. ii Texas Tech University, Alejandro Echeverry, December 2007 TABLE OF CONTENTS ACKNOWLEDGEMENT..ii ABSTRACT...ix LIST OF TABLES.xi LIST OF FIGURES.xiii CHAPTER I1 LITERATURE REVIEW...1 Escherichia coli .......................................................................................... 2 Escherichia coli O157:H7 ................................................................ 4 Human Disease caused by Escherichia coli O157:H7 .................... 5 The Survival of Escherichia coli O157:H7 in foods.......................... 7 Salmonella ............................................................................................... 10 Human disease caused by Salmonella ......................................... 11 The survival Of Salmonella In Foods ............................................ 12 Isolation and enumeration methods ......................................................... 15 Pre-enrichment step ...................................................................... 17 Enumeration and Isolation ............................................................ 18 Interventions ............................................................................................ 24 Lactic acid ..................................................................................... 25 Acidified sodium chlorite ............................................................... 27 Lactic acid bacteria ....................................................................... 29 iii Texas Tech University, Alejandro Echeverry, December 2007 Meat tenderness and processing ............................................................. 31 Needle tenderization ..................................................................... 32 Microbial Issues of Meat Tenderization ......................................... 34 Meat Enhancement ....................................................................... 38 Microbial Issues of Enhanced Meat .............................................. 39 Disease Outbreaks related to needle tenderized/enhanced meat ........... 42 Outbreak 1 .................................................................................... 43 Outbreak 2 .................................................................................... 44 Outbreak 3 .................................................................................... 44 Conclusions ............................................................................................. 45 Literature Cited ........................................................................................ 46 CHAPTER II70 VALIDATION OF INTERVENTION STRATEGIES TO CONTROL ESCHERICHIA COLI O157:H7 IN NEEDLE TENDERIZED AND INJECTED BEEF PROCESSED UNDER PACKER AND PURVEYOR SETTINGS...70 Introduction .............................................................................................. 70 Packer And Purveyor Settings ...................................................... 71 Materials And Methods ............................................................................ 74 Experimental Design and Analysis ................................................ 74 Microbiological Cultures ................................................................ 75 Preparation Of Cocktail Cultures ................................................... 75 Meat Preparation.......................................................................... 76 iv Texas Tech University, Alejandro Echeverry, December 2007 Microbial Challenge....................................................................... 78 Equipment Sanitation .................................................................... 78 Processing of Subprimals: Needle Tenderization and Enhancement ................................................................................ 83 Microbiological Analysis: High Inoculation Levels ......................... 87 Microbiological Analysis: Low Inoculation Levels .......................... 90 Results ..................................................................................................... 91 Application Of Interventions At Packer Setting High Inoculation Levels ......................................................................... 91 Application Of Interventions at Purveyor Setting High Inoculation Levels ....................................................................... 104 Application Of Interventions At Packer Setting Low Inoculation Levels ....................................................................... 114 Application Of Interventions At Purveyor Setting Low Inoculation Levels ....................................................................... 120 Discussion ............................................................................................. 127 Surface High Inoculation results - Packer ................................... 128 Surface High Inoculation Results - Purveyor ............................... 129 Top Subsection - High Inoculation Results (Packer) ................... 133 Top Subsection - High Inoculation Results (Purveyor) ................ 134 A And B Subsections High Inoculation Results (Packer And Purveyor) ................................................................ 136 v Texas Tech University, Alejandro Echeverry, December 2007 Low Inoculation Results .............................................................. 136 Acknowledgements ................................................................................ 139 Literature Cited ...................................................................................... 140 CHAPTER III.143 VALIDATION OF INTERVENTION STRATEGIES TO CONTROL SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM DT 104 IN NEEDLE TENDERIZED AND INJECTED BEEF PROCESSED UNDER PACKER AND PURVEYOR SETTINGS.143 Introduction ............................................................................................ 143 Materials And Methods .......................................................................... 145 Experimental Design and Analysis .............................................. 145 Microbiological Cultures .............................................................. 146 Preparation Of Cocktail Cultures ................................................. 146 Meat Preparation........................................................................ 147 Microbial Challenge..................................................................... 148 Equipment Sanitation .................................................................. 148 Processing of Subprimals: Needle Tenderization and Enhancement ....................................................................... 149 Microbiological Analysis: High Inoculation Levels ....................... 150 Microbiological Analysis: Low Inoculation Levels ........................ 152 Results ................................................................................................... 153 Application Of Interventions At Packer Setting High Inoculation Levels ....................................................................... 153 vi Texas Tech University, Alejandro Echeverry, December 2007 Packer Setting Top Counts ...................................................... 157 Packer Setting Subsection A Counts ....................................... 158 Packer Setting Subsection B Counts ....................................... 162 Application Of Interventions at Purveyor Setting High Inoculation Levels .................................................................................. 165 Purveyor Setting Surface Counts ............................................. 165 Purveyor Setting Top Counts ................................................... 165 Purveyor Setting Subsection A Counts .................................... 170 Purveyor Setting Subsection B Counts .................................... 171 Application Of Interventions At Packer Setting Low Inoculation Levels ....................................................................... 175 Application Of Interventions At Purveyor Setting Low Inoculation Levels ....................................................................... 181 Discussion ............................................................................................. 188 High Inoculation Results ............................................................. 189 Low Inoculation Results .............................................................. 197 Acknowledgements ................................................................................ 200 References ............................................................................................ 201 CHAPTER IV.204 FINAL SUMMARY AND IMPLICATIONS.204 Literature Cited ...................................................................................... 211 vii Texas Tech University, Alejandro Echeverry, December 2007 APPENDICES...212 Appendix A. Effectiveness Of Interventions in Reducing E. coli O157:H7 Counts Significantly When Compared To The Control ............................................................................ 213 Appendix B. Effectiveness Of Interventions in Reducing Salmonella Counts Significantly When Compared To The Control ............................................................................ 214 Appendix C. The Jaccard H Tenderizer. ..................................... 215 Appendix D. The Koch Injectamatic......................................... 217 Appendix E. Raw Plate Counts ................................................... 219 Appendix F. SAS Input Procedures............................................. 241 Appendix G. Federal Register 9 CFR Part 417 ........................... 252 VITA256 viii Texas Tech University, Alejandro Echeverry, December 2007 ABSTRACT After investigation by state health departments and the Center for Disease Control and Prevention, three different outbreaks between 2000 and 2004 were linked to the consumption of non-intact products contaminated with Escherichia coli O157:H7. Following this, in May 2005, the USDA-FSIS published notice that establishments who produce mechanically tenderized and moisture enhanced beef products were required to reassess their HACCP plans due potential contamination risk to consumers. The purpose of this study was to determine the effectiveness of different intervention strategies to control E. coli O157:H7 and Salmonella enterica serotype Typhimurium definitive phage type 104 in needle tenderized and enhanced beef strip loins. Treatments included a) lactic acid producing bacteria (LAB; ~107 cfu/g), acidified sodium chlorite (ASC; 1000 ppm), and lactic acid (LA; 3%) which were evaluated under two application scenarios: packer and purveyor. Packer samples were treated immediately after fabrication, whereas purveyor samples were treated after aging. Samples were aged for 14 or 21 days prior to needle tenderization or enhancement followed by microbial enumeration on the surface and at two internal subsections within the product. Salmonella and E. coli O157:H7 were significantly reduced by all interventions upon initial application. At the packer setting, Salmonella surface counts on day 14 were significantly reduced by all interventions in enhanced and needle tenderized beef. On day 21 all interventions were significantly effective in reducing pathogen levels in needle tenderized beef; however, only LAB and LA ix Texas Tech University, Alejandro Echeverry, December 2007 were significant in enhanced samples. At the purveyor setting, Salmonella surface counts on day 14 were significantly reduced by LA (needle tenderized beef) and LAB (enhanced beef). At the purveyor setting no differences between interventions were observed on day 21. E. coli O157:H7 surface counts at the packer setting were significantly reduced in needle tenderized beef on day 14 by the application of LA; however, no differences were observed on enhanced samples. On day 21 all interventions were significantly effective in reducing pathogen counts. At the purveyor setting, E. coli O157:H7 was reduced significantly from needle tenderized and enhanced beef only by application of LAB (Day 14 and 21). Internal E. coli O157:H7 counts were significantly reduced by 90% or more by using LAB, LA and ASC. A > 2 log reduction in the E. coli O157:H7 counts were observed after treatment with the interventions on day 14 in needle tenderized and injected beef. On day 21, ASC and LA reduced E. coli O157:H7 by up to 3 logs while LAB showed 2.0 log reductions. Internal Salmonella counts were reduced by > 2.0 log after 14 days using LAB and LA, and were significantly reduced by all interventions by day 21 of aging. Results from this study indicate that application of LAB, ASC, and LA sprays reduced E. coli O157:H7 and Salmonella enterica serotype Typhimurium definitive phage type 104 in beef subprimals with varying degrees of efficacy depending on time, location, and application setting. x Texas Tech University, Alejandro Echeverry, December 2007 LIST OF TABLES Table 1. ELISA Results For E. coli O157:H7 in Needle Tenderized Control Samples (Packer)...115 Table 2. ELISA Results For E. coli O157:H7 in Needle Tenderized LAB Samples (Packer)115 Table 3. ELISA Results For E. coli O157:H7 in Needle Tenderized ASC Samples (Packer)116 Table 4. ELISA Results For E. coli O157:H7 in Needle Tenderized LA Samples (Packer)...117 Table 5. ELISA Results For E. coli O157:H7 in Enhanced Control Samples (Packer).118 Table 6. ELISA Results For E. coli O157:H7 in Enhanced LAB Samples (Packer).119 Table 7. ELISA Results For E. coli O157:H7 in Enhanced ASC Samples (Packer).119 Table 8. ELISA Results For E. coli O157:H7 in Enhanced LA Samples (Packer).120 Table 9. ELISA Results For E. coli O157:H7 in Needle Tenderized CTRL Samples (Purveyor)..121 Table 10. ELISA Results For E. coli O157:H7 in Needle Tenderized LAB Samples (Purveyor).122 Table 11. ELISA Results For E. coli O157:H7 in Needle Tenderized ASC Samples (Purveyor)122 Table 12. ELISA Results For E. coli O157:H7 in Needle Tenderized LA Samples (Purveyor)...123 Table 13. ELISA Results For E. coli O157:H7 in Enhanced CTRL Samples (Purveyor).124 Table 14. ELISA Results For E. coli O157:H7 in Enhanced LAB Samples (Purveyor).125 Table 15. ELISA Results For E. coli O157:H7 in Enhanced ASC Samples (Purveyor).126 Table 16. ELISA Results For E. coli O157:H7 in Enhanced LA Samples (Purveyor).126 Table 17. ELISA Results For Salmonella in Needle Tenderized Control Samples (Packer)...176 Table 18. ELISA Results For Salmonella in Needle Tenderized LAB Samples (Packer) ...176 Table 19. ELISA Results For Salmonella in Needle Tenderized ASC Samples (Packer)177 Table 20. ELISA Results For Salmonella in Needle Tenderized LA Samples (Packer)...178 xi Texas Tech University, Alejandro Echeverry, December 2007 Table 21. ELISA Results For Salmonella in Enhanced Control Samples (Packer).179 Table 22. ELISA Results For Salmonella in Enhanced LAB Samples (Packer).180 Table 23. ELISA Results For Salmonella in Enhanced ASC Samples (Packer).180 Table 24. ELISA Results For Salmonella in Enhanced LA Samples (Packer).181 Table 25. ELISA Results For Salmonella in Needle Tenderized Control Samples (Purveyor)...182 Table 26. ELISA Results For Salmonella in Needle Tenderized LAB Samples (Purveyor).183 Table 27. ELISA Results For Salmonella in Needle Tenderized ASC Samples (Purveyor)184 Table 28. ELISA Results For Salmonella in Needle Tenderized LA Samples (Purveyor)...184 Table 29. ELISA Results For Salmonella in Enhanced Control Samples (Purveyor).185 Table 30. ELISA Results For Salmonella in Enhanced LAB Samples (Purveyor).186 Table 31. ELISA Results For Salmonella in Enhanced ASC Samples (Purveyor).187 Table 32. ELISA Results For Salmonella in Enhanced LA Samples (Purveyor).187 xii Texas Tech University, Alejandro Echeverry, December 2007 LIST OF FIGURES Figure 1. Generic Slaughter and Fabrication Process Flow Diagram72 Figure 2A. Strip Loins Trimming And Fabrication of Subprimals....................................................................................77 Figure 2B. Detail Of Fabricated Subprimals...................................................77 Figure 3. The Intralox Conveyor Belt System79 Figure 4. Vacuum Packaging Of Subprimals Under Refrigerated Conditions.80 Figure 5. Cleaning and Sanitizing Process Of Pathogen Laboratory81 Figure 6. Cleaning And Sanitizing Of Conveyor Belt82 Figure 7. The Jaccard Manual Tenderizer Model H ....84 Figure 8. Manual Tenderization Of Subprimals.84 Figure 9. Steaks Surface After Tenderization (Detail) 85 Figure 10. The Koch Injectamatic Pi21 Brine Injector..85 Figure 11. Injection Process.86 Figure 12. Injection Process (Detail)...86 Figure 13. Inoculated Surface Swab...87 Figure 14. Schematic of Sections Analyzed For Each Subsample...89 Figure 15. Removal Of Sections Within The Steak..89 Figure 16. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 0, PA).92 Figure 17. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 14, PA)...93 Figure 18. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 21, PA)...95 Figure 19. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 14, PA)...96 Figure 20. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 21, PA)...97 Figure 21. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 14, PA).99 Figure 22. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 21, PA).........100 Figure 23. Effects of water (CTRL), lactic acid bacteria (LAB), acidified xiii Texas Tech University, Alejandro Echeverry, December 2007 sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 14, PA).102 Figure 24. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 21, PA)..103 Figure 25. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 14, PU).105 Figure 26. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 21, PU).106 Figure 27. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 14, PU)....108 Figure 28. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 21, PU)....109 Figure 29. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 14, PU)..110 Figure 30. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 21, PU)..111 Figure 31. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 14, PU)..112 Figure 32. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 21, PU)..113 Figure 33. Summary of surface E. coli O157:H7 counts on enhanced subprimals over time (Packer)129 Figure 34. Summary of surface E. coli O157:H7 counts on needle tenderized subprimals over time (Packer)...131 Figure 35. Summary of surface E. coli O157:H7 counts on enhanced subprimals over time (Purveyor)132 Figure 36. Summary of surface E. coli O157:H7 counts on needle tenderized subprimals over time (Purveyor)133 Figure 37. Summary of TOP E. coli O157:H7 counts on EN and NT subprimals over time (Packer)...134 Figure 38. Summary of TOP E. coli O157:H7 counts on EN and NT subprimals over time (Purveyor)135 Figure 39. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 0, PA)...154 xiv Texas Tech University, Alejandro Echeverry, December 2007 Figure 40 Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 14, PA).155 Figure 41. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 21, PA)..156 Figure 42. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 14, PA).157 Figure 43. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 21, PA).159 Figure 44. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 14, PA)..160 Figure 45. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 21, PA)..161 Figure 46. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 14, PA)..163 Figure 47. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 21, PA)..164 Figure 48. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 14, PU)....166 Figure 49. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 21, PU)..167 Figure 50. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 14, PU).168 Figure 51. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 21, PU).169 Figure 52. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 14, PU)...170 Figure 53. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 21, PU)...172 Figure 54. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in xv Texas Tech University, Alejandro Echeverry, December 2007 subsection B of inoculated beef strip loins (Day 14, PU)..173 Figure 55. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 21, PU)..174 Figure 56. Summary of surface Salmonella counts on enhanced subprimals over time (Packer)190 Figure 57. Summary of surface Salmonella counts on needle tenderized subprimals over time (Packer)191 Figure 58. Summary of surface Salmonella counts on enhanced subprimals over time (Purveyor)192 Figure 59. Summary of surface Salmonella counts on needle tenderized subprimals over time (Purveyor)193 Figure 60. Summary of TOP Salmonella counts on EN and NT subprimals over time (Packer)...194 Figure 61. Summary of TOP Salmonella counts on EN and NT subprimals over time (Purveyor)195 xvi Texas Tech University, Alejandro Echeverry, December 2007 CHAPTER I LITERATURE REVIEW Worldwide diarrheal diseases are believed to be the cause of approximately 1.8 million human deaths in 2004 alone (1). In the United States, the Center for Disease Control and Prevention (CDC) estimates that the number of foodborne illnesses to be around 76 million, resulting in approximately 325,000 hospitalizations and 5,000 deaths. Of those, almost 14 million cases of foodborne illness, 60,854 hospitalizations and 1,800 deaths are caused by known foodborne pathogens (196). Many of these outbreaks have been associated with the consumption of contaminated food products, including those of animal origin, such as ground beef products. The cost of human illness, medical expenses and productivity losses associated with the six most dominant foodborne pathogenic bacteria has been estimated to be at least $2.9 and $6.7 billion per year (49, 129). Bacteria from the environment or that are harbored in the gastrointestinal tract of different herbivorous animal species used in food production can contaminate the carcass during the slaughter process. Meat contamination can also occur at any other point during fabrication due to inadequate cleaning and sanitation procedures or failure of employees to follow good hygiene and manufacturing practices. 1 Texas Tech University, Alejandro Echeverry, December 2007 Escherichia coli Escherichia coli is a microorganism described and characterized for the first time by Dr. Theodor Escherisch in 1885. It belongs to the Enterobacteriaceae family, with other microorganisms including Salmonella, Yersinia, Shigella, Citrobacter, Klebsiella, Enterobacter, and Proteus genera. The term Escherichia refers to the genus that is composed of gram-negative, aerobic, facultative anaerobic, non spore-forming rods, and the term coli to the species within the family. The food industry has been using non-pathogenic E. coli since the earliest 1900s as an indicator of fecal contamination in water and milk (30), as well as an indicator of the sanitary conditions in the food processing environment. Non-pathogenic strains of E. coli are part of the normal enteric flora of humans and warm-blooded animals intestines, living as commensals in the bowel and being the predominant facultative anaerobe organism in the human gastro-intestinal tract (81). Most strains are harmless to their host and their role in the body remains uncertain. Some studies suggest that E. coli serves a beneficial function in the body by synthesizing vitamins and by outcompeting other pathogenic bacteria that may be ingested with food or water (91). While most strains of E. coli are non-pathogenic, some strains can cause several different forms of gastroenteritis, each with its own symptoms and epidemiology. Pathogenic strains are divided into six different categories 2 Texas Tech University, Alejandro Echeverry, December 2007 according to the mechanisms by which diarrhea is produced, virulence properties, pathogenicity mechanisms, clinical syndromes and manifestations, and serological subgroups (79, 81). The categories of the pathogenic strains E. coli are enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enterohemorrhagic E. coli (EHEC). The mechanisms by which diarrhea is produced vary for each type of E. coli and include attachment of the bacteria to the intestinal cells, invasion, and production of enterotoxins (91). Pathogenic E. coli isolates are classically differentiated on the basis of three surface antigens: the somatic lipopolysaccharide or cell wall antigens (O), the flagellar antigens (H), and the capsular antigens (K). To date, approximately 174 O antigens, 56 H antigens, and 103 K antigens have been identified (81, 91), with the combination of the different antigens defining the E. coli serotype. Nowadays, the use of serogroups together with other characteristics like biotype or enterotoxin production help in the differentiation of those strains that cause infections and illness in both humans and animals (30). 3 Texas Tech University, Alejandro Echeverry, December 2007 Escherichia coli O157:H7 Among the hundreds of strains that cause disease, those in the enterohemorrhagic E. coli (EHEC) group are the most notorious of all due the severity of the disease and number of cases and outbreaks related to it. The EHEC group was identified for the first time as a cause of human disease when two consecutive outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in the United States were linked to the serotype O157:H7 in 1982 after consumption of undercooked hamburger patties in a chain of fast food restaurants (207). During the past twenty years, E. coli O157:H7 has emerged as a major disease causing pathogen, capable of causing high morbidity and mortality numbers among humans that become infected (216). The EHEC group is also referred to in the literature as verocytotoxigenic E. coli (VTEC) or shiga toxin-producing E. coli (STEC) due the production of toxins that are closely homologous to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (203), also called verotoxins (VTs) or Shiga-like toxin (SLT). Escherichia coli O157:H7 was first described in 1975 in California after it was isolated from a woman with bloody diarrhea, but its identification as an enteropathogen was not until two, nearly simultaneous, U.S. outbreaks during 1982 (131, 207). It is considered a serious threat to public health in developed countries. In the United States alone, it is the single greatest cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS) (12, 83, 190, 216). 4 Texas Tech University, Alejandro Echeverry, December 2007 Human Disease caused by Escherichia coli O157:H7 E. coli O157:H7 causes the majority and most severe outbreaks of gastrointestinal illnesses related to E. coli (91, 190) from infections that range from asymptomatic conditions to mild bloody diarrhea or even severe hemorrhagic colitis. Severity of symptoms usually depends on status of the person infected with the pathogen, with the very young or immunocompromised suffering the most severe episodes. Most of the strains of the EHEC group possess several virulence determinant factors that allow an intimate attachment or adherence to the intestinal cells within the host. In addition, the EHEC posse genes that encode for the production of very powerful verotoxins, which are immunologically and genetically related to those produced by Shigella dysenteriae (76, 81). Moreover, these toxins inhibit protein synthesis, causing damage to vascular endothelial cells in certain organs and even cellular death. Infection with E. coli O157:H7 can cause a wide variety of outcomes (91, 130, 190, 237), with cases being reported worldwide. Bloody diarrhea (or hemorrhagic colitis, HC) caused by E. coli O157:H7, where infection of the large intestine occurs, is clinically different from that produced by other gastrointestinal pathogens. Clinical symptoms range from one to eight days, with an average incubation period of three days (190). Initially, patients develop abdominal cramps and watery diarrhea, with a varying percentage of these patients diarrhea resolving without further complications. The cramps can be very severe, 5 Texas Tech University, Alejandro Echeverry, December 2007 with the cecum and ascending colon as the most affected areas that can mimic an acute abdomen inflammation and lead to exploratory laparotomy. Fever is usually absent or mild but occasionally can exceed 102 F (38.9C). In mild disease without bloody diarrhea, patients have less abdominal cramps, vomiting, and fever, and are less likely to develop systemic sequelae, hemolytic uremic syndrome (HUS), or to die (130, 231). The occurrence of bloody diarrhea can happen as often as 15 to 30 minutes. Vomiting is also reported in about 30% to 50% of cases. Approximately 95% of the cases of HC resolve completely without further complication, however, the remaining 5% develop hemolytic uremic syndrome. Hemolytic uremic syndrome, a term used for the first time in 1955, is defined as a disorder where kidney failure, hemolytic anemia, and thrombocytopenia (platelet deficiency) develops, usually after 7 days of the onset of diarrhea (236, 249). These symptoms are also accompanied by coagulation defects and variable nervous system signs (123, 234, 249). The pathogen avoids expulsion from the body through its virulence factors causing an attachment that induces the transfer of verotoxin to the mucosa where it is transported by the epithelial cells and absorbed by the gut wall (123, 146, 153). Most people recover from the HC without the use of antibiotics or any other specific treatment in 5 to 10 days with the exception of the individuals who develop HUS. However, HUS has no specific treatment once the illness started. Supportive care is usually necessary to prevent further complications, including 6 Texas Tech University, Alejandro Echeverry, December 2007 careful fluid administration in order to prevent overhydration, hyponatremia, and seizures (234, 248, 249). The use of antibiotics for treatment of HC caused by an E. coli O157:H7 infection is not recommended since some studies have found that antimicrobials are a risk factor and patients under antibiotic treatment are more likely to develop HUS (123, 130, 165, 209, 231, 234). The Survival of Escherichia coli O157:H7 in foods After recognition of E. coli O157:H7 as a causation of food-borne illness, studies have focused specifically in the growth, survival, and inactivation characteristics of this pathogen (30, 136, 194). Studies have been performed to understand the behavior of E. coli O157:H7 in different substrates and foods for varying periods of time, as well as the effect that different intrinsic and extrinsic factors such as hot and cold temperatures, pH, organic acids, water activity (Aw), salt, control of reduction-oxidation potential (RO), fat content, irradiation, and preservatives will have on this specific pathogen. E. coli O157:H7 can be controlled by proper cooking of the food product to a specific temperature and time. Escherichia coli O157:H7 is a cause for concern especially if present in foods that do not go through a treatment process to eliminate the pathogen, or that could be contaminated after such process and before packaging as in the case of ready-to-eat (RTE) products. After the outbreak in 1993 (15, 31), the USDA considered E. coli O157:H7 an adulterant if present in ground beef, setting for the first time in the United States history a zero tolerance policy for presence 7 Texas Tech University, Alejandro Echeverry, December 2007 of a microorganism in raw meat product (121, 126, 142, 235, 238). Some examples of the foods involved with E. coli O157:H7 outbreaks include ground beef (15, 31, 44) and meat products (16, 128, 145); apple juice (18, 34, 65); radish (168, 244); raw sprouts (19, 46, 173); lettuce (4, 122); and other type of fresh vegetables including baggy salads (4, 24, 25, 219), as confirmed by the recent spinach outbreak in California. Microorganisms are not killed instantly when exposed to a lethal agent, but rather, the population decreases exponentially. The D value or decimal reduction time is used in food microbiology to describe at any given temperature the time required in minutes to reduce 90% (or 1 log) of a specific microbial population in a specific food, and it is affected by factors such as pH, water activity (Aw), content of preservatives, product composition, and the size of the microbial population, among others. Studies have revealed that cooking ground beef with 17-20% fat at 57.2C and 62.8C have D values of 4.5 and 0.40, respectively. Cooking hamburgers to an internal temperature of 71.1C (160F) for 15 seconds is required to assure adequate cooking and prevent outbreaks (47, 81, 137, 192, 230, 250). Pasteurization is also an accepted heating method to destroy this pathogen in milk, fruit juices and ciders. Treatment of milk for 15 seconds at 71.7C (161F) allows a 5-log reduction of E. coli O157:H7, and the same reduction is achieved in apple cider when it is pasteurized at 68.1 for 14 seconds (7, 154). Other studies have shown recovery of E. coli O157:H7 in artificially 8 Texas Tech University, Alejandro Echeverry, December 2007 inoculated foods after frozen storage. In one study, E. coli O157:H7 was recovered from inoculated strawberries, radishes, and cabbage after 2 and 4 weeks of storage at -20C (114). Ground beef used in the manufacturing of hamburger patties is often produced in a central location and distributed under frozen conditions to fast food restaurants in different locations. In the 1993 E. coli O157:H7 multistate outbreak involving undercooked hamburgers, contaminated frozen patties produced by a single plant in California were involved with illness 6 weeks after the production date (31, 238). Studies performed after that outbreak in inoculated ground beef patties (20% fat) revealed that E. coli O157:H7 can survive for up to 4 weeks after storage at -2C with a 1.5 log reduction in the population. Storage of ground beef at -20C for 12 months established recovery of the pathogen with an approximate reduction of 1.0 log (26), demonstrating the ability of E. coli O157:H7 to survive in hamburgers for long periods of time at frozen temperatures with little decline in numbers of viable cells. As seen in the examples above, E. coli O157:H7 displays a unique ability to survive in a wide variety of products subjected to different process conditions for long periods of time, allowing the foods to serve as vehicles in the transmission of infections. 9 Texas Tech University, Alejandro Echeverry, December 2007 Salmonella Salmonella is the genus name of a microorganism discovered for the first time by Theobald Smith in 1886; however, the credit of its discovery was claimed by his employer, Dr. Daniel Elmer Salmonella. This bacterium belongs to the Enterobacteriaceae family, with other microorganisms including Yersinia, Shigella, Citrobacter, Klebsiella, Enterobacter, and the Proteus genera. The genus Salmonella is composed of mesophilic heterotrophic, Gram-negative bacilli that are facultative anaerobic, straight and flagellated rods, usually between 0.7-1.5 x 2.0-5.0 m in size (11, 125). There are two generally accepted species: S. enterica and S. bongori (66); with the first one divided into six subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica (11). Salmonella can be distinguished antigenically by formation of clumps when exposed to homologous antisera, with differences based on either the somatic (O), flagella (H) or capsular (Vi) antigens, which is unique to each of the serotypes (73, 97, 127). With the use of this approach there are almost 2600 recognized serotypes (179). The subspecies that causes the most concerns is Salmonella enterica subsp. enterica, with serotypes Enteritidis, Typhi, Thiphimurium, Dublin, and Agona as the most isolated from foods of animal origin and major causes of foodborne related outbreaks (60, 67, 127). 10 Texas Tech University, Alejandro Echeverry, December 2007 Human disease caused by Salmonella Foodborne illnesses attributed to Salmonellae are one of the major causes of morbidity and mortality troubling health officials today. Infection with this pathogen has a large socioeconomic impact on human and animal health. Despite a declining trend in the number of foodborne cases in the last decade, preliminary FoodNet data from 2006 confirmed an increase in the number of foodborne illnesses due to Salmonella, leading with 39% of attributable outbreaks (172). It is estimated that foodborne illnesses due to Salmonellae result in approximately 1.3 million cases each year in the United States alone (172, 196). In addition, of the total number of deaths from known foodborne etiology, Salmonella cases are estimated to cause 553 (30.6%) of them. Most food-associated Salmonella infections involved non-typhoidal Salmonella (NTS) serovars, which tend to cause a self-limiting gastroenteritis outcome, including symptoms such as nausea, vomiting, headache, fever, chills, myalgia and tenesmus. In addition, if the infection aggravates, it could lead to bacterimia, septicemia and death (257). The gastroenteritis onset can start as early as 5 hours (usually 12 36 hours) and can last up to five days after consumption of contaminated food depending on the ingested dose. Invasion of the intestinal mucosa occurs leading to an inflammatory response and diarrhea (11). A short onset is usually indicative of either high doses of the pathogen or consumption by an immunocompromised or susceptible person (125), but other factors such as serovar causing the infection and antibiotic resistance patterns 11 Texas Tech University, Alejandro Echeverry, December 2007 also contribute to the course of infection. Appropriate Salmonella infection treatment usually includes fluid and electrolyte replacement. The use of antimicrobial therapy is only required if there is evidence of bacterimia or invasion from the intestinal tract (257); however, susceptibility testing against the drugs most commonly used (chloramphenicol, ampicillin, trimethoprimsulfamethoxazole and fluoroquinolones) is recommended because of the emergence of multidrug resistant (MDR) strains, such as Salmonella Typhimurium definitive phage type (DT) 104, which can reduce the treatment options in case of infection and is associated with exacerbated episodes of illness (109, 110, 198, 205, 256, 257). The survival Of Salmonella In Foods Salmonella species are adapted to a large number of hosts, including food and farm animals such as poultry (chickens, turkeys, fowls), swine, cattle, goats, sheep, horses, rabbits and many other mammals (125). Because of this, foods of animal origin serve as vehicles of transmission for this pathogen, allowing its presence in food processing facilities. Other sources also include rodents, flies, cockroaches and others pests that can normally be found in plant environments. Some foodhandlers can get infected with the pathogen and become asymptomatic carriers. If good hygiene practices are not followed, Salmonella can be transmitted easily through the foods as they are handled and prepare. Some examples of the foods involved with nontyphoid salmonellosis outbreaks 12 Texas Tech University, Alejandro Echeverry, December 2007 include poultry and poultry salads (108, 112, 148); meat products (17, 48, 75); milk (14, 21, 124, 220); cheese (9, 32, 88, 201); shell eggs and egg products (20, 22, 50, 120, 161, 213); peanut butter (13, 221); tomatoes (23, 69, 118); cocoa and chocolate products (68, 245); raw sprouts and other type of fresh produce (52, 141, 173). Even though Salmonella is not capable of forming spores, this microorganism has the ability to survive in foods for long periods of time as it is a very resistant microorganism capable of adapting to extreme conditions. Differences in pH, water activity, substrates, and food composition affect the D values and the recovery of this pathogen in specific food products (43, 134, 135, 162, 217). Studies have shown the ability of this microorganism to acquired thermal resistance when exposed to sublethal temperatures. When Salmonella has been exposed to temperatures 50 C for 15 to 30 minutes, its thermal resistance increases as some studies have shown (38, 39). In addition, cells exposed to elevated growth temperatures, heat shock, or starvation during the growth phase have also shown increase in the thermal resistance, as noted in a study of turkey products (246). Thermal inactivation of Salmonella has been determined in different meat products (177, 184, 197). In one study, the D-values for Salmonella were 43.10 minutes (55 C) and 0.096 minutes (70 C) in ground turkey; and 37.05 minutes (55 C) and 0.066 minutes (70 C) in beef (177). In a thermal lethality validation study in breaded pork patties, the average calculated D-value for Salmonella at 13 Texas Tech University, Alejandro Echeverry, December 2007 a temperature range of 55 C to 70 C were 69.48 minutes to 0.29 minutes, respectively (185). In another study using Salmonella typhimurium DT104, it was observed that an increase in the fat levels in the beef resulted in longer lag periods and lower D-values (134). In this experiment, the lag period increased from 4:43 min in beef containing 7% fat to 28:12 min in beef with 24% fat; and the D-values at 58 C were 3:22, 2:46, 2:49 and 1:61 minutes in beef with 7, 12, 18 and 24% fat, respectively. An increase in the lag time period due to injury of the cells can also lead to failure to detect the pathogen in a food product when traditional microbial methods are used (158). The reported minimum pH value at which Salmonella can grow in food products is 3.3; with its growth being codependent on the type of acidulant or organic acid used to decrease the pH in the food product (61, 125). Survival of Salmonella in mayonnaise and salad dressing (containing lemon juice, wine vinegar or acetic acid) have shown that the pathogen dies when introduced to these products but its death rate varies not only on the concentration of the acid, but also the storage temperature and organism adaptability (51, 61, 187, 223). In addition, other studies have shown that exposure of Salmonella to low pH values can trigger an adaptive pH protective system that will protect cells from more severe acid stress encounters. This homeostatic system allows the bacteria to control its internal pH (to levels of 5.0 to 5.5) in order to obtain a stable condition that allows its survival in acidic foods (89, 90), a critical advantage when compared to other bacteria. In one study, Salmonella cells exposed to pH 14 Texas Tech University, Alejandro Echeverry, December 2007 between 4.0 to 5.0 showed an increased acid resistance (survival) when compared to other cells exposed to a pH of 5.5 to 6.0 (143). In another study, Salmonella colonies isolated from food and humans were tested for acid resistance. The study indicated that after exposure for two hours to a 5.0 pH, cells had a strong variation of acid survival depending on the type of isolate. All human isolates were high acid resistant (with surviving rates > 10% at pH = 2.5) when compared to food isolates that were only intermediate or low acid resistant (33), indicating a possible positive correlation between acid resistance and pathogenicity (98, 150). As it can be seen, this stress response might also enhance the survival of the pathogen against additional environmental factors, such as acid-based interventions or sanitizers commonly used in the meat industry. Isolation and enumeration methods During a foodborne outbreak, early detection and recognition of the causative agent is required to allow for proper treatment of the patients (74). Isolation of the causative agent is also required not only to administrate the proper medication, but also to promptly identify other possible cases and routes of transmission, and limit the number of deaths that may occur (31). Sensitive detection methods for E. coli O157:H7 and Salmonella allow surveillance nets to be activated at health department levels in order to initiate procedures to recall food products if necessary. The food industry also requires and demands rapid 15 Texas Tech University, Alejandro Echeverry, December 2007 and accurate identification methods to verify the processes and the environmental sanitation conditions of the plant in order to provide a safe food product. To allow for this, culture media containing nutrients such as aminonitrogen compounds, energy sources, minerals, salts, buffers and growth promoting factors are necessary to allow detection of the pathogen of interest. Isolation of E. coli O157:H7 from animal, food, environmental, and clinical cases is based on the unique phenotypic and genotypic characteristics of this microorganism that allows its detection and confirmation with the use of serological and biochemical tests. Escherichia coli O157:H7 differs from generic E. coli in some unique aspects, including the absence or slow sorbitol fermentation (214), no haemolysis (or disintegration of red cells with the release of hemoglobin) on sheep and rabbit blood agar (207), and no manifestation of glucuronidase activity (155, 202). A wide variety of methods and protocols describing the culture techniques for the isolation of the pathogen using these properties have been reported (57, 58, 163, 166). Usually, three general steps are required: enrichment of the sample, isolation of the presumptive colonies, and final confirmation of the bacteria. Most of the studies conducted follow these accepted steps, but may differ due to the media used or the supplements added to the media (171). 16 Texas Tech University, Alejandro Echeverry, December 2007 Pre-enrichment step Pre-enrichment of E. coli O157 can be done by addition of buffered peptone water (BPW) supplemented with vancomycin to the sample followed by an incubation period of 6 h at 37C (82). One study used BPW supplemented with vancomycin, cefsulodin, and cefixime (BPW-VCC) in ground beef samples to compare recovery of heat-injured and freeze-injured cells (204). Other studies have used Gram-negative (GN) broth, a selective medium normally used for the microbiological examination of foods that allows the growth of coliforms while inhibiting competitive flora such as Gram-positive bacteria (159). GN broth is usually supplemented with vancomycin, cefixime and cefsulodin (GN-VCC broth) and used in the detection of E. coli O157:H7 from fecal samples, feed and water (45, 78, 151). Another medium commonly used for pre-enrichment is tryptic soy broth (TSB), a solution that supports and promotes the growth of different microorganisms and has been used in several studies (96, 144, 159). TSB also can be used in addition of antibiotics such as cefixime and vancomycin (TSBcv) and has been proved to be very sensitive in the detection of E. coli O157:H7 from bovine feces (71, 211). Isolation of Salmonella from food products is considered a complex procedure because of all the steps involved. Usually detection of this pathogen involves the following steps: pre-enrichment, enrichment, selective-differential plating, isolation and identification (125). The pre-enrichment step (also know as resuscitation step) can be done depending on the likelihood that the bacterial 17 Texas Tech University, Alejandro Echeverry, December 2007 cells present in the product are at very low levels and/or that they have been injured during processing of the food product or collection and transporting of the samples. Salmonella pre-enrichment usually involves buffered peptone water if the food product is high in nutrients (such as proteins) or lactose broths in food with less nutrients (125), with neutralization being required if the pH is either too high or too low. Selective pre-enrichment can be used for the detection of Salmonella in meat and dairy products by transferring the sample of interest into rappaport vassiliadis (RV) broth (180, 186) and into tetrathionate (TT) broth (226) followed by incubation of the tubes at 35C for 24 hours. Both mediums selectively allow Salmonella to growth while suppressing other coliform or gram positive microorganisms (159). Enumeration and Isolation Classically, detection of E. coli O157:H7 has been based on the use of selective and differential media that allows the use of phenotypic characteristics to recognize the pathogen from other bacteria. E. coli O157:H7 shows typical reactions for generic E. coli except it lacks the capacity to ferment sorbitol within 24 h. When plated on MAC, E. coli O157:H7 ferments lactose rapidly and cannot be differentiated from other generic E. coli strains and enteric microorganisms that may also grow in the agar (228). This finding was followed with the introduction of sorbitol-MacConkey agar (SMAC), where lactose was replaced with sorbitol as a carbohydrate source (159, 214) and where non-sorbitol fermenting (NSF) cultures appear either colorless or pale yellow as compared 18 Texas Tech University, Alejandro Echeverry, December 2007 with sorbitol-fermenting microorganisms which appear pink, allowing a fast, easier and more reliable recognition of presumptive E. coli O157:H7 colonies (35, 74, 214). However, SMAC plates are not 100% effective for the detection of this pathogen, since it has been reported that some E. coli O157:H7 isolates can ferment sorbitol (182), leading to false negative results on this test. The use of antibiotics in this media can improve the detection methods as seen when cefixime, a potent antimicrobial with a broad in vitro activity against pathogens, was added to SMAC. The use of cefixime on SMAC agar enhances the detection of E. coli O157:H7 while inhibiting the growth of both generic E. coli and Proteus , another NSF bacteria that may also be found in the samples (188). Addition of potassium tellurite can also increase the detection and selectivity of SMAC to E. coli O157:H7, and some studies have used both cefixime and tellurite together with SMAC (CT-SMAC) agar successfully in fecal (29, 45, 189, 254) and environmental samples (95, 203, 212, 233) in order to inhibit background flora. Another commonly used differential method for the detection of E. coli O157:H7 is based on the biochemical use of chromogenic compounds, substances that remain colorless until acted upon by an enzyme. Fluorocult E. coli O157 agar, a medium that contains 4-methylumbelliferyl-b-D-glucuronide (MUG), is used to take advantage of this characteristic. When MUG is cleaved by -D-glucuronidase (GUD), an enzyme produce by E. coli strains, a compound called 4-methylumbelliferone (MU) is produced (155). The presence of MU in the media can be confirmed with long-wave ultraviolet light (366nm), yielding a blue 19 Texas Tech University, Alejandro Echeverry, December 2007 fluorescence color; however, E. coli O157:H7 do not possess the enzyme and cannot hydrolyze this substrate. Since no fluorescence is produced under the UV light, identification of presumptive E. coli O157:H7 colonies is facilitated with the use of this differential agar (155, 156, 228). A chromogenic media called CHROMagarTM O157 is another selective medium that can be used for the isolation, differentiation and presumptive identification of Escherichia coli O157:H7 from food, veterinary and environmental samples and has been proved to be more sensitive than SMAC in the detection of presumptive E. coli O157. It is based on the production of colonies with a mauve color (E. coli O157) as compared to other non-O157 strains that exhibit a blue color instead (77, 242). The presence of Salmonellae is usually determined by subjecting the samples that have been pre-enriched previously into rappaport vassiliadis (RV) broth or tetrathionate (TT) broth and plate them into selective media, such as xylose Lysine Desoxycholate (XLD) agar or Xylose Lysine Tergitol 4 (XLT4), which allows for differentiation of Salmonella from other enteric pathogens based on color changes (125, 159, 169). The formation of colonies with black centers is indicative of microorganisms that can decarboxylate lysine and must be confirmed with additional biochemical test. Enumeration of Salmonella is usually performed by the use of the most probable number (MPN) technique, a method that can become very expensive and time consuming if a high number of samples need to be analyzed. This method allows the estimation of the number of original concentration of undiluted 20 Texas Tech University, Alejandro Echeverry, December 2007 bacteria in the original sample, assuming they are distributed randomly within the sample, not clustered together and they dont repel each other (84, 125). Use of non-selective media for direct enumeration of Salmonella is not recommended as background flora can overgrow in the plates leading to overestimation of the pathogen in the sample. In the food processing environment, Salmonella cells can become injured due several factors such as heat shock (temperature fluctuations due cooking or holding during processing), cold stress (chilling, freezing) or acid stress (such as organics acids used during interventions). Besides causing injury to the cells, these stress factors might affect the metabolism leading to a viable but non-culturable state (VNC), where the ability of the pathogen to be recovered on agar medium is diminished while remaining intact and retaining viability (37, 59, 206). Additionally, direct plating of samples onto selective media can result in underestimation of the numbers of this pathogen, as injured cells might not grow in the presence of the selective supplements and inhibitors that are part of the media formulation (255). Previous experiments have shown differences in the recovery of bacteria when subjected to differential media, a culture method where some of the components allow for differentiation of the species based on biochemistry properties (227). In one study involving irradiated comminuted turkey, cultures obtained from heat-, cold-, and starvation-injured Salmonella had significantly different counts between XLD agar plates (selective media) and tryptic soy agar with yeast extract (TSAYE) plates (non-selective media) (246). In order to 21 Texas Tech University, Alejandro Echeverry, December 2007 prevent this underestimation of the actual pathogen counts some procedures have been developed to obtain more accurate results, allowing the injured cells to recover and repair before the selective agents start to inhibit and affect the cells (28, 86, 170). Selective media is a culture method where selective agents such as salts, dyes, and antibiotics agents are incorporated into the formulation allowing the growth of the targeted microorganism, while other bacteria that are not resistant to the selective compounds or concentrations are inhibited (159, 227). One example of the use of selective media is one technique known as overlay resuscitation (OV), where cultures are plated initially in a non selective medium for 2-4 hours followed by a overlay of selective media that inhibits background flora for an additional 20 22 hours. This procedure has been found to be an effective method to enumerate injured bacteria in food samples. Less difficult methods based in this principle known as thin agar layer or agar underlay method have been proved to be effective in recovering pathogens that have been subjected to different types of stress factors. In the thin agar layer method, originally developed to recover sublethally heat-injured L. monocytogenes, a thin layer of selective medium is allowed to solidify in the Petri dish followed by the application of a two-thin layers with non-selective media (TSA) (138). This procedure allowed injured and uninjured cells to grow in TSA before the selective agents start to diffuse and cause inhibition of bacteria other than the one of interest. It is worth noticing that this procedure produced L. monocytogenes 22 Texas Tech University, Alejandro Echeverry, December 2007 colonies similar to those observed when plated in the selective media alone. Other studies have used variations on this technique with different types of selective media and have been proved successful for enumeration of pathogens other than L. monocytogenes. Kang and Fung (139) used xylose lysine decarboxylase (XLD) as a selective agent in conjunction with tryptic soy agar (a non selective agar) to facilitate the recovery of heat-injured Salmonella Typhimurium. In another study, cold-injured L. monocytogenes, S. Typhimurium and Y. enterocolitica had better recovery capacity when the thin agar layer method was used when compared to plating directly onto selective media (253). In an additional study using acidinjured cells (previously exposed to 2 % acetic acid for 1, 2 and 4 minutes), the same authors found that the recovery of E. coli O157:H7, Staphylococcus aureus, S. Typhimurium and Yersinia enterocolitica was significantly higher when colonies were plated using the thin agar layer technique than those obtained when directly plated onto selective media (251). A study by Wu and Fung (252) used a four compartment Petri dish for the simultaneous recovery of four different heat injured pathogens. The system consisted of a four compartment Petri dish were MacConkey sorbitol agar (MSA), modified Oxford medium (MOX), xylose lysine desoxychlolate (XLD), and cefsulodin irgasan novobiocin (CIN) were used as the selective medium for the recovery and enumeration of E. coli O157:H7, L. monocytogenes, S. Typhimurium, and Y. enterocolitica from inoculated milk and ground beef, respectively. 23 Texas Tech University, Alejandro Echeverry, December 2007 Interventions Microbial contamination of meat with pathogens such as E. coli O157:H7 and Salmonella is a public health concern due to the outbreaks of foodborne illness commonly associated with the consumption of these products. The need to prevent these unfortunate incidents has prompted the incorporation of different types of control measures in the processing facilities in order to reduce and eliminate these pathogens from the food products and to prevent them from entering the food supply. Contamination of the carcasses can occur in different steps during the slaughter process, especially during de-hiding and evisceration of the animal. As part of the adoption of the Hazard Analysis and Critical Control Point (HACCP) system, all beef processors and plants need to develop a plan that identifies the hazards that are associated to their respective process and the control measures that can be implemented in each step to reduce their likelihood in the food product. In the meat industry some of these control measures are known as interventions, with many of them being used in sequence as part of a multiple hurdle approach. These control measures can be categorized into a) physical (hot water spray, steam pasteurization, steam-vacuuming, water wash cabinet and knife trimming); b) chemical (organic acids, polyphosphates, chlorine, acidified sodium chlorite, ozone, peroxyacetic acid, nisin, and lactoferrin); c) emerging technologies (hydrostatic pressure, irradiation, pulsed electric fields, and microwaves) (210); and d) biological (lactic acid bacteria and 24 Texas Tech University, Alejandro Echeverry, December 2007 bacteriophages). Use of the previous interventions and their effectiveness on beef hides (5), carcasses (140), beef trim/variety meats and ground beef (224) have been reported by previous authors. The effectiveness of the interventions and the levels of bacterial reduction that are obtained vary according to the testing methodologies that are used and the type of meat surface that has been tested, often leading to diverse results (239). Additionally, the concentration of the acid and its pH also determines the effectiveness of the compound against bacterial loads (224). It is worth noting that even though interventions can reduce the risk of pathogens to be transferred to meat surfaces and their final products, in no case they can be a substitute for good manufacturing practices and good cleaning and sanitation procedures in the processing facility. Lactic acid Lactic acid is a non toxic, generally recognized as safe (GRAS) organic compound commonly used in the meat industry as a chemical treatment to decontaminate beef and pork carcasses due to its immediate (bactericidal) and delayed (bacteriostatic) effects (70). This compound is naturally produced by lactic acid bacteria during fermentation of foods and its use as an antimicrobial for decontamination of beef carcasses has been approved by the U.S. Department of Agriculture-Food Safety and Inspection Service (FSIS) (195). Lactic acid also offers a residual effect over time that prevents pathogen growth in the meat intended to be grinded (208). Several different studies have tested 25 Texas Tech University, Alejandro Echeverry, December 2007 the efficacy of this compound as agent to reduce bacterial contamination in beef and meat products. In one of the earliest studies, experiments were conducted on veal, pig, and beef slaughter lines to assess the microbial loads on carcasses surfaces after treatment with lactic acid (80). The authors reported that when a 1% lactic acid solution was sprayed on hot carcasses it was more effective against bacteria than when chilled carcasses were sprayed. Additionally, the authors reported that the APC and Enterobacteriaceae colony counts were significantly lower (P < 0.05) when treated with lactic acid when compared to the controls. In another study, the efficacy of lactic acid to control E. coli O157:H7 attached to beef carcasses was tested at three different concentrations. Lean and adipose meat tissues were inoculated with a cocktail containing three different strains of this pathogen. The authors reported a log reduction of 1.0, 1.7, and 2.5 cfu/cm2 when using 1%, 3%, and 5% lactic acid, respectively (240). In another study, the effects of lactic acid (1.5 % concentration) on bacteria were tested by spraying the solution on beef strip loins before and after 14, 28, 56, 84, and 126 days of vacuum-packaged storage (85). The authors reported that the total microbial counts (log CFU/cm2) of all cuts were lower in the loins treated with acid when compared to the control samples. In a study conducted by Rose and others (116), aerobic plate counts (APC), total coliform counts (TCC), and generic E. coli counts (ECC) were tested in beef short plate (BSP) pieces subjected to lactic acid at concentrations of 1.25 %, 2.00 % and 2.50 %. The authors reported that 26 Texas Tech University, Alejandro Echeverry, December 2007 the use of 2.00% or 2.50% LA resulted in lower APC than that of the control at 0, 2, and 5 days of storage. Use of LA at 1.25 % resulted in higher APC, TCC and ECC than those obtained with 2.00% or 2.50%. Recently, as part of a validation of an acid wash in a HACCP plan, Dormedy and others (176) used 2% lactic acid as a critical control point in a large meat processing facility. The authors reported that the use of lactic acid at 2% concentration resulted in significant lower mesophilic, psychrotrophic, lactic acid bacteria, coliforms and generic E. coli counts in the beef carcasses. After further processing, ground beef made from these carcasses also presented lower mesophilic aerobic plate counts (APC) by more than 1 log when compared to the USDA Baseline study (55). Acidified sodium chlorite Acidified sodium chlorite (ASC) is a generally recognized as safe (GRAS) antimicrobial agent approved by the Food and Drug Administration for the treatment of poultry, seafood and meat (200), especially when it is used on prechill and post-chill meat products. ASC is produced by adding an acidulant to a solution of sodium chlorite (NaClO2). The mode of action of ASC, which is a broad spectrum antimicrobial, is due to a non-specific attack on the bacterial cells. Besides bacteria, ASC also has capacity to inhibit viruses, yeasts, molds and some protozoa (113). The use of ASC in poultry meats, red meats, and processed meat products is approved in solutions containing from 500 to 1200 ppm at a pH of 2.3 to 2.9. Several different studies have tested the efficacy of this compound as agent to reduce bacterial contamination in poultry, beef and 27 Texas Tech University, Alejandro Echeverry, December 2007 meat products; however, its antibacterial activity can greatly be affected by the presence of organic matter (115). Besides the concentration level to which it is used, its antimicrobial properties are dependent upon the type of activation method (type of acid used), the application method (sprays/dipping) and the contact time with the carcass or meat product. In one study, the efficacy of acidified sodium chloride (activated by either phosphoric acid or citric acid) sprayed on beef carcasses to reduce E. coli O157:H7 and Salmonella Typhimurium was tested (152). The authors reported that by using a phosphoric acid-activated ASC, E. coli O157:H7 and Salmonella Typhimurium were reduced by 3.8 to 3.9 log units, respectively. When they used citric acid-activated ASC, an increase in the reduction for both pathogens was reported (4.5 to 4.6 log units, respectively). In another study, the effectiveness of ASC in reducing E. coli O157:H7 counts on beef carcass adipose tissue and beef trimmings was tested (6, 27). When using a 0.02 % ASC solution, E. coli O157:H7 was significantly (P < 0.05) reduced by 1.9 log cfu/cm2 in beef carcass adipose tissue and by 1.8 log10 CFU/g in beef trimmings, respectively. In another study, Hajmeer and others (175) tested an ASC spray application to observe its effectiveness against E. coli O157:H7 on beef briskets. The authors reported that ASC was effective (P < 0.05) in reducing this pathogen (average reduction of 0.7 log10 CFU/mL) when compared to the control. In one study conducted at Texas Tech University, ASC (1000 ppm) was tested on beef trim prior to and after grinding in a simulated industrial processing environment (56). The authors 28 Texas Tech University, Alejandro Echeverry, December 2007 reported that the use of ASC on the surface of beef trim did not showed any reduction on the levels of E. coli O157:H7; however, there was a significant reduction (P < 0.05) on the levels of Salmonella. Additionally, the authors reported that ground beef obtained from the ASC-treated trim had significant reduced levels of E. coli O157:H7 (6 hours after processing) and Salmonella (6 and 24 hours after processing) when compared to the control samples. One study on inoculated fresh beef cuts also proved the effectiveness of ASC (final concentration of 0.12% sodium chlorite) in reducing E. coli O157:H7, where this intervention resulted in a 2.50 log CFU/cm2 reduction of the pathogen at day 0 (178). Consistently, all ASC treated samples had lower E. coli O157:H7 during the 14 day storage period (4 C) when compared to the control samples. Lactic acid bacteria Lactic acid bacteria (LAB) are defined as a group of gram-positive, catalase-negative, acid tolerant, non-sporulating, microaerophilic, rods known for the production of lactic acid as the final metabolic product obtained from the fermentation of carbohydrates. The LAB is compromised, among others, from bacteria of the Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus genera (10). The use of LAB in fermentation of foods could easily be described as one of the oldest methods of food preservation (222). Lactic acid bacteria and their products are generally recognized as safe (GRAS), and their use and application in food products is approved by the U.S. Food and Drug Administration (FDA) 29 Texas Tech University, Alejandro Echeverry, December 2007 (2). The application of LAB, lactic acid, and other metabolic products (bacteriocins) to enhance the safety of non-fermented food products has been tested in different studies throughout the years. Microbial antagonism of different species of LAB toward food-borne pathogens such as Listeria monocytogenes, Salmonella, and E. coli O157:H7 in ground beef and ready-to-eat (RTE) meat products has been tested successfully in different studies. A study conducted by Muthukumarasamy and others (2, 133, 147, 160) tested Lactobacillus reuteri as a competitive inhibitor against E. coli O157:H7 on refrigerated ground beef stored under modified atmosphere packaging. The authors reported that before the end of the storage period (20 days) the Lactobacillus reuteri reduced viability of the pathogen for up to 6.0 log CFU/g. In another study, LAB were tested against L. monocytogenes in Ready-to-Eat Meat Products (72, 133). In this study, the authors identified 3 LAB strains (Pediococcus acidilactici, Lactobacillus casei, and Lactobacillus paracasei) that exhibited the most antilisterial activity. When using a cocktail of the previous strains in L. monocytogenes inoculated RTE meat products, a bacteriostatic activity was observed in cooked ham (2.6 log10 units lower than controls); whereas, bactericidal activity was observed in frankfurters (4.2 to 4.7 log10 lower than the controls). The authors also reported that after the 28-day refrigerated storage period the numbers of LAB only increased by 1.0 log10 cycle. When the authors conducted a sensory panel (triangle test) with non-pathogen inoculated RTE meat products over a eight week evaluation period, it was reported that the 30 Texas Tech University, Alejandro Echeverry, December 2007 use of LAB had no significant effect on the sensory quality of the product. Additionally, no signs of spoilage were observed when using LAB. In a recent study conducted at Texas Tech University, the authors used a cocktail of four different LAB strains (NP 51, NP 25, NP 7, and NP 3) to inhibit E. coli O157:H7 and Salmonella in ground beef (229). The authors reported that the combined LAB cocktail was significantly effective in reducing E. coli O157:H7 (2.0 log10 units) and Salmonella (> 3.0 log10 units) in ground beef (P < 0.05) after 3 days of refrigerated storage. A sensory panel was also conducted in this study, with no panelists being able to detect differences between cooked product with and without added LAB cultures. From the previous examples, it can be seen that the use of LAB as an intervention is an effective method to reduce levels of pathogenic microflora in meat and meat products without detrimental effects on the eating quality of the products. Meat tenderness and processing Eating meat is not only a necessary part of a healthy diet, but an enjoyable experience due to the sensory and organoleptic factors involved during its consumption, such as aroma, flavor, tenderness and juiciness (133) . Even though the importance of each of these factors varies among individuals, when consuming whole muscle cuts such as steaks, tenderness is possibly one of the major factors consumers use to judge the overall eating quality, experience and acceptability of the product (53, 167) 31 Texas Tech University, Alejandro Echeverry, December 2007 Factors that affect the degree of tenderness in an animal include species, sex, age, type of muscle (connective tissue, myofibril, sarcoplasm), as well as external factors such as diet, holding temperature and postmortem electrical stimulation (87, 183). Methods to improve the degree of tenderness have been studied for several years by processors in order to increase the value of those cuts with low marbling that fail to grade USDA choice or that simply are not desired by consumers. One of the most common methods to increase tenderness in the meat cuts is by aging the carcass under refrigerated temperatures (157), a process where endogenous enzymes degrade the proteins reducing the strength of the myofibrillar structure (215); however, this process can be costly to the processors due to carcass weight loss, microbial spoilage, energy and space required to maintain the refrigerated conditions of the meat (72). Some additional processes used to increase tenderization of meat cuts include muscle stretching, shock-wave pressure, dry and wet aging, and enzymatic treatment with proteases. Tenderness of a product can be assessed usually by measuring the relative shear force of the meat or by sensory evaluation with a trained panel (62). Needle tenderization Needle tenderization is the process in which the meat is penetrated by very sharp blades without stretching or tearing apart the muscle fibers of the meat. The product is usually placed on a conveyor belt system, and as the 32 Texas Tech University, Alejandro Echeverry, December 2007 product moves down the line, it is penetrated with the blades. The use of this technique is a common practice to improve the quality of whole muscle cuts, such as chucks, ribs, tenderloins, and strip loins. Other terms used in the industry to refer to this process include needling or Jaccarding (name used after the first machine was invented, designed and built by Jaccard in New York) (41). The process of needle tenderization varies with the manufacturer and specifications required by the processor. Needles can vary in size, thickness, and number of blades per square cm, depending on the appearance and acceptability required by the processor and the consumer; and the number of passes through the blades can vary depending on the conveyors speed. In one of the earliest studies on this process, the effects of mechanically tenderize blade size on quality properties were tested (62-64). The authors reported that there werent any differences on cooking loss or thawing loss percentage due to mechanical tenderization or blade size. No differences in flavor, juiciness and Warner Bratzler shear forces due to the treatments were found; however, the process greatly improved the tenderness ratings (P < 0.05) of the treated steaks when compared to the controls. In another study, authors also observed that mechanically tenderized meat was more tender than untreated controls (42). Additionally, the authors reported that there was not an increase in tenderness when using slower conveyor speeds, even though these resulted in more needle punctures in the meat. In another study, the authors found significant cooking losses in mechanically tenderized loin steaks when compared to the controls; 33 Texas Tech University, Alejandro Echeverry, December 2007 however, a taste panel found no differences in juiciness and tenderness between treated and non-treated steaks, suggesting that this process offers no advantages when using meat that is already of acceptable tenderness. Microbial Issues of Meat Tenderization Sensory and quality attributes of tenderized meat have been studied extensively by many authors before; however, the microbiological aspects of this process have not received much attention until very recently. It is generally accepted that bacteria associated with meat are derived from the ingesta, the environment and the instruments used in the fabrication of the carcass, occurring only in the surface of the meat (62). The internal muscles and deep tissues of the carcass are sterile unless they are subjected to a considerable breakdown of the connective tissue structure and muscle fibers. Similarly, during the process of carcass fabrication, needle tenderization introduces bacteria into the deep tissues of the subprimals (104-106), which can become a problem if the meat is undercooked. In an early study, it was determined that passing a roast through the tenderizer for four times increased significantly (P < 0.05) the number of aerobic and anaerobic bacteria when compared to those samples subjected to only one or two passes or to untenderized controls (42). In another study, raw, lean beef rounds inoculated with Salmonella were tested after use of needle tenderization. In this study, authors reported that despite decontamination with boiling water for 20 minutes, core samples still contained Salmonella (around one cell per 400 34 Texas Tech University, Alejandro Echeverry, December 2007 grams) (133). Additionally, when dipping the inoculated roasts in a 50 ppm chlorine solution for 30 seconds and then subject them to tenderization, no reduction in the levels of Salmonella on the surface was observed. In 1979 two additional studies also analyzed microbial levels in needle tenderized meat. In the first one by Petersohn and others (191), the authors mechanically tenderized boneless beef loins, vacuum packed them in a barrier film and analyzed them over a ten day period. Total aerobic, anaerobic, and psychrotrophic plate counts were obtained from the surface and internal parts of the steaks. The authors reported that none of the total aerobic plate counts for tenderized or non-tenderized beef steaks differed significantly on any of the five sampling days (0, 1, 2, 5, and 10 days of storage); however, tenderized samples had consistently higher aerobic microbial counts than controls. In addition, tenderized samples had significantly higher surface psychrotrophic counts than controls on day 2 and 5 of storage. When the interior of the meat was analyzed, the authors reported that the total aerobic, anaerobic and psychrotrophic bacterial counts for tenderized and nontenderized samples were not significantly different on any of the sampling days, but in general, tenderized samples presented higher numbers when compared to the controls. In a second study, Raccach and others (199) analyzed the bacterial quality of needle tenderized, electrically stimulated beef. The authors reported non-significant differences in the amount of aerobic bacteria that were recovered from the interior of the tenderized and non-tenderized rounds. The authors 35 Texas Tech University, Alejandro Echeverry, December 2007 pointed out that when using needle tenderization (an inoculating machine), good sanitary and environmental conditions clearly played a role in the low number of bacteria that was recovered from the meat; and that there was always the possibility of microbial proliferation in the meat if these aspects are not controlled. Other authors have recently focused on the microbial conditions of different types of mechanically tenderized meat. In one study, samples from tenderized meat (surface and internal) obtained from four different retail stores (A, B, C, and D) were analyzed for aerobes, coliforms, E. coli, and organisms that formed black or grey colonies in HarlequinTM agar (a medium for the recovery of Listeria) (101). For all retail stores, counts were recovered from the surfaces of samples with means ranging between 3.5 to 4.0 log10 cfu/cm2 (aerobic); < 0.0 to 2.0 log10 cfu/25cm2 (coliforms), and < 0.0 to 0.5 log10 cfu/cm2. For the interior of the samples, the counts varied greatly from store to store, with reported counts ranging from 1.5 to 2.8 log10 cfu/gr for aerobic microorganism and 0.3 log10 cfu/ 25 gr for E. coli, respectively. In an E. coli O157:H7 risk assessment for blade-tenderized beef conducted at Kansas State University, beef top sirloin subprimals were inoculated with high levels of the pathogen (106 cfu/cm2) and subjected to one pass through a needle tenderization unit (193). After evaluation of core samples, the needle tenderization process resulted in about 3.0 logs of the pathogen being translocated into the deep tissues (6 cm from the surface). Samples inoculated at 36 Texas Tech University, Alejandro Echeverry, December 2007 low levels also resulted in a similar trend, with approximately 1.8 logs of the pathogen being transferred into the center of the meat cut. When determining adequate cooking temperatures for the steaks using an oven, the authors also reported that internal temperatures of 140F and higher were needed to eliminate E. coli O157:H7 by broiling. In another study conducted by Gill and others (103), the microbiological conditions of the surface and deep tissues of beef mechanically tenderized at a packing plant were determined. The authors reported that the tenderizing process did not significantly the numbers of bacteria (aerobes, coliforms and E. coli) on the surfaces of striploins and that none of them were recovered from the deep tissues of treated cuts. When these results are compared to those obtained by Gill and others (101), the results of the packing plant study revealed that the surface counts at retail stores were 2.0 log10 units more than those obtained in the plant. The authors suggested that not only storage was a factor on the high surface numbers obtained at retail stores, but that the cleanliness of the tenderizing equipment at the packing plant was a major aspect affecting the numbers of bacteria recovered from deep tissues. Other studies have also confirmed that the numbers of bacteria recovered from deep tissues of needle tenderized meat are significantly affected by the number of bacteria in the surface and the penetration depth but not by the number of incising events (passes) to which the meat is subjected (102). 37 Texas Tech University, Alejandro Echeverry, December 2007 Meat Enhancement Enhancement is a process used by the industry to improve the quality, tenderness and juiciness by injecting a solution into the deep tissues of the meat. This process can be used to 1) incorporate curing agents (salt, nitrites, ascorbates and seasonings) throughout the entire product; 2) improve flavor by the use of spices (deep marination) (36, 54, 174); or 3) to improve the water holding capacity and tenderness of the product by introducing binding agents (salt, phosphates, dextrose, proteins), acids and proteolytic enzymes (papain, bromelin) as part of the formulation (8, 99, 149, 164, 181, 218, 232, 241, 243). Other additional terms used in the industry to describe this process are injection, deep basting, and "pump marinating". At the retail level, products that have been subjected to this process must declare the percentage of injection and must be labeled as moisture added. There are two main methods used to pump the brine to the predetermined percentage rate desired by the producer. In the first one, also known as stitch pumping, a hollow needle is manually used to penetrate the meat in different parts and to pump the solution to the desired level (3). One of the major problems when using this process is that when not doing it properly, parts of the tissue might not be reached with the brine, failing to diffuse it completely through the muscle. The second method to pump the brine uses a multiple injector, a variation of the first one, where several needles (with holes on them through which brine can come out) are used simultaneously by automatic action. The 38 Texas Tech University, Alejandro Echeverry, December 2007 needle marks are usually visible in the surface of raw meat, but not noticeable once it has been cooked. Effects of this process on the quality attributes of meat have been reported extensively. In one study, pork loins pumped to 110 % of original weight with a solution containing salt (5.5%) and sodium tripolyphosphate (3.3%) were compared to non-enhanced control loins (117). After sensory analysis, the authors reported that the pork chops enhanced with salt and phosphates had a significantly increased tenderness, juiciness, and a higher overall flavor rating when compared to the controls. In another study, enhanced beef strip steaks (108%) were tested for sensory and quality attributes before and after aging under refrigerated conditions after injection with a brine containing alkaline phosphate, salt, and natural flavors (247). The authors reported that enhanced samples had higher tenderness and juiciness scores than controls; and tenderness was significantly higher when the process was performed before the aging period. Enhanced meat had significant lower Warner-Bratzler shear force values (P < 0.05) when compared to non-enhanced samples by day 7 confirming greater tenderness in treated samples. Microbial Issues of Enhanced Meat As well as with needle tenderization, injection of meats can pose the risk of translocating microbial flora and pathogens that are in the surface of the meat 39 Texas Tech University, Alejandro Echeverry, December 2007 into sterile deep tissues of the cut. Some studies have tested survival of different pathogens in the brine, a solution that is usually recirculated and that if contaminated can subsequently inoculate additional cuts; however, just a few studies have focused on the surface-deep tissues translocation levels that can occur while enhancing meat products. Introduction of pathogenic microorganisms into the deep tissues of meat can result in a shorter shelf life and an increase of the risk of foodborne illness (132). In a study conducted in Canada, the brine used to pump moistureenhanced pork was microbiologically analyzed for up to 2.5 hours after recirculation (111). The authors reported significant increases in the numbers of bacteria obtained from the brine after 1.75 hours of recirculation. After 2.5 hours of recirculation, the reported log CFU/ml counts were 4.50 (total plate count), 2.99 (lactic acid bacteria), 3.95 (pseudomonas), 2.79 (B. thermosphacta) and 3.01 (enterics); indicating that these solutions can harbor significant numbers of spoilage bacteria and can be distributed easily in the meat. In a recent study, the impact of a commercial injection process in the microbial flora of pork loins was studied (40). The authors reported that moistureenhanced loin samples (comprised of both surface and subsurface muscle) presented significantly larger (P < 0.05) numbers of total psychrotrophic bacteria, pseudomonas, lactic acid bacteria, and Enterobacteriaceae when compared with noninjected loins. In a similar study, aerobic bacteria recovered from recirculated 40 Texas Tech University, Alejandro Echeverry, December 2007 brine were >3.5 log10 units more than those obtained from the preparation tank after 30 minutes of processing (100). Similarly, other authors have tested the survival of pathogens in the brine and its effects on enhanced products. In one study, brine used to enhance eye of round primal cuts was inoculated with cultures of Listeria innocua, and portions of meat and brine analyzed after injection (107). The authors reported that the levels of this pathogen in the meat were about 0.72 log10 units less than those obtained in the brine. The authors also suggested that factors such as pumping pressure and number of strokes per minute can also affect the amount of brine (and therefore, pathogens) retained by the meat. Additionally, the authors suggested that if a meat product is subjected to both needle tenderization and injection with brine, the enhancement process must be performed prior to the tenderization process to reduce the levels of possible contamination retained by the meat. In a study conducted at Colorado State University, decontamination methods for E. coli O157:H7 were tested on beef subprimal cuts intended for moisture enhancement. Inoculated meat cuts were treated with hot water, lactic acid, and activated lactoferrin among other interventions and then injected with a brine solution containing 0.5 % sodium chloride, 0.25% sodium tripolyphosphate and 2.5 % sodium lactate (119). The authors reported that treatment of the meat cuts with the interventions resulted in 0.9 to 1.1 log10 cfu/100 cm2 reduction (a significant reduction when compared to the control samples); however, no 41 Texas Tech University, Alejandro Echeverry, December 2007 significant differences among treatments were found. When internal swab surfaces were analyzed, the process resulted in <1.05% cfu/cm2 of surface pathogen transferred into the meat. Disease Outbreaks related to needle tenderized/enhanced meat In October 1994 under the Federal Meat Inspection Act (FMIA), the Food Safety and Inspection Service of the U.S. Department of Agriculture (FSISUSDA) declared E. coli O157:H7 to be an adulterant in raw ground beef. According to the FSIS, an adulterated product is any food that contains any poisonous or deleterious substance which may render it injurious to health (93, 94). This decision occurred in response to a multi-state outbreak due to consumption of contaminated beef patties, which resulted in 400 illnesses and four deaths (31). Afterwards, the FSIS established new provisions for all meat and poultry plants requiring the mandatory implementation of a Hazard Analysis and Critical Control Point (HACCP) system in order to identify risky and potentially hazardous practices that account for microbial contamination. According to the FSIS, a non-intact beef product is defined as ground beef; beef that has been injected with solutions; beef that has been mechanically tenderized by needling, cubing, Frenching, or pounding devices; and beef that has been reconstructed into formed entrees (92). In May 2005, FSIS-USDA published notice that establishments who produce mechanically tenderized beef were required to reassess their HACCP plans because recent outbreaks 42 Texas Tech University, Alejandro Echeverry, December 2007 indicated that E. coli O157:H7 was a hazard reasonably likely to occur in these type of products . Even though meat marination and moisture enhanced meat are different processes, they have similarities to meat tenderization and can also pose a health risk if contamination is not addressed properly. As Johnston and others previously predicted (133), undercooking of contaminated meat products subjected to these processes were likely to have played a major role in the occurrence of these outbreak episodes. A summary of the outbreaks that prompted the previous actions are explained below: Outbreak 1 The first outbreak episode identified by the FSIS occurred in Michigan in August 2000 (92). After laboratory analysis with pulsed field gel electrophoresis (PFGE), a technique used to determine the relatedness of bacteria, the Michigan Department of Community Health (MDCH) identified two human E. coli O157:H7 strains with matching patterns. After epidemiological studies, consumption of steaks prepared to rare-medium degree of doneness was identified as the possible cause of the episode. This outbreak did not end in a product recall but resulted in the steak supplier implementing changes in the sanitizing procedures used in their tenderizer machine. Additionally, the supplier implemented an E. coli O157:H7 testing program for the beef intended for tenderization. 43 Texas Tech University, Alejandro Echeverry, December 2007 Outbreak 2 A second outbreak involved products produced by company A between March 17 and March 22, 2003 in Chicago, Illinois and sold by door-to-door vendors. After epidemiological studies, eleven cases of E. coli O157:H7 infections were identified by state health departments from Minnesota, Michigan, Kansas, Iowa, and North Dakota (92). All cases were strongly linked to tenderized steaks injected with a marinade solution (through multiple passes), a process that likely transferred the pathogen from the surface to the interior of the steaks. Establishment A voluntary recalled 739,000 pounds of frozen beef product and implemented changes in their standard sanitation operation procedures by dismantling, washing and sanitizing the equipment that were used in these processes on a daily basis instead of once per week (92, 145). Outbreak 3 The third outbreak linked to tenderized meat occurred in Denver, Colorado in August 2004. After microbial analysis with PFGE, the Colorado Department of Public Health and Environment confirmed four cases of human infection with E. coli O157:H7. An epidemiological case control study determined that consumption of tenderized, marinated beef steaks was the only related product ate by those infected (92). These findings resulted in the recall of approximately 406,000 pounds of frozen beef products produced on June 23, 2004 by establishment B located in Bolingbrook, Illinois. 44 Texas Tech University, Alejandro Echeverry, December 2007 Conclusions As observed from previous studies and the reported outbreaks (92, 103, 145, 199, 225), the processes of needle tenderization, blade tenderization, moisture enhancement, and marinade injection have the potential of transferring microbial flora and pathogens from the surface of meat cuts into the deep tissues of meat. The use of organic acids and other killing steps have been validated to reduce pathogen loads on beef carcasses, trim and ground beef products; however, there is a lack of data of the potential uses of these interventions of non-intact beef products. The objective of this study was to validate the effectiveness of using interventions as control measures to reduce E. coli O157:H7 and Salmonella Typhimurium DT 104 in two different types of nonintact beef products (needle tenderized and moisture enhanced steaks). Additionally, the use of the interventions was validated under two different application industrial contexts (Packer and Purveyor settings). The data obtained from this study has the potential to benefit the industry by 1) providing scientific evidence that can be used to reassess the HACCP plans as mandated by the FSIS (92), and 2) validate the use of current interventions as effective methods to reduce the pathogen load in meat intended for non-intact beef products. 45 Texas Tech University, Alejandro Echeverry, December 2007 Literature Cited 1. (WHO), W. H. O. 2004. The World Health Report 2004. In WHO. 2. Aberle, E. D., J. C. Forrest, D. E. Gerrard, and E. W. Mills. 2001. Palatability and cookery of meat. p. 233-246. 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Marcel Dekker, Inc., New York, NY. 69 Texas Tech University, Alejandro Echeverry, December 2007 CHAPTER II VALIDATION OF INTERVENTION STRATEGIES TO CONTROL ESCHERICHIA COLI O157:H7 IN NEEDLE TENDERIZED AND INJECTED BEEF PROCESSED UNDER PACKER AND PURVEYOR SETTINGS Introduction Escherichia coli O157:H7 is a foodborne pathogen capable of causing high morbidity and mortality numbers among humans that become infected. Gastrointestinal illness related to infections with this pathogen range from asymptomatic conditions, cramps, and vomiting, to mild bloody diarrhea and even more severe outcomes. In the United States, infection with this pathogen is considered as the major cause of hemorrhagic colitis and hemolytic uremic syndrome (18, 21). It is estimated that infections with this bacteria causes more than 73,000 illnesses and 61 deaths each year in the United States (19), with many of the outbreaks linked to the consumption of beef and beef products. In 1994 under the Federal Meat Inspection Act (FMIA) the Food Safety and Inspection Service of the U.S. Department of Agriculture (FSIS-USDA) declared E. coli O157:H7 to be an adulterant in raw ground beef, meaning that its presence on this product contains any poisonous or deleterious substance which may render it injurious to health(12). The decision to make E. coli O157:H7 an adulterant occurred after a multi-state outbreak between December 1992 and January 1993, when more than 700 people were infected and 4 70 Texas Tech University, Alejandro Echeverry, December 2007 children died after consumption of undercooked hamburgers from different restaurants of the same fast-food chain (2, 3). Recently in May 2005, the FSIS published notice that establishments who produce mechanically tenderized beef were required to reassess their HACCP plans because three recent outbreaks indicated that E. coli O157:H7 was a hazard reasonably likely to occur in these type of products. Processors of other type of non-intact beef products as defined by the FSIS (injected with marinades, moisture enhanced, mechanically tenderized by needling, cubing, Frenching, or pounding devices; and beef that has been reconstructed into formed entrees) also need to comply with the new regulations (11, 12, 17). Packer And Purveyor Settings In the U.S. the needle tenderization and injection process can occur at different points during the meat supply chain continuum. In a meat packing industry system (PA), a company usually handles the slaughtering of the animals and its further processing and distribution. After slaughtering, hide removal, evisceration and splitting, the carcasses are usually subjected to one (or to a combination of) intervention such as steam vacuum, hot water, organic acids, or steam pasteurization in order to reduce pathogen loads (6-9, 22). Many processors use a multiple hurdle approach to decrease significantly the microbial contamination and improve the safety of their products (1). Finally, carcasses/variety meats are chilled and stored under refrigerated conditions during an aging period (from 7 to 21 days) before shipping (Figure 1). Needle 71 Texas Tech University, Alejandro Echeverry, December 2007 Receiving Cattle / Holding Stunning/Holding Dehiding / Skinning / Head Removal Knife Trimming, steam vacuum, hot water carcass wash, acid wash and other interventions can be applied at one or multiple different steps throughout the flow Chilling Carcass Fabrication Carcass Shipping Primals Packaging / Labeling Packaging / Labeling Wash / Intervention Trim Rail Splitting Evisceration P A C K E R Cold Storage / Aging / Shipping Cold Storage / Aging / Shipping Receiving / Cold Storage/Aging P U R V E Y O R Receiving / Cold Storage/Aging Finished Product Fabrication Finished Product Fabrication Tenderization/Enhancement Potential Intervention Application Tenderization/Enhancement Cold Storage / Display / Retail Figure 1. Generic Slaughter and Fabrication Process Flow Diagram. 72 Texas Tech University, Alejandro Echeverry, December 2007 tenderization and/or injection of steaks and beef cuts can occur in the packing plant at this point in the process. Purveyors (PU) in the other hand include merchandisers, retailers, supermarket and restaurateurs that provide primal cuts and steaks to the consumers. Usually these cuts have been obtained previously from the packers after the aging period. At this point in the process meat is not subjected to any type of intervention; however, in the case of those cuts considered to be tough, they are subjected to needle tenderization and moisture enhancement (or both) to increase the tenderness and juiciness of the final product. Many times the USDA wants additional control measures to be added to the HACCP plan to prevent reoccurrence of microbial contamination. Currently, processors who produce mechanically tenderized beef products have very little control over their situation. This data will give them alternatives to control pathogens in the products they produce. Finally, this technology would help address current food safety and public health concerns by providing an added assurance of safety in mechanically tenderized or enhanced beef products. The objective of this study was to validate the use of different interventions to control E. coli O157:H7 in USDA choice strip loins (longissimus lumborum) steaks intended for either blade/needle tenderization or injection with brine solution under simulated packer and purveyor settings. Translocation of bacteria from the inoculated surface into the interior of the steaks was analyzed to 73 Texas Tech University, Alejandro Echeverry, December 2007 determine the potential microbial safety hazard to which consumers might be exposed when consuming these products. Materials And Methods Experimental Design and Analysis The experiment was a randomized complete block split split plot design with individual steaks defined as experimental units. Experiments were carried out in triplicate. Sample site within individual steaks (surface swab, top and core) were analyzed individually from the others. Process (EN and NT) as well as high and low inoculated samples were analyzed separately. For each set of treatments at high inoculation levels duplicate plates were obtained for each dilution at each sampling time and averages obtained. Average surface swabs counts were transformed into log10 cfu/cm2 while top and core counts were transformed into log10 cfu/g of meat, respectively in order to control and stabilize statistical variance and fulfill the requirements for normality prior to the analysis. Log10 counts were considered a dependent variable of interest; while process, treatment and sampling day were independent variables. All data were imported into a commercially available software package and analyzed using the mixed procedure (20). Comparisons of least square means were obtained. Low inoculation levels were recorded as categorical data (either negative or positive cultures) and analyzed using logistic regression techniques with the same 74 Texas Tech University, Alejandro Echeverry, December 2007 statistical package. In all cases replication, treatment and aging period were considered random effects. Microbiological Cultures Experiments involving high and low pathogen levels were conducted at separate occasions. A three-strain cocktail mixture of E. coli O157:H7 (strains 922, 944, and 966) (Texas Tech University Food Microbiology Laboratory Stock Collection, Lubbock, TX) were used to inoculate the meat subprimals. Frozen stock cultures were grown individually in trypticase soy broth (TSB) at 37 C for 24 h and passed three times prior to experimental use. The final cocktail concentration was approximately 5.76 x 108 cfu/ml (high inoculum level) and 2.58 x 103 cfu/ml (low inoculum level). Preparation Of Cocktail Cultures Three - 200 ml portions of TSB was prepared for each strain, after which cells were centrifuged and resuspended into 30 ml of TSB (20 % glycerin followed by freezing at -80 C) to create a concentrated culture. On the day of the experiment vials containing each of the pathogens were thawed at room temperature and transferred to 10 ml tubes of sterile tryptic soy broth. These tubes were transported to the pathogen processing facility where they were combined into 1000 ml of buffered peptone water (BPW) to form a cocktail used to inoculate the meat as described below. 75 Texas Tech University, Alejandro Echeverry, December 2007 Meat Preparation All the experiments were conducted in the in the Biosafety Level 2 pathogen processing facility in the Food Technology Building at Texas Tech University. This 750 ft2 facility allows validation and processing studies of food inoculated with pathogens under simulated industrial conditions. USDA select, boneless beef strip loins (longissimus lumborum) obtained from a commercial processor were transported to the pathogen laboratory, trimmed and fabricated for uniformity into subprimals measuring approximately 8 x 5 x 3 inches (steaks) (Figures 2A and 2B). A concentrated cocktail culture was prepared to facilitate inoculation of large quantities of the meat subprimals. Steaks were inoculated by dipping each of the subprimals into a sanitized container containing the pathogen with a buffer solution. Inoculated subprimals were placed on sterile stainless steel mesh racks and held at refrigerated temperatures for one hour to facilitate attachment before processing. After attachment, subprimals were randomly assigned to one of the industrial application settings, either packer (PA) or purveyor (PU). Immediately after assignment to either application the PA samples were carried from the pathogen labs cooler into the processing facility (room temperature) where they were treated with the interventions as described below; inoculated PU samples were vacuum packaged in high-barrier Cryovac bags and stored under refrigerated conditions during the aging process (14 or 21 days) prior to the use of the antimicrobials. 76 Texas Tech University, Alejandro Echeverry, December 2007 a e n r r Fig gure 2A. Strip Loins Tr rimming An nd Fa abrication of Subprimals. f Figu 2B. Det Of Fabr ure tail ricated Subprimals. 77 Texas Tech University, Alejandro Echeverry, December 2007 Microbial Challenge In the processing facility 6 randomly assigned subprimals were fed to an Intralox conveyor belt system (series 800, Intralox, Inc., Harahan, La.) (Figure 3) similar to those used in the meat industry and treated by spraying one of the antimicrobial interventions onto the surface as they moved down the belt. The following interventions were evaluated: 1) control (sterile distilled water; CTRL); 2) lactic acid bacteria (LAB); 3) 1000-1200 ppm acidified sodium chlorite (ASC); 4) 3% lactic acid (LA). This spraying process was performed at room temperature. Sprayed PA subprimals were collected at the end of the line on stainless mesh racks and packaged under vacuum in high-barrier Cryovac bags in a refrigerated room (Figure 4). PA samples were then held under refrigerated conditions for either 14 or 21 days; PU samples were not treated with the antimicrobials until the end of the aging period. In addition to the samples challenged with the antimicrobials, inoculated (INOC) subprimals not subjected to any intervention were also analyzed after aging and processing. Equipment Sanitation Prior to experimentation the pathogen processing laboratory was subjected to a full 3-day cleaning and sanitation process with a quaternary ammonium sanitizer (Bi-QuatTM, Birko Corp., Henderson, Colo.) of all walls, ceilings, processing equipments, racks and other utensils and instruments to 78 Texas Tech University, Alejandro Echeverry, December 2007 guarantee absence of any pathogens and background flora with the potential of misleading results. Figure 3. The Intralox Conveyor Belt System. 79 Texas Tech University, Alejandro Echeverry, December 2007 Figure 4. Vacuum Packaging Of Subprimals Under Refrigerated Conditions. Additionally, swab samples of the equipment were obtained between interventions to validate the cleaning and sanitation process (Figure 5). For quality assurance and once the subprimals were processed, the conveyor belt system and all equipment were cleaned with a commercial detergent and sanitized with a three-way quaternary ammonium disinfectant (AlaQuat, Birko Corp., Henderson, Colo.) between interventions within a replication followed by a rinse with hot water (150 -180 F) prior to the exposure of the subprimals to each of the interventions (Figure 6). Additionally, swab samples of the equipment were obtained between interventions to validate the sanitation process. 80 Texas Tech University, Alejandro Echeverry, December 2007 Figure 5. Cleaning and Sanitizing Process Of Pathogen Laboratory. 81 Texas Tech University, Alejandro Echeverry, December 2007 Figure 6. Cleaning And Sanitizing Of Conveyor Belt. To guarantee pureness of the sprayed solutions the conveyors tank was emptied between treatments and the system was operated with 1) hot water for 2 minutes followed by 2) sterile distilled water for one minute before refilling with the next intervention. Similarly, the multi-needle injector as well as the manual needle tenderizer were cleaned and sanitized following the previous procedure after processing of each of the subprimals. The brine used in the enhancement process was not recirculated (as occurs in the industry) in order to prevent cross 82 Texas Tech University, Alejandro Echeverry, December 2007 contamination of the subsamples with the potential of misleading microbial results. Processing of Subprimals: Needle Tenderization and Enhancement After the aging period under refrigerated conditions, PA and PU samples were transported to the pathogen laboratory for further processing. For each treatment at any given aging period and application, two subprimals were analyzed. Steaks were randomly assigned to one of the following process: 1) needle tenderization (NT) with a manual tenderizer (Jaccard Manual Tenderizer Model H, Orchard Park, NY) (Figures 7 - 9) or 2) enhancement with brine (EN) formulated to provide 0.3% sodium chloride and 0.3% sodium tripolyphosphate in the final product using a multi needle injector (Injectamatic Pi21 Automatic Brine Injector, Koch Equipment LLC, Kansas City, MO) (Figures 10 -12). Enhanced samples were pumped to approximately 110% of their original weight. After processing all subprimals were vacuum packaged under refrigerated condition, stored in a plastic cooler with ice packs, and transported directly to the Food Microbiology Laboratory in the Experimental Sciences Building at Texas Tech University and examined within 30 to 60 min after collection. Subprimals were analyzed for microbial counts on day 0, 14, and 21. 83 Texas Tech University, Alejandro Echeverry, December 2007 Figure 7. The Jaccard Manual Tenderizer Model H Figure 8. Manual Tenderization Of Subprimals 84 Texas Tech University, Alejandro Echeverry, December 2007 Figure 9. Steaks Surface After Tenderization (Detail). Figure 10. The Koch Injectamatic Pi21 Brine Injector. 85 Texas Tech University, Alejandro Echeverry, December 2007 Figure 11. Injection Process. Figure 12. Injection Process (Detail). 86 Texas Tech University, Alejandro Echeverry, December 2007 a e n r r Microbiological Analysis: High Ino oculation Levels e Samp ples held fo 0 days we transpo or ere orted to the food micro obiology lab b im mmediately after being vacuum p y g packaged. F each sa For ample, a 50 cm2 surfac 0 ce area was sw wabbed by u using a sterile cotton t and a ste tip erile templa (USDA -50 ate te emplate, Biotrace International) (Figure 13). The tip wa placed in a tube as n containing 9 milliliters o buffered peptone wa and se of ater erial dilution were ns performed. Figure 13 Inoculated Surface Swab. 3. 87 Texas Tech University, Alejandro Echeverry, December 2007 A100 l of each dilution were plated (duplicate plates for each dilution) using the thin agar layer method which allows injured cells (due to stress conditions such as acid environments or cold temperatures) to resuscitate and grew on the media while inhibiting other microorganisms and native background flora that can growth in the plates leading to overestimation of the pathogen (5, 16, 24, 25). Similarly, viable E. coli O157:H7 cells that were injured with the spray interventions and the refrigeration conditions might not be recovered adequately if plated directly onto selective media, an agar containing strong supplements and inhibitors, possibly resulting in underestimation of the numbers of E. coli O157:H7. Samples were plated on an overlay of MacConkey agar (approximately 7 ml) with two successive 7-ml layers of tryptic soy agar (total 14 ml) and incubated for 24 hours at 37 C. This method was chosen as the presence of native background flora can overgrowth in the plates leading to overestimation of the pathogen in the sample. After incubation, plates were counted using the Spiral Biotech Q CountTM (Version 2.0, Spiral Biotech, Norwood, MA). For both applications, PA and PU, previously treated samples that were vacuum packed and held under refrigeration for 14 or 21 days were subjected to either injection with brine (EN) or needle tenderization (NT) after the aging process. Microbiological surface analysis for these subprimals were performed similarly as described before on day 0. In addition to the surface counts, for each of the processed steaks another 3 sections were analyzed (top, A and B) (Figures 14 - 15): The external surface of each subprimal (top) was trimmed 88 Texas Tech University, Alejandro Echeverry, December 2007 Figure 14. Schematic of sections analyzed for each subsample Figure 15. Removal Of Sections Within The Steak. 89 Texas Tech University, Alejandro Echeverry, December 2007 approximately 0.25 inch deep using aseptic procedures before slicing to determine internalized pathogen loads on the product (13). For the remaining internal surfaces (A and B) meat was collected aseptically and evaluated to determine pathogen loads on the interior of the product. The subsections were placed in stomacher bags, the weights were recorded and 99 ml of buffered peptone water were added. Samples were then stomached for 2 minutes and serial dilutions were performed. Plating and other microbiological analysis was done similarly as explained before. Microbiological Analysis: Low Inoculation Levels In order to mimic the expected levels of contamination that could be faced by processors, steaks were inoculated at low E. coli O157:H7 levels. Subprimal steaks were then randomly assigned to one of the applications, treated with interventions, and held under refrigerated conditions in a similar way to those inoculated at high levels as described above. The use of rapid test kits for detection of pathogens is a convenient method used by processors to guarantee the safety of their products. To simulate microbiological techniques used in the food industry, an enzyme-linked immunosorbent assay (ELISA) test were used to detect the pathogen in the inoculated product. At time of analysis surface swabs were obtained from each subsample in order to enumerate the actual inoculation level; however, no results were obtained as they were below the detection level. In addition, approximately 90 Texas Tech University, Alejandro Echeverry, December 2007 25 grams of meat were obtained for each of the subsections within the steak (top, A and B) and analyzed using a rapid test kit specific for E. coli O157:H7 (Reveal, Neogen Co., Lansing, MI). Manufacturers directions were followed for this type of analysis and results recorded as positive/negative for the pathogen. Results Application Of Interventions At Packer Setting High Inoculation Levels Packer Setting Surface Counts Microbial surface counts on day 0 revealed that there were no significant differences in the levels of E. coli O157:H7 due to the use of interventions (P = 0.1116). However, all treated subsamples and the control (CTRL, LAB, ASC, and LA) had significant lower surface counts when compared to the inoculated, nontreated sample (INOC) (P < 0.05), indicating that the water and the interventions were effective in reducing the levels of microbial contamination between 0.57 to 0.96 log10 units. Although there was no statistical difference among treatments, samples treated with lactic acid exhibited the largest reduction at this point (Figure 16). On day 14 there were no significant intervention/process interactions for surface counts on either enhanced or needle tenderized samples (P = 0.1678). Only after analyzing each of the interventions individually a reduction (P < 0.05) is observed on NT samples that were subjected to LA when compared to those treated with LAB (Figure 17). 91 Texas Tech University, Alejandro Echeverry, December 2007 7.00 a 6.00 b 5.00 LAB INOC b b b CTRL Log 10 cfu/cm2 4.00 ASC 3.00 LA 2.00 1.00 Intervention Figure 16. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 0, PA). ab Means with different superscripts differ (P < 0.05). Samples were not significantly different after interventions (P = 0.1116) (Standard Error = 0.3923). For comparison purposes, inoculated but non treated samples are also included (INOC). Samples were inoculated and treated on day 0 and microbiologically analyzed on the same day 92 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a 5.00 b b b b a INOC CTRL b b b c LAB ASC log 10 cfu/cm2 4.00 3.00 LA 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 17. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 14, PA). Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.3647). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. abc 93 Texas Tech University, Alejandro Echeverry, December 2007 Surface samples on day 21 only showed significant reduction in only the NT samples treated with LA (P = 0.0160). A reduction on the pathogens surface count was also observed with the use of ASC (P = 0.0539) when compared to the control (Figure 18). Enhanced samples showed no differences among interventions; however, they were significantly lower when compared to both inoculated, non-treated (INOC) and control samples. Packer Setting Top Counts No TOP sections were enumerated on day 0 as the samples were not processed with NT or EN until after the end of the aging period. On day 14 subsection TOP didnt show an intervention/process interaction. Needle tenderized samples didnt show differences in the E. coli O157:H7 populations as a consequence of the interventions; however, all interventions resulted in significantly lower pathogen counts when compared to INOC and CTRL samples. Samples sprayed with LA and subjected to the enhancement process showed a significant reduction in the counts (P = 0.0302) when compared to non-treated steaks (Figure 19). On day 21 there were no intervention/process interactions (P = 0.624) for top subprimals. Samples subjected to injection with brine didnt show difference among treatments and control samples; however, they were significantly lower than INOC samples. In contrast, the top section of needle tenderized samples exhibited a significant reduction on microbiological counts on samples treated with ASC and LA (P < 0.05) (Figure 20). 94 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a 5.00 a b b b a b b b a INOC CTRL LAB ASC LA Log 10 cfu/cm2 4.00 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 18. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 21, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.5366). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 95 Texas Tech University, Alejandro Echeverry, December 2007 7.00 INOC a 6.00 ab b 5.00 b b 4.00 b ASC LA 3.00 a a b b CTRL LAB log 10 cfu/g 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 19. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 14, PA).. ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.3463). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed 96 Texas Tech University, Alejandro Echeverry, December 2007 7.00 a 6.00 b 5.00 b b b a a b bc c INOC CTRL LAB Log 10 cfu/g 4.00 ASC LA 3.00 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 20. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 21, PA). abc Means for each process with different superscripts differ (P < 0.05). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 97 Texas Tech University, Alejandro Echeverry, December 2007 Packer Setting Subsection A Counts No A subsections were enumerated on day 0 as the samples were not processed with needle tenderization or enhancement until after the end of the aging period. On day 14 Subsection A from EN samples showed lower counts (ca. 1.7 log10 units) on those samples subjected to LA when compared to the control (P = 0.0301). A smaller reduction (approximately 1.3 log10 cycle) was also observed on samples subjected to ASC but the difference was not significant (P = 0.1496) when compared to control. No differences on the pathogens counts were observed due to the interventions on subsection A for those samples subjected to needle tenderization (Figure 21); however, all treated samples were significantly lower than the INOC and the control. On day 21subsection A from subprimals subjected to Enhancement presented a statistical significant difference among interventions, with those samples treated with LA having the lowest counts (P < 0.05). No differences were observed in the levels of the pathogen between LAB and ASC interventions, even though these treatments also had lower counts than control samples. Needle Tenderized samples did not showed significant differences in E. coli O157:H7 counts on this subsection when treated with either LAB, ASC or LA (Figure 22). 98 Texas Tech University, Alejandro Echeverry, December 2007 4.00 a 3.50 3.00 b ab a a INOC CTRL LAB b bc ASC LA c b b Log 10 cfu/g 2.50 2.00 1.50 1.00 0.50 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 21. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 14, PA). abc Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.5046). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 99 Texas Tech University, Alejandro Echeverry, December 2007 5.00 a a a a INOC CTRL LAB ASC b b b b c b LA 4.00 Log 10 cfu/g 3.00 2.00 1.00 0.00 ENHANCEMENT Process NEEDLE TENDERIZATION Figure 22. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 21, PA). abc Means for each process with different superscripts differ (P < 0.05). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 100 Texas Tech University, Alejandro Echeverry, December 2007 Packer Setting Subsection B Counts No B subsections were enumerated on day 0 as the samples were not processed with needle tenderization or enhancement until after the end of the aging period. After analysis of enhanced and needle tenderized samples on day 14, subsection B did show significant differences on the pathogens levels when treated samples were compared to controls; however, there was no differences in the levels of pathogens due to interventions. Enhanced samples subjected to the interventions had counts that were up to 3.0 log10 units lower than the control or the inoculated samples. Needle tenderized samples presented a similar trend, with samples subjected to the interventions presenting counts that were between 2.0 to 2.5 log10 cfu/g lower than the control. The amount of bacteria recovered for treated samples were very similar for both processes, withj levels varying between 1.0 to 2.0 log10 cfu/g of meat (Figure 23). On day 21 EN samples showed significant lower counts on samples treated with LAB, ASC, and LA when compared to the control or the inoculated samples. Needle tenderized samples treated with LAB, ASC, and LA also presented significant lower pathogen levels (from 2.0 to 2.5 log10 units) for this subsection when compared to INOC samples or from 1.0 to 1.5 log10 units when compared to the controls. For both the enhanced and needle tenderized samples control were significantly higher than interventions (P < 0.05; Figure 24). 101 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a a a a INOC CTRL LAB 5.00 log 10 cfu/g 4.00 b b b b b b ASC LA 3.00 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 23. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 14, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.4944). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 102 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a 5.00 a 4.00 LAB a ASC 3.00 b b b LA b INOC a CTRL Log 10 cfu/g b 2.00 b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 24. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 21, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.7141). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement (EN) or needle tenderization after which microbiological samples were collected and analyzed. 103 Texas Tech University, Alejandro Echeverry, December 2007 Application Of Interventions at Purveyor Setting High Inoculation Levels Purveyor Setting Surface Counts Microbial surface counts on day 0 for PU were the same as the results obtained for the PA application (Figure 16). On day 14 enhanced Surface samples showed a reduction on the levels of E. coli O157:H7 contamination (Approximate 0.7 log10 units) when treated with LAB; however, this intervention is not considered significantly different from the CTRL or the other interventions. A similar trend was observed in those samples subjected to needle tenderization, where no differences between treatments and control were observed on day 14 Contamination levels of enhanced and samples needle tenderized beef ranged between 3.52 to 4.36 and 3.55 to 4.21 log10 units, respectively (Figure 25). On day 21 enhanced Surface samples showed a reduction on the levels of E. coli O157:H7 contamination (Approximate 0.7 logs) when treated with LAB; however, this intervention is not considered significantly different from the CTRL. A similar, but most notorious trend was observed in those samples subjected to needle tenderization (Figure 26). There was a significantly lower (P < 0.05) recovery of the pathogen in the surface of the subsamples that were sprayed with LAB when compared to the water control. The difference in this case was approximately 1.3 log10 cfu/cm2 between LAB and CTRL subsamples. 104 Texas Tech University, Alejandro Echeverry, December 2007 5.00 CTRL a a a a a a a LAB ASC LA 4.00 a log 10 cfu/cm2 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 25. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 14, PU). Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.4313). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. a 105 Texas Tech University, Alejandro Echeverry, December 2007 CTRL a 5.00 a Log 10 cfu/cm2 4.00 a a a a ab b LAB ASC LA 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 26. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in the surface of inoculated beef strip loins (Day 21, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.5747). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 106 Texas Tech University, Alejandro Echeverry, December 2007 Purveyor Setting Top Counts The outer top surfaces (TOP) of EN and NT samples at day 14 presented statistically significant reductions in the levels of E. coli O157:H7 after treatment with a LAB spray (P < 0.05). Enhanced samples treated with lactic acid bacteria had > 1.2 log10 units reduction in the top subsection when compared to the control; the reduction achieve by LAB in needle tenderized samples was > 0.70 log10 units (Figure 27). For both processes there was no difference in the E. coli O157:H7 contamination levels when after applying ASC or LA sprays. On day 21 the microbial counts of the top subsection were significantly lower on those samples treated with LAB and ASC and subjected to enhancement. Enhanced samples treated with ASC showed reductions of approximately 0.9 log10 units. In the top subsection needle tenderized samples presented significant reductions of the pathogen when sprayed with LAB and LA (P < 0.05), with reductions greater than 1.1 log10 units (Figure 28). Purveyor Setting Subsection A Counts For those samples subjected to enhancement on day 14, subsection A didnt exhibit any differences among treatments; however, there was a significant lower pathogen recovery on those subprimals that were treated with LAB. Similarly, needle tenderized samples showed similar microbial counts on this subsection, all of them with counts between 1.6 to 2.0 log10 units lower than controls treated only with water (Figure 29). 107 Texas Tech University, Alejandro Echeverry, December 2007 a 5.00 a a CTRL a b ab ab LAB ASC LA 4.00 Log 10 cfu/g b 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 27. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.3536). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 108 Texas Tech University, Alejandro Echeverry, December 2007 a 6.00 b 5.00 Log 10 cfu/g 4.00 3.00 2.00 1.00 ENHANCEMENT b ab CTRL a ab b b LAB ASC LA NEEDLE TENDERIZATION Figure 28. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection TOP of inoculated beef strip loins (Day 21, PU). Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.4177). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. ab 109 Texas Tech University, Alejandro Echeverry, December 2007 5.00 a a CTRL LAB ASC 4.00 Log 10 cfu/g 3.00 b 2.00 ab ab b b LA b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 29. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7635). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 110 Texas Tech University, Alejandro Echeverry, December 2007 On day 21 subsection A counts presented significant statistical differences among treatments (P < 0.05) for both processes. Enhanced samples subjected to ASC presented counts that were lower by more than 2.0 log10 units; needle tenderized samples subjected to LA also showed counts that were lower by > 2.6 log10 units when compared to the controls. Needle tenderized samples subjected to LAB also had significant lower counts than the controls by more than 1.7 log10 units (Figure 30). a 4.00 a a ab CTRL LAB 3.00 ab bc bc c ASC Log 10 cfu/g LA 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 30. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection A of inoculated beef strip loins (Day 21, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7065). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 111 Texas Tech University, Alejandro Echeverry, December 2007 Purveyor Setting Subsection B Counts For those samples subjected to enhancement on day 14, subsection B didnt exhibit any differences among the interventions; however, there was a significant lower pathogen recovery on those subprimals that were treated with ASC (P < 0.05). Similarly, needle tenderized samples showed significant lower recovery rates when comparing interventions and controls with > 2.0 log10 units differences. The largest reduction was observed in those samples treated with LAB (Figure 31). 5.00 a 4.00 ab Log 10 cfu/g a CTRL LAB ASC LA 3.00 ab b b 2.00 b bc 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 31. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.8614). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 112 Texas Tech University, Alejandro Echeverry, December 2007 In both enhancement and tenderized samples, the subsection B did present statistical significant differences among interventions on day 21. In NT samples the greatest reduction was observed with samples treated with ASC, with pathogen levels being > 1.8 log10 units lower than the controls. Similarly, needle tenderized samples subjected to LAB and LA showed the lowest contamination level in this subsection, with pathogen counts being > 2.2 log10 lower than the controls (Figure 32). 5.00 a a ab b b c b c CTRL LAB ASC LA 4.00 Log 10 cfu/g 3.00 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 32. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing E coli O157:H7 in subsection B of inoculated beef strip loins (Day 21, PU). abc Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7383). After the intervention, samples were immediately processed with either brine enhancement or needle tenderization. Microbiological samples were then collected and analyzed. 113 Texas Tech University, Alejandro Echeverry, December 2007 Application Of Interventions At Packer Setting Low Inoculation Levels For each process and treatment surface swabs and serial dilutions were obtained on every sampling day; however, no microbial counts were obtained from the plates as the E. coli O157:H7 populations were likely below the detection limit (<1.0 x 102 cfu/cm2). Detection with the ELISA test was performed for subsections TOP, A and B on day 14 and 21, and results recorded as positive/negative (1 or 0, respectively) for E. coli O157:H7. Needle Tenderized Samples CTRL (PA) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for CTRL samples after 21 days of aging under refrigerated conditions. ELISA results for CTRL samples subjected to needle tenderization can be observed on Table 1. Needle Tenderized Samples LAB (PA) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LAB samples after 21 days of aging under refrigerated conditions. ELISA results for LAB samples subjected to needle tenderization can be observed on Table 2. 114 Texas Tech University, Alejandro Echeverry, December 2007 Table 1. ELISA Results For E. coli O157:H7 in Needle Tenderized Control Samples (Packer) Controlling For ELISA=1 Intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling For ELISA=0 Intervention=CTRL Table 2. ELISA Results For E. coli O157:H7 in Needle Tenderized LAB Samples (Packer) Controlling For ELISA=1 Intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling For ELISA=0 Intervention=LAB Needle Tenderized Samples ASC (PA) After processing and microbial analysis there were no pathogens detected on any of the sampled locations within the subprimal on day 14. Similar results were 115 Texas Tech University, Alejandro Echeverry, December 2007 obtained for ASC samples after 21 days of aging under refrigerated conditions. ELISA results for ASC samples subjected to needle tenderization can be observed on Table 3. Table 3. ELISA Results For E. coli O157:H7 in Needle Tenderized ASC Samples (Packer) Controlling For ELISA=1 Intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling For ELISA=0 Intervention=ASC Needle Tenderized Samples LA (PA) After processing and microbial analysis there were no pathogens detected on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LA samples after 21 days of aging under refrigerated conditions. ELISA results for LA samples subjected to needle tenderization can be observed on Table 4. 116 Texas Tech University, Alejandro Echeverry, December 2007 Table 4. ELISA Results For E. coli O157:H7 in Needle Tenderized LA Samples (Packer) Controlling For ELISA=1 Intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling For ELISA=0 Intervention=LA Enhanced Samples CTRL (PA) After processing and microbial analysis, the E. coli O157:H7 was detected once on one of the samples (33.3%) on day 14 in the top subsection; however, there was no detection of the pathogen on either location A or B within the subprimal on this day. Similar results were obtained after 21 days of aging under refrigerated conditions, with only one sample testing positive for E. coli O157:H7 in the top subsection. ELISA results for CTRL samples subjected to enhancement can be observed on Table 5. 117 Texas Tech University, Alejandro Echeverry, December 2007 Table 5. ELISA Results For E. coli O157:H7 in Enhanced Control Samples (Packer) Controlling For ELISA=1 Intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 2 2 TOP 1 1 Location A 0 0 Location A 3 3 B 3 3 Total 8 8 B 0 0 Total 1 1 Controlling For ELISA=0 Intervention=CTRL Enhanced Samples LAB (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples (33%) on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. In contrast, after 21 days of aging under refrigerated conditions and processing one of the subsections (A) resulted in a positive result for the pathogen, even though the top (containing the inoculated surface) yielded negative results. The ELISA results for LAB samples subjected to enhancement can be observed on Table 6. Enhanced Samples ASC (PA) After processing and microbial analysis, the pathogen was detected once on one of the top and A subsamples, respectively on day 14. No detection of the pathogen occurred on either location B on this day. After 21 days of aging under 118 Texas Tech University, Alejandro Echeverry, December 2007 refrigerated conditions none of the subsections resulted in a positive result for the pathogen. The ELISA results for ASC samples subjected to enhancement can be observed on Table 7. Table 6. ELISA Results For E. coli O157:H7 in Enhanced LAB Samples (Packer) Controlling For Elisa=1 Intervention=LAB Day Frequency 14 21 Day Frequency 14 21 Top 2 3 Top 1 0 Location A 0 1 Location A 3 2 B 3 3 Total 8 8 B 0 0 Total 1 1 Controlling For Elisa=0 Intervention=LAB Table 7. ELISA Results For E. coli O157:H7 in Enhanced ASC Samples (Packer) Controlling For Elisa=1 Intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 1 0 Location A 2 3 B 3 3 Total 7 9 B 0 0 Total 2 0 Controlling For Elisa=0 Intervention=ASC 119 Texas Tech University, Alejandro Echeverry, December 2007 Enhanced Samples LA (PA) After processing and microbial analysis, the E. coli O157:H7 was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. After 21 days of aging under refrigerated conditions none of the subsections resulted in a positive result for the pathogen. The ELISA results for LA samples subjected to enhancement can be observed on Table 8. Table 8. ELISA Results For E. coli O157:H7 in Enhanced LA Samples (Packer) Controlling For ELISA=1 Intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling For ELISA=0 Intervention=LA Application Of Interventions At Purveyor Setting Low Inoculation Levels Needle Tenderized Samples CTRL (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for CTRL samples after 21 days of aging under refrigerated 120 Texas Tech University, Alejandro Echeverry, December 2007 conditions. ELISA results for CTRL samples subjected to needle tenderization can be observed on Table 9. Table 9. ELISA Results For E. coli O157:H7 in Needle Tenderized CTRL Samples (Purveyor) Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 intervention=CTRL Needle Tenderized Samples LAB (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LAB samples after 21 days of aging under refrigerated conditions. ELISA results for LAB samples subjected to needle tenderization can be observed on Table 10. Needle Tenderized Samples ASC (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for ASC samples after 21 days of aging under refrigerated 121 Texas Tech University, Alejandro Echeverry, December 2007 conditions. ELISA results for ASC samples subjected to needle tenderization can be observed on Table 11. Table 10. ELISA Results For E. coli O157:H7 in Needle Tenderized LAB Samples (Purveyor) Controlling for ELISA=1 Intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 Intervention=LAB Table 11. ELISA Results For E. coli O157:H7 in Needle Tenderized ASC Samples (Purveyor) Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 intervention=ASC 122 Texas Tech University, Alejandro Echeverry, December 2007 Needle Tenderized Samples LA (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LA samples after 21 days of aging under refrigerated conditions. ELISA results for LA samples subjected to needle tenderization can be observed on Table 12. Table 12. ELISA Results For E. coli O157:H7 in Needle Tenderized LA Samples (Purveyor) Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 intervention=LA Enhanced Samples CTRL (PU) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. After 21 days of aging under refrigerated conditions there was no detection of E. coli O157:H7 123 Texas Tech University, Alejandro Echeverry, December 2007 in any of the subsections within the sample. ELISA results for CTRL samples subjected to enhancement can be observed on Table 13. Table 13. ELISA Results For E. coli O157:H7 in Enhanced CTRL Samples (Purveyor) Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=CTRL Enhanced Samples LAB (PU) After processing and microbial analysis, the pathogen was detected once on one of the top, A, and B subsections within the subprimal, respectively (Day 14). However, after 21 days of aging under refrigerated conditions none of the subsections resulted in a positive result for the pathogen in the analyzed samples. The ELISA results for LAB samples subjected to enhancement can be observed on Table 14. 124 Texas Tech University, Alejandro Echeverry, December 2007 Table 14. ELISA Results For E. coli O157:H7 in Enhanced LAB Samples (Purveyor) Controlling for ELISA=1 intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 1 0 Location A 2 3 B 2 3 Total 6 9 B 1 0 Total 3 0 Controlling for ELISA=0 intervention=LAB Enhanced Samples ASC (PU) After processing and microbial analysis, the pathogen was detected once on one of the top, A, and B subsections within the subprimal, respectively (Day 14). However, after 21 days of aging under refrigerated conditions none of the subsections resulted in a positive result for the pathogen. The ELISA results for ASC samples subjected to enhancement can be observed on Table 15. Enhanced Samples LA (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LA samples after 21 days of aging under refrigerated conditions. ELISA results for LA samples subjected to needle tenderization can be observed on Table 16. 125 Texas Tech University, Alejandro Echeverry, December 2007 Table 15. ELISA Results For E. coli O157:H7 in Enhanced ASC Samples (Purveyor) Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 1 0 Location A 2 3 B 2 3 Total 6 9 B 1 0 Total 3 0 Controlling for ELISA=0 intervention=ASC Table 16. ELISA Results For E. coli O157:H7 in Enhanced LA Samples (Purveyor) Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 intervention=LA 126 Texas Tech University, Alejandro Echeverry, December 2007 Discussion According to the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) and the FSIS, an intact beef steak is defined as [a] cut of whole muscle(s) that has not been injected, mechanically tenderized, or reconstructed (10). Recently contaminated non-intact meat products have been implicated in several foodborne outbreaks and product recalls, prompting the FSIS to address the safety of these types of products (11, 17). Previous studies have characterized levels of contamination that can be internalized into the internal muscle of meats with either tenderization (23) or moisture enhancement process (4). Additional studies have analyzed samples that have been tenderized at the processing plant (15) or at the retail level (14, 15); however, they have focused on total aerobic counts, coliform counts, and other native meat microflora. In this study we characterized the translocation levels of Escherichia coli O157:H7 that occur from contaminated meat surfaces of strip loins into the interior of the muscle after processing with either needle tenderization (NT) or moisture enhancement (EN). Two different settings where the interventions can be applied were analyzed. In the packer setting (PA) application of interventions to the meats surface occur after the animal has been slaughtered and split. These samples are held under refrigerated conditions for an aging period (that varies according to producer and needed requirements) before the subprimals 127 Texas Tech University, Alejandro Echeverry, December 2007 are subjected to a process to increase the tenderness/juiciness of the final product. In the second setting, purveyor application (PU), the establishments receive the different meat cuts and proceed to process them in-house with either NT or EN (or both). We analyzed this situation as a potential point for application of the intervention to the subprimals. Surface High Inoculation results - Packer At high inoculation levels the effectiveness of the interventions varied according to the process (NT or EN) or the day of application. Based solely on the results obtained from the surface, the subprimals treated with the interventions and then subjected to enhancement yielded approximately 1.0 log10 units less by day 14 and 21 when compared to day 0 (Figure 33). At day 0 and 14 no differences were observed among treatments and controls; however, interventions resulted in statistical lower numbers (ca. 1 log10 cycle cfu/cm2) when compared to the inoculated, non-treated samples (INOC). On day 21the interventions resulted in lower counts when compared to INOC and CTRL samples. Based solely on the results obtained from the surface, the subprimals treated with the interventions followed by needle tenderization yielded approximately 0.6 log10 units less than inoculated samples on day 0; however, no differences were observed between interventions and control (Figure 34). By day 14 reported E. coli O157:H7 counts were approximately 1.0 log10 units less 128 Texas Tech University, Alejandro Echeverry, December 2007 6.00 5.00 a b b b b a b b b b a a b b INOC CTRL LAB b Log 10 cfu/cm2 4.00 3.00 ASC LA 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 33. Summary of surface E. coli O157:H7 counts on enhanced subprimals over time (Packer). ab Means for each day with different superscripts differ (P < 0.05). than inoculated samples on day 0, with the greatest reduction (ca. 1.4 log10 cfu/cm2 units) obtained in those subprimals treated with LA. On day 21 a similar trend was observed, with LA samples yielding the lowest pathogen count; however, there was no statistical difference between LA and the other interventions at this time (Figure 34). Surface High Inoculation Results - Purveyor Based solely on the results obtained from the surface, the subprimals treated with the interventions followed by enhancement did not present differences due to the treatments on day 0. By day 14 only those samples treated with LAB were significantly lower when compared to the controls (Figure 129 Texas Tech University, Alejandro Echeverry, December 2007 35). The same trend was also observed on day 21. On day 14 the samples treated with LAB presented pathogenic counts that were approximately 1.5 log10 units less than those obtained on day 0. Based solely on the results obtained from the surface, the subprimals treated with the interventions followed by needle tenderization did not present differences due to the treatments on day 0. By day 14 only those samples treated with LAB were significantly lower when compared to the controls (Figure 36). No significant differences were observed on day 14 due to the application of ASC or LA, The same trend was observed on day 21, with contamination level for LAB samples about 1.5 log10 cycle units less than the controls (about 2.0 log10 units if compared to the LAB levels on day 0). The LAB surface results of NT samples had a similar trend to those obtained with enhanced subprimals previously and that can be observed on Table 35. 130 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a b b b b a b b b c a b b b a INOC CTRL LAB ASC LA 5.00 log 10 cfu/g 4.00 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 34. Summary of surface E. coli O157:H7 counts on needle tenderized subprimals over time (Packer). abc Means for each day with different superscripts differ (P < 0.05) 131 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a a 5.00 a b a a b a a a ab a CTRL LAB ASC LA 4.00 log 10 cfu/cm2 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 35. Summary of surface E. coli O157:H7 counts on enhanced subprimals over time (Purveyor). ab Means for each day with different superscripts differ (P < 0.05) 132 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a 5.00 a a a a a 4.00 a b a a b CTRL LAB ASC LA log 10 cfu/cm2 b 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 36. Summary of surface E. coli O157:H7 counts on needle tenderized subprimals over time (Purveyor). ab Means for each day with different superscripts differ (P < 0.05) Top Subsection - High Inoculation Results (Packer) When Top subsamples were analyzed on day 14 and 21, a similar trend to those surface samples was observed. On day 14 EN samples did not present any difference among treatments, although a reduction of more than 1,5 log10 units was observed on LA samples when compared to INOC. On day 21 EN samples did not show differences among treatments. For those subprimals processed with NT there was no statistical difference among treatments; 133 Texas Tech University, Alejandro Echeverry, December 2007 however, they decrease the microbial counts by up to 1.0 log cycle on day 14. On day 21 NT samples treated with LA presented a significant reduction (P < 0.05) of more than 1.4 log10 units when compared to the CTRL (Figure 37). INOC 6.00 a ab b 5.00 b b 4.00 b b b b b a a a a b b b bc c a CTRL LAB ASC LA Log 10 cfu /g 3.00 2.00 1.00 0.00 EN 14 EN 21 NT 14 NT 21 Figure 37. Summary of TOP E. coli O157:H7 counts on EN and NT subprimals over time (Packer). abc Means for each day and process with different superscripts differ (P < 0.05). Top Subsection - High Inoculation Results (Purveyor) When Top subsamples (EN) were analyzed on day 14 only those samples treated with LAB presented significant E. coli O157:H7 reductions (>1.0 134 Texas Tech University, Alejandro Echeverry, December 2007 log10 units). On day 21 there was no difference among treatment on EN samples; however, the LAB and ASC interventions resulted in effective pathogen reductions. On day 14 NT samples did not present differences among interventions; however, LAB samples were significantly different from CTRL samples. On day 21 NT samples treated with LAB and LA presented a significant reduction (P < 0.05) of more than 1.0 log10 units when compared to the CTRL (Figure 38). a 6.00 ab a 5.00 a a b b a ab b b ab b b a a CTRL LAB ASC 4.00 LA Log 10 cfu/g 3.00 2.00 1.00 0.00 EN 14 EN 21 NT 14 NT 21 Figure 38. Summary of TOP E. coli O157:H7 counts on EN and NT subprimals over time (Purveyor). ab Means for each day and process with different superscripts differ (P < 0.05). 135 Texas Tech University, Alejandro Echeverry, December 2007 A And B Subsections High Inoculation Results (Packer And Purveyor) E. coli O157:H7 counts in subsections A and B resulted in mixed results in the degree of effectiveness. In the packer application for subsection A the contamination levels varied from < 1.00 log10 to 3.0 log10 units in enhanced samples (day 14) and from < 1.00 log10 to up to 4.0 log units on day 21. In subsection B of needle tenderized subprimals the E. coli O157:H7 contamination levels also varied dramatically, with pathogen recovery levels of up to 4.0 log10 cfu/g. In general, for both needle tenderized and enhanced subprimals, those samples subjected to LAB, ASC and LA resulted in lower contamination levels on subsections A and B when compared to the control or the INOC samples, in some cases by 1.0 to 3.0 log10 units. In the purveyor application for both subsections A and B obtained from EN and NT subprimals the E. coli O157:H7 varied from < 1.00 log (LA, ASC) to >4.0 log10 cfu/g in INOC and CTRL samples. Low Inoculation Results Packer Results At low inoculation levels the interventions used to spray the subprimals resulted very effective in eliminating E. coli O157:H7 from the surfaces. No positive ELISA test was obtained from samples subjected to needle tenderization on subsections top, A, or B (day 14 and 21). Data obtained for steaks subjected to enhancement resulted in one positive E. coli O157:H7 for each of the interventions; however, these results were obtained on the top subsections of the steaks and not in the A and B subsections within the subprimal (day 14). On day 136 Texas Tech University, Alejandro Echeverry, December 2007 21, only one positive was obtained in an enhanced sample treated with LAB on subsection B. These results could be an indication that 1) internalization of contamination into the internal muscle with needle tenderization did not occur in this experiment, or 2) if translocation occurred, the numbers were so small that not enough cells were present for the ELISA test to be positive even after incubation of the samples. Detection of translocated bacteria in these sections was dependent upon the ELISAs test detection limit. An additional explanation for negative A and B results might be that translocated E coli O157:H7 occurred into parts within the subsection that were not analyzed. As a reminder, only 25 grams were collected and analyzed for each subsection with the ELISA test, leaving plenty of muscle material out of the analysis where the pathogen (if present) could have been internalized with the process. Just because subsections A and B were negative for the pathogen it doesnt necessarily mean that the microorganism was absent of the interior of the muscle. In the case of the positive E. coli O157:H7 in subsection B of EN samples on day 21, it could have happened that the brine served as a vehicle facilitating the transportation of the pathogen to the interior of the muscle, confirming that even at low levels the translocation of bacteria still occurs. Purveyor Results At low inoculation levels, for all needle tenderized samples, all the interventions resulted very effective in eliminating E. coli O157:H7 from the surfaces and internal muscles. No positive ELISA test results were obtained 137 Texas Tech University, Alejandro Echeverry, December 2007 from samples subjected to needle tenderization on subsections top, A, or B on day 14 or 21. On the other hand, at this application setting, enhanced samples subjected to CTRL, LAB, and ASC resulted in one positive ELISA result on the top of the subprimal on day 14. Subsections A and B that were treated with LAB an ASC also resulted in one positive E. coli O157:H7 results; however, no positives were obtained for the CTRL subprimal (day 14). No positive E. coli O157:H7 results were obtained for any of the subsections on day 21. Only enhanced samples treated with LA were consistently negative for the pathogen for all the subsections (Day 14 and 21), indicating the effectiveness of its use in this application setting. We hypothesize that the variation on the contamination levels for each subsection was a function of how deep inside the muscle the needles and the blades cut throughout the meat. Microbial contamination is internalized in the muscle, but this doesnt occur at a constant rate. We observed higher microbial counts on the top subsection as expected, but in some cases the numbers on subsection A were not higher than those on subsection B, which was located closer to the conveyor belt. It could happen that when the blades and the needles pierce the meat all the way to the bottom (another external surface contaminated with the pathogen) they might carry some bacteria back in into subsections B and A (in that order). Other possible causes include carriage of the pathogens inside the meat using the brine as a vehicle through the pierced locations of the meat. 138 Texas Tech University, Alejandro Echeverry, December 2007 From the results obtained in this study it was found that the use of interventions such as LAB, ASC, and LA in inoculated subprimals was very effective in reducing the levels of E. coli O157:H7 contamination in the surface and internal muscle of meat after 14 and 21 day aging. Spraying the steaks with ASC and LA resulted in the lowest contamination levels when compared to the control spray. At low levels the interventions were generally effective; however, as they are not 100 % effective in eliminating the pathogen, some positives were obtained in the top, A, and B subsections. Still, processors should take notice and must try to adapt their process to implement interventions in subprimals intended for needle tenderization/ enhancement steaks. Acknowledgements This study was Funded by the Beef Checkoff. 139 Texas Tech University, Alejandro Echeverry, December 2007 Literature Cited 1. Bacon, R. T., K. E. Belk, J. N. Sofos, R. P. Clayton, J. O. Reagan, and G. C. Smith. 2000. Microbial populations on animal hides and beef carcasses at different stages of slaughter in plants employing multiple-sequential interventions for decontamination. J Food Prot. 63:1080-6. 2. Barrett, T. J., H. Lior, J. H. Green, R. Khakhria, J. G. Wells, B. P. Bell, K. D. Greene, J. Lewis, and P. M. Griffin. 1994. 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DL. 1997. - Emerging foodborne diseases. Emerg Infect Dis. 3:285-93. 141 Texas Tech University, Alejandro Echeverry, December 2007 22. Siragusa, G. R. 1995. The Effectiveness Of Carcass Decontamination Systems For Controlling The Presence Of Pathogens On The Surfaces Of Meat Animal Carcasses. Journal of Food Safety. 15:229-238. 23. Sporing, S. 1999. Escherichia coli O157:H7 risk assessment for production and cooking of blade tenderized beef steaks. p. vii, 102 leaves : ill. ; 28 cm. In, Food Science, vol. Master Thesis. Kansas State University, Manhattan, Kansas. 24. Wu, V. C., D. Y. Fung, D. H. Kang, and L. K. Thompson. 2001. Evaluation of thin agar layer method for recovery of acid-injured foodborne pathogens. J Food Prot. 64:1067-71. 25. Wu, V. C. H., D. Y. C. Fung, and D. H. Kang. 2001. Evaluation Of Thin Agar Layer Method For Recovery Of Cold-Injured Foodborne Pathogens. Journal of Rapid Methods and Automation in Microbiology. 9:11-25. 142 Texas Tech University, Alejandro Echeverry, December 2007 CHAPTER III VALIDATION OF INTERVENTION STRATEGIES TO CONTROL SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM DT 104 IN NEEDLE TENDERIZED AND INJECTED BEEF PROCESSED UNDER PACKER AND PURVEYOR SETTINGS Introduction Salmonella are an important cause of morbidity and mortality, with approximately 95 % of infections cases linked to consumption of contaminated foods. Data from the Center for Disease Control and Prevention estimates that foodborne illness due to Salmonella results in approximately 1.3 million infection cases and 553 deaths each year in the United States alone (18). The incidence of S. Typhimurium infections has decreased significantly since 1996 even though the overall estimated annual incidence of Salmonella infections has not (16), indicating that steps taken to control this pathogen in animals and food products have not been completely successful. One of the clones of this genus, Salmonella Typhimurium definitive phage type (DT) 104, is well known for being an important human and animal pathogen due to its multidrug resistance which prevents effective treatment of infections. For the sake of conciseness this serovar will be referred as Salmonella in this and following chapters. 143 Texas Tech University, Alejandro Echeverry, December 2007 In May 2005, the FSIS published notice that establishments who produce mechanically tenderized beef were required to reassess their HACCP plans because three recent outbreaks indicated that E. coli O157:H7 was a hazard reasonably likely to occur in these type of products. Processors of other type of non-intact beef products as defined by the FSIS are also required to comply with the new regulations (6, 7, 14). Even though the outbreaks in which this decision was based do not involve Salmonella, measures taken to reduce this pathogen at all stages of the meat production system might prove to be beneficial in decreasing contamination of meats with other pathogenic microorganism of public health concern (5). Needle tenderization, moisture enhancement and marination are technologies commonly used in the meat industry to increase the tenderness, juiciness and value of meat products (3, 4, 15, 21); however, their use have the potential to translocate contamination from the surface of the cut into the internal muscle by becoming an inoculating machine (19) as some studies have shown previously (9, 10, 12, 17). With this in mind, the objective of this study was to validate the use of different interventions to control Salmonella in USDA choice strip loins (longissimus lumborum) steaks intended for either blade/needle tenderization or injection with brine solution under simulated packer and purveyor settings. Translocation of bacteria from the inoculated surface into the interior of the 144 Texas Tech University, Alejandro Echeverry, December 2007 steaks was analyzed to determine the potential microbial safety hazard to which consumers might be exposed when consuming these products. Materials And Methods Experimental Design and Analysis The experiment was a randomized complete block split split plot design with individual steaks defined as experimental units. Experiments were carried out in triplicate. Sample site within individual steaks (surface swab, top and core) were analyzed individually from the others. Process (EN and NT) as well as high and low inoculated samples were analyzed separately. For each set of treatments at high inoculation levels duplicate plates were obtained for each dilution at each sampling time and averages obtained. Average surface swabs counts were transformed into log10 cfu/cm2 while top and core counts were transformed into log10 cfu/g of meat, respectively in order to control and stabilize statistical variance and fulfill the requirements for normality prior to the analysis. Log10 counts were considered a dependent variable of interest; while process, treatment and sampling day were independent variables. All data were imported into a commercially available software package and analyzed using the mixed models methodologies (20). Comparisons of least square means were obtained. Low inoculation levels were recorded as categorical data (either negative or 145 Texas Tech University, Alejandro Echeverry, December 2007 positive cultures) and analyzed using logistic regression techniques with the same statistical software. Microbiological Cultures Experiments involving high and low pathogen levels were conducted at separate occasions. A three strains cocktail mixture of Salmonella enterica serotype Typhimurium definitive phage type 104 (strains 205, 206, and 214) (Texas Tech University Food Microbiology Laboratory Stock Collection, Lubbock, TX) were used to inoculate the meat subprimals. Frozen stock cultures were grown individually in trypticase soy broth (TSB) at 37 C for 24 h and passed three times prior to experimental use. The final cocktail concentration was approximately 5.76 x 108 cfu/ml (high inoculum level) and 2.58 x 103 cfu/ml (low inoculum level). Preparation Of Cocktail Cultures Three - 200 ml portions of TSB was prepared for each strain, after which cells were centrifuged and resuspended into 30 ml of TSB (20 % glycerin followed by freezing at -80 C) to create a concentrated culture. On the day of the experiment vials containing each of the pathogens were thawed at room temperature and transferred to 10ml tubes of sterile tryptic soy broth. These tubes were transported to the pathogen processing facility where they were combined into 1000 ml of buffered peptone water (BPW) to form a cocktail used to inoculate the meat as described below. 146 Texas Tech University, Alejandro Echeverry, December 2007 Meat Preparation All the experiments were conducted in the in the Biosafety Level 2 pathogen processing facility in the Food Technology Building at Texas Tech University. USDA select, boneless beef strip loins (longissimus lumborum) obtained from a commercial processor were transported to the pathogen laboratory, trimmed and fabricated for uniformity into subprimals measuring approximately 8 x 5 x 3 inches (steaks). A concentrated cocktail culture was prepared to facilitate inoculation of large quantities of the meat subprimals. Steaks were inoculated by dipping each of the subprimals into a sanitized container containing the pathogen with a buffer solution. Inoculated subprimals were placed on sterile stainless steel mesh racks and held at refrigerated temperatures for one hour to facilitate attachment before processing. After attachment, subprimals were randomly assigned to one of the industrial application settings, either packer (PA) or purveyor (PU). Immediately after assignment to either application the PA samples were carried from the pathogen labs cooler into the processing facility (room temperature) where they were treated with the interventions as described below; inoculated PU samples were vacuum packaged in high-barrier Cryovac bags and stored under refrigerated conditions during the aging process (14 or 21 days) prior to the use of the antimicrobials. 147 Texas Tech University, Alejandro Echeverry, December 2007 Microbial Challenge In the processing facility 6 randomly assigned subprimals were fed to an Intralox conveyor belt system (series 800, Intralox, Inc., Harahan, La.) similar to those used in the meat industry and treated by spraying one of the antimicrobial interventions onto the surface as they moved down the belt. The following interventions were evaluated: 1) control (sterile distilled water; CTRL); 2) lactic acid bacteria (LAB); 3) 1000-1200 ppm acidified sodium chlorite (ASC); 4) 3% lactic acid (LA). This spraying process was performed at room temperature. Sprayed PA subprimals were collected at the end of the line on stainless mesh racks and packaged under vacuum in high-barrier Cryovac bags in a refrigerated room. PA samples were then held under refrigerated conditions for either 14 or 21 days; PU samples were not treated with the antimicrobials until the end of the aging period. In addition to the samples challenged with the antimicrobials, inoculated (INOC) subprimals not subjected to any intervention were also analyzed after aging and processing. Equipment Sanitation Prior to experimentation the pathogen processing laboratory was subjected to a full 3-day cleaning and sanitation process with a quaternary ammonium sanitizer (Bi-QuatTM, Birko Corp., Henderson, Colo.) of all walls, ceilings, processing equipments, racks and other utensils and instruments to guarantee absence of any pathogens and background flora with the potential of 148 Texas Tech University, Alejandro Echeverry, December 2007 misleading results. Additionally, swab samples of the equipment were obtained between interventions to validate the cleaning and sanitation process. For quality assurance and once the subprimals were processed, the conveyor belt system and all equipment were cleaned with a commercial detergent and sanitized with a three-way quaternary ammonium disinfectant (AlaQuat, Birko Corp., Henderson, Colo.) between interventions within a replication followed by a rinse with hot water (150 -180 F) prior to the exposure of the subprimals to each of the interventions. Additionally, swab samples of the equipment were obtained between interventions to validate the sanitation process. To guarantee pureness of the sprayed solutions the conveyors tank was emptied between treatments and the system was operated with 1) hot water for 2 minutes followed by 2) sterile distilled water for one minute before refilling with the next intervention. Similarly, the multi-needle injector as well as the manual needle tenderizer were cleaned and sanitized following the previous procedure after processing of each of the subprimals. The brine used in the enhancement process was not recirculated (as occurs in the industry) in order to prevent cross contamination of the subsamples with the potential of misleading microbial results. Processing of Subprimals: Needle Tenderization and Enhancement After the aging period under refrigerated conditions, PA and PU samples were transported to the pathogen laboratory for further processing. For each 149 Texas Tech University, Alejandro Echeverry, December 2007 treatment at any given aging period and application, two subprimals were analyzed. Steaks were randomly assigned to one of the following process: 1) needle tenderization (NT) with a manual tenderizer (Jaccard Manual Tenderizer Model H, Orchard Park, NY) or 2) enhancement with brine (EN) formulated to provide 0.3% sodium chloride and 0.3% sodium tripolyphosphate in the final product using a multi needle injector (Injectamatic Pi21 Automatic Brine Injector, Koch Equipment LLC, Kansas City, MO). Enhanced samples were pumped to approximately 110% of their original weight. After processing all subprimals were vacuum packaged under refrigerated condition, stored in a plastic cooler with ice packs, and transported directly to the Food Microbiology Laboratory in the Experimental Sciences Building at Texas Tech University and examined within 30 to 60 min after collection. Subprimals were analyzed for microbial counts on day 0, 14, and 21. Microbiological Analysis: High Inoculation Levels Samples held for 0 days were transported to the food microbiology lab immediately after being vacuum packaged. For each sample, a 50 cm2 surface area was swabbed by using a sterile cotton tip and a sterile template (USDA -50 template, Biotrace International). The tip was placed in a tube containing 9 milliliters of buffered peptone water and serial dilutions were performed. A100 l of each dilution were plated (duplicate plates for each dilution) using the thin agar layer method which allows injured cells (due to stress 150 Texas Tech University, Alejandro Echeverry, December 2007 conditions such as acid environments or cold temperatures) to resuscitate and grew on the media while inhibiting other microorganisms and native background flora that can growth in the plates leading to overestimation of the pathogen (2, 13, 24, 25). Similarly, viable Salmonella cells that were injured with the spray interventions and the refrigerated tempertature conditions might not be recovered adequately if plated directly onto selective media, an agar containing strong supplements and inhibitors, possibly resulting in underestimation of the numbers of the pathogen. Samples were plated on an overlay of xylose lysine decarboxylase (XLD) medium (approximately 7 ml) with two successive 7-ml layers of tryptic soy agar (total 14 ml) and incubated for 24 hours at 37 C. This method was chosen as the presence of native background flora can overgrowth in the plates leading to overestimation of the pathogen in the sample. After incubation, plates were counted using the Spiral Biotech Q CountTM (Version 2.0, Spiral Biotech, Norwood, MA). For both applications, PA and PU, previously treated samples that were vacuum packed and held under refrigeration for 14 or 21 days were subjected to either injection with brine (EN) or needle tenderization (NT) after the aging process. Microbiological surface analysis for these subprimals were performed similarly as described before on day 0. In addition to the surface counts, for each of the processed steaks another 3 sections were analyzed (top, A and B). The external surface of each subprimal (top) was trimmed approximately 0.25 inch deep using aseptic procedures before slicing to determine internalized 151 Texas Tech University, Alejandro Echeverry, December 2007 pathogen loads on the product (8). For the remaining internal surfaces (A and B) meat was collected aseptically and evaluated to determine pathogen loads on the interior of the product. The subsections were placed in stomacher bags, the weights were recorded and 99 ml of buffered peptone water were added. Samples were then stomached for 2 minutes and serial dilutions were performed. Plating and other microbiological analysis was done similarly as explained before. Microbiological Analysis: Low Inoculation Levels In order to mimic the expected levels of contamination that could be faced by processors, steaks were inoculated at low Salmonella levels. Subprimal steaks were then randomly assigned to one of the applications, treated with interventions, and held under refrigerated conditions in a similar way to those inoculated at high levels as described above. The use of rapid test methods for detection of pathogens is a convenient method used by processors to guarantee the safety of their products. To simulate microbiological techniques used in the food industry, an enzyme-linked immunosorbent assay (ELISA) test were used to detect the pathogen in the inoculated product. At time of analysis surface swabs were obtained from each subsample in order to enumerate the actual inoculation level; however, no results were obtained as they were below the detection limit. In addition, approximately 25 grams of meat were obtained for each of the subsections within the steak (top, A and B) and analyzed using a rapid test kit specific for Salmonella (Reveal, 152 Texas Tech University, Alejandro Echeverry, December 2007 Neogen Co., Lansing, MI). Manufacturers directions were followed for this type of analysis and results recorded as positive/negative for the pathogen. Results Application Of Interventions At Packer Setting High Inoculation Levels Packer Setting Surface Counts Microbial surface counts on day 0 revealed that there were no significant differences in the levels of Salmonella due to the use of interventions and when compared to the control samples. However, all treated subsamples (CTRL, LAB, ASC, and LA) had significant lower surface counts when compared to the inoculated, non-treated sample (INOC) (P < 0.05), indicating that the interventions were effective in reducing the initial levels of microbial contamination (Figure 39). On day 14 there was a statistically significant reduction on surface counts of both enhanced and needle tenderized samples. Enhanced samples treated with ASC showed the most significant Salmonella reduction by more than 1.0 log10 cfu/cm2 units; however, there were no differences among interventions. Needle tenderized samples treated with LAB, ASC and LA had significant lower Salmonella counts; however, there was no difference among treatments. Again, samples treated with ASC showed the largest reduction in this setting and process by exactly 2.0 log 10 cfu/cm2 units (Figure 40). 153 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a b b b b INOC CTRL LAB ASC 5.00 Surface log 10 cfu/cm2 4.00 LA 3.00 2.00 1.00 1 Figure 39. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 0, PA). ab Means with different superscripts differ (P < 0.05) (Standard error = 0.1651). Samples were inoculated and treated on day 0. Surface swabs were collected and microbiologically analyzed within two hours after collection. 154 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a INOC CTRL a 5.00 a a LAB ASC LA log 10 cfu/cm2 4.00 b b b b b b 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 40 Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 14, PA). Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.3850). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. ab 155 Texas Tech University, Alejandro Echeverry, December 2007 On day 21 surface counts presented significant statistical differences among treatments (P < 0.05) for both processes. Enhanced samples treated with LAB and LA presented lower counts when compared to the control by more than 1.0 log10 cycle; however, the use of ASC did not present significant differences. Needle tenderized samples presented almost an identical behavior when compared to the EN process: Significant reductions were achieve by the use of LAB and LA in the order of 1.0 log10 cfu/cm2 (Figure 41). 6.00 INOC CTRL a 5.00 a ab a a ab b b 3.00 b LAB ASC LA log 10 cfu/cm2 4.00 b 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 41. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 21, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.2486). At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 156 Texas Tech University, Alejandro Echeverry, December 2007 Packer Setting Top Counts On day 14, subsection top presented significant statistical differences among treatments (P < 0.05) for both the enhancement and needle tenderization processes (Figure 42). Enhanced samples treated with LAB and presented significant lower counts when compared to the control; however, the use of ASC 6.00 a 5.00 a b b a ab ab a a ab INOC CTRL LAB ASC LA log 10 cfu/gr 4.00 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 42. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 14, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.3083). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 157 Texas Tech University, Alejandro Echeverry, December 2007 and LA did not present significant reductions in the pathogen level. Needle tenderized samples presented almost an identical behavior when compared to the EN process: Significant reductions were achieved by the use of LAB by more than 1.0 log10 cfu/cm2 when compared to CTRL and INOC; however, no significant differences were found between the control and the other two interventions (ASC, LA). On day 21 there was a significant pathogen reduction in enhanced samples treated with LAB when compared to the control; however, no statistical differences were found among CTRL and ASC or CTRL and LA on this day. The Top samples subjected to enhancement injection presented a similar trend as EN samples, with only LAB treated samples presenting significant reductions in the levels of Salmonella (Figure 43). Packer Setting Subsection A Counts No A subsections were enumerated on day 0 as the samples were not processed with NT or EN until after the end of the aging period. On day 14 Subsection A obtained from EN samples showed significant lower counts (ca. 1.5 logs) on those samples subjected to LAB and LA when compared to the control. No differences between CTRL and ASC were found for this location. Samples subjected to needle tenderization and treated with LA presented a significant lower pathogen counts (greater than 2.0 log10 cfu/g units) when compared to the recovery levels obtained from control (Figure 44). 158 Texas Tech University, Alejandro Echeverry, December 2007 a 5.00 a a a a a a a INOC CTRL LAB b 4.00 b ASC LA log 10 cfu/g 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 43. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 21, PA). ab Means for each process with different superscripts differ (P < 0.05). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 159 Texas Tech University, Alejandro Echeverry, December 2007 5.00 a a a INOC CTRL LAB 4.00 a a ab a ASC LA Log 10 cfu/g 3.00 b 2.00 b b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 44. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 14, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7061). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. On day 21 subsection A from subprimals subjected to enhancement presented a statistical significant difference among treatments, with those samples treated with LA having the lowest counts (P < 0.05) by 2.0 log10 cfu/g 160 Texas Tech University, Alejandro Echeverry, December 2007 units when compared to the controls. Needle tenderized samples did not show significant differences in E. coli O157:H7 counts on this subsection when treated with either LAB, ASC or LA (Figure 45), even though reductions > 0.5 log10 cfu/g units were found in samples treated with LAB and LA . INOC CTRL 4.00 a a a a 3.00 a ab ab b a a LAB ASC LA Log 10 cfu/g 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 45. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 21, PA). abc Means for each process with different superscripts differ (P < 0.05)(Standard Error = 0.7310). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 161 Texas Tech University, Alejandro Echeverry, December 2007 Packer Setting Subsection B Counts No B subsections were enumerated on day 0 as the samples were not processed with NT or EN until after the end of the aging period. After analysis of enhanced samples a significant lower recovery (from 0.8 to 1.0 log10 cfu/g) was obtained from those samples subjected to the interventions (figure 46). On day 14, needle tenderized samples treated with LAB showed significant lower Salmonella counts when compared to the controls (>2.0 log10 cfu/g). Needle tenderized samples did not present significant reductions due to the application of ASC or LA in this subsection. On day 21 neither EN nor NT samples showed significant lower counts among treatments and control. Only when recovery rates for both processes are compared to their respective inoculated samples a difference in the pathogen levels (of up to 2.0 log10 units) can be observed (Figure 47). 162 Texas Tech University, Alejandro Echeverry, December 2007 a a 4.00 a INOC CTRL LAB ASC 3.00 LA a a ab Log 10 cfu/g 2.00 b b b b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 46. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 14, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.7106). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 163 Texas Tech University, Alejandro Echeverry, December 2007 a a INOC CTRL 3.00 b LAB ASC log 10 cfu/g b 2.00 b b b b b LA b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 47. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 21, PA). ab Means for each process with different superscripts differ (P < 0.05) (Standard Error = 0.5366). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed 164 Texas Tech University, Alejandro Echeverry, December 2007 Application Of Interventions at Purveyor Setting High Inoculation Levels Purveyor Setting Surface Counts Microbial surface counts on day 0 for PU were the same results obtained for the PA application. On day 14 enhanced Surface samples showed a significantly reduction on the levels of E. coli O157:H7 contamination (approximate 0.7 logs) when treated with LA; however, no differences were observed between this intervention and ASC and LA. Only surface counts on needle tenderized samples treated with LA showed a significant difference when compared to the controls (> 0.6 log10 cfu/cm2 units); however, no differences were observed between results obtained after treatment with LA and those samples treated with LAB and ASC (Figure 48). On day 21 there were no statistical differences among treatments in the amounts of Salmonella recovered from samples surfaces subjected to either EN or NT (Figure 49). Purveyor Setting Top Counts Enhanced samples did not show significant differences in Salmonella counts on this subsection when treated with LAB, ASC or LA. Similar recovery levels for the pathogen as well as for the treatment effects were observed in needle tenderized samples (Figure 50). 165 Texas Tech University, Alejandro Echeverry, December 2007 CTRL 5.00 a ab b a ab ab ab b LAB ASC LA log 10 cfu/cm2 4.00 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 48. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.2718). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 166 Texas Tech University, Alejandro Echeverry, December 2007 a 5.00 a a a a a a a CTRL LAB ASC LA log 10 cfu/cm2 4.00 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 49. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in the surface of inoculated beef strip loins (Day 21, PU). a Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.3954). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed 167 Texas Tech University, Alejandro Echeverry, December 2007 6.00 a 5.00 a a a a a a a CTRL LAB ASC 4.00 log 10 cfu/g LA 3.00 2.00 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 50. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.2690). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 168 Texas Tech University, Alejandro Echeverry, December 2007 On day 21 the microbial counts of the top subsection presented a similar trend to those results obtained on day 14: For both enhanced and needle tenderized samples no significant differences in the levels of Salmonella were obtained as a result of applying the interventions when compared to controls. For both processes the recovery levels ranged between 4.52 to 4.85 log10 cfu/g units (Figure 51). CTRL 5.00 a a a a a a a a LAB ASC LA log 10 cfu/g 4.00 3.00 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 51. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection TOP of inoculated beef strip loins (Day 21, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.1406). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 169 Texas Tech University, Alejandro Echeverry, December 2007 Purveyor Setting Subsection A Counts For those samples subjected to enhancement on day 14, subsection A presented significant differences between treatments and control. Samples subjected to LAB presented almost 3.0 log10 cfu /g lower counts to those subjected to distilled sterile water. Similarly, needle tenderized samples showed similar microbial counts on this subsection, all of them with counts between 2.16 to 2.75 log10 units lower than controls (Figure 52). a 5.00 a CTRL LAB ASC log 10 cfu/g 4.00 b b 3.00 b 2.00 b b b LA 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 52. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.8274). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 170 Texas Tech University, Alejandro Echeverry, December 2007 On day 21 subsection A counts presented significant statistical differences among treatments (P < 0.05) for both processes. Enhanced samples subjected to ASC presented counts that were lower by more than 2.5 log10 units; needle tenderized samples subjected to LA also showed counts that were lower by >2.70 log10 units when compared to the control. Needle tenderized samples subjected to LAB and LA also had statistically significant lower counts than the controls by more than 2.20 log10 units when compared to the control (Figure 53). Purveyor Setting Subsection B Counts For those samples subjected to enhancement on day 14, subsection B didnt exhibit any differences among treatments; however, all samples subjected to the interventions presented significantly lower Salmonella levels to those subjected to the control (P < 0.05). Needle tenderized samples treated with LAB showed significant lower recovery rates when compared to the control. No statistical differences in the levels of the pathogen were found among ASC, LA and CTRL (Figure 54). In both enhancement and tenderized samples, subsection B did present statistical significant differences among treatments on day 21. Enhanced samples presented recovery levels that were > 1.20 log10 cfu/g lower than the control. In needle tenderized samples the greatest reduction for this subsection was observed in those samples treated with LA, with pathogen levels being approximately 2.0 log10 units lower than the controls (Figure 55). 171 Texas Tech University, Alejandro Echeverry, December 2007 a 5.00 a CTRL LAB ASC Log 10 cfu/g 4.00 b b b LA 3.00 b b b 2.00 1.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 53. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection A of inoculated beef strip loins (Day 21, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7214). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 172 Texas Tech University, Alejandro Echeverry, December 2007 a a a ab CTRL LAB ASC LA 4.00 Log 10 cfu/g 3.00 b 2.00 b b b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 54. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 14, PU). ab Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.8614). Samples were inoculated and treated on day 0, and then aged under refrigeration for 14 days. At day 14 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 173 Texas Tech University, Alejandro Echeverry, December 2007 CTRL 4.00 a a LAB ASC 3.00 b ab ab b b LA Log 10 cfu/g 2.00 b 1.00 0.00 ENHANCEMENT NEEDLE TENDERIZATION Figure 55. Effects of water (CTRL), lactic acid bacteria (LAB), acidified sodium chlorite (ASC) and lactic acid (LA) in reducing Salmonella in subsection B of inoculated beef strip loins (Day 21, PU). abc Means for each process with different superscripts differ (P < 0.05) (Standard error = 0.7234). Samples were inoculated and treated on day 0, and then aged under refrigeration for 21 days. At day 21 samples were processed with either brine enhancement or needle tenderization after which microbiological samples were collected and analyzed. 174 Texas Tech University, Alejandro Echeverry, December 2007 Application Of Interventions At Packer Setting Low Inoculation Levels For each process and treatment surface swabs were obtained on every sampling day; however, no microbial counts were obtained as the Salmonella populations were likely below the detection limit (< 1.0 x 102 cfu/cm2). Detection with the ELISA test was performed for subsections top, A and B on day 14 and 21 and results recorded as positive (1) or negative (0) for the pathogen. Needle Tenderized Samples CTRL (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. After 21 days of aging under refrigerated conditions, two out of the three samples tested positive for Salmonella on the top subsection. None of the samples obtained from subsections A or B yielded positive results. ELISA results for CTRL samples subjected to needle tenderization can be observed on Table 17. Needle Tenderized Samples LAB (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There were no pathogens detected on either location A or B within the subprimal on day 14. On day 21 there was no detection of the pathogen on any of the sampled locations after treatment with LAB. ELISA results for LAB samples subjected to needle tenderization can be observed on Table 18. 175 Texas Tech University, Alejandro Echeverry, December 2007 Table 17. ELISA Results For Salmonella in Needle Tenderized Control Samples (Packer). Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 2 1 TOP 1 2 Location A 0 0 Location A 3 3 B 3 3 Total 8 7 B 0 0 Total 1 1 Controlling for ELISA=0 intervention=CTRL Table 18 . ELISA Results For Salmonella in Needle Tenderized LAB Samples (Packer). Controlling for ELISA=1 intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=LAB 176 Texas Tech University, Alejandro Echeverry, December 2007 Needle Tenderized Samples ASC (PA) After processing and microbial analysis, the pathogen was detected twice on the top subsection of samples treated with ASC (day 14); however, no positives were obtained on subsections A or B. On day 21 there were no detection of the pathogens on any of the sampled locations after treatment with ASC. ELISA results for ASC samples subjected to needle tenderization can be observed on Table 19. Table 19. ELISA Results For Salmonella in Needle Tenderized ASC Samples (Packer). Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 1 3 TOP 2 0 Location A 0 0 Location A 3 3 B 3 3 Total 7 9 B 0 0 Total 2 0 Controlling for ELISA=0 intervention=ASC Needle Tenderized Samples LA (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection) after treatment with LA spray. There was no detection of the pathogen on either location A or B within the subprimal on day 14. On day 21 there was no detection of the pathogen on any 177 Texas Tech University, Alejandro Echeverry, December 2007 of the sampled locations after treatment with this intervention. ELISA results for LA samples subjected to needle tenderization can be observed on Table 20. Table 20. ELISA Results For Salmonella in Needle Tenderized LA Samples (Packer). Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=LA Enhanced Samples CTRL (PA) After processing and microbial analysis, the pathogen was detected twice on the top subsection of control samples (day 14). There were no pathogens detected on either location A or B within the subprimal on day 14. After 21 days of aging under refrigerated conditions, two out of the three analyzed subsamples tested positive in the top subsection. Additionally, subsection A and B tested positive for Salmonella ssp once. ELISA results for CTRL samples subjected to brine enhancement can be observed on Table 21. 178 Texas Tech University, Alejandro Echeverry, December 2007 Table 21. ELISA Results For Salmonella in Enhanced Control Samples (Packer). Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 1 1 TOP 2 2 Location A 0 1 Location A 3 2 B 3 2 Total 7 5 B 0 1 Total 2 4 Controlling for ELISA=0 intervention=CTRL Enhanced Samples LAB (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection) after treatment with LA B. There was no detection of the pathogen on either location A or B within the subprimal on day 14. On day 21 there were no pathogens detected on any of the sampled locations after treatment with this intervention. ELISA results for LAB samples subjected to needle tenderization can be observed on Table 22. Enhanced Samples ASC (PA) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection) after treatment with ASC. There was no detection of the pathogen on either location A or B within the subprimal on day 14. Exactly the same results were obtained on day 21. The 179 Texas Tech University, Alejandro Echeverry, December 2007 ELISA results for ASC samples subjected to enhancement can be observed on Table 23. Table 22. ELISA Results For Salmonella in enhanced LAB Samples (Packer). Controlling for ELISA=1 intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=LAB Table 23. ELISA Results For Salmonella in Enhanced ASC Samples (Packer). Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 2 2 TOP 1 1 Location A 0 0 Location A 3 3 B 3 3 Total 8 8 B 0 0 Total 1 1 Controlling for ELISA=0 intervention=ASC 180 Texas Tech University, Alejandro Echeverry, December 2007 Enhanced Samples LA (PA) After processing and microbial analysis, Salmonella was detected once on one of the samples on day 14 (top subsection) after treatment with LA. There was no detection of the pathogen on either location A or B within the subprimal on day 14. On day 21 there was no detection of the pathogen on any of the sampled locations after treatment with this intervention. ELISA results for LA samples subjected to needle tenderization can be observed on Table 24. Table 24. ELISA Results For Salmonella in Enhanced LA Samples (Packer). Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 8 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=LA Application Of Interventions At Purveyor Setting Low Inoculation Levels Needle Tenderized Samples CTRL (PU) After microbial analysis of samples held under refrigerated conditions for 14 days, subsections top and A tested positive once for Salmonella . No positives were obtained for subsection B. After 21 days of aging under 181 Texas Tech University, Alejandro Echeverry, December 2007 refrigerated conditions, each one of the subsections tested positive once for the pathogen. ELISA results for CTRL samples subjected to needle tenderization can be observed on Table 25. Table 25. ELISA Results For Salmonella in Needle Tenderized CTRL Samples (Purveyor). Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 2 2 TOP 1 1 Location A 1 1 Location A 2 2 B 3 2 Total 7 6 B 0 1 Total 2 3 Controlling for ELISA=0 intervention=CTRL Needle Tenderized Samples LAB (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. After 21 days of refrigerated storage the pathogen was detected once on one of the top subsections treated with LAB. There was no detection of the pathogen on either location A or B within the subprimal on day 21. ELISA results for LAB samples subjected to needle tenderization can be observed on Table 26. 182 Texas Tech University, Alejandro Echeverry, December 2007 Table 26. ELISA Results For Salmonella in Needle Tenderized LAB Samples (Purveyor). Controlling for ELISA=1 intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 3 2 TOP 0 1 Location A 0 0 Location A 3 3 B 3 3 Total 9 8 B 0 0 Total 0 1 Controlling for ELISA=0 intervention=LAB Needle Tenderized Samples ASC (PU) After processing and microbial analysis, the pathogen was detected twice on the top subsection of ASC samples (day 14). There was no detection of the pathogen on either location A or B within the subprimal on day 14. After 21 days of aging under refrigerated conditions the same results were obtained for ASC samples. ELISA results for ASC samples subjected to needle tenderization can be observed on Table 27. Needle Tenderized Samples LA (PU) After microbial analysis of samples held under refrigerated conditions for 14 days, subsections top, A , and B tested positive once for Salmonella ; however, none of the subsections tested positive for this pathogen after 21 days of aging under refrigerated conditions. ELISA results for LA samples subjected to needle tenderization can be observed on Table 28. 183 Texas Tech University, Alejandro Echeverry, December 2007 Table 27. ELISA Results For Salmonella in Needle Tenderized ASC Samples (Purveyor). Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 1 1 TOP 2 2 Location A 0 0 Location A 3 3 B 3 3 Total 7 7 B 0 0 Total 2 2 Controlling for ELISA=0 intervention=ASC Table 28. ELISA Results For Salmonella in needle Tenderized LA Samples (Purveyor). Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 1 0 Location A 1 0 Location A 3 3 B 3 3 Total 9 9 B 1 0 Total 3 0 Controlling for ELISA=0 intervention=LA 184 Texas Tech University, Alejandro Echeverry, December 2007 Enhanced Samples CTRL (PU) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. Similar results were obtained after 21 days of aging under refrigerated conditions. ELISA results for CTRL samples subjected to enhancement can be observed on Table 29. Table 29. ELISA Results For Enhanced Salmonella in CTRL Samples (Purveyor). Controlling for ELISA=1 intervention=CTRL Day Frequency 14 21 Day Frequency 14 21 TOP 2 2 TOP 1 1 Location A 0 0 Location A 3 3 B 3 3 Total 8 8 B 0 0 Total 1 1 Controlling for ELISA=0 intervention=CTRL Enhanced Samples LAB (PU) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. Similar results were obtained after 21 days of aging under refrigerated conditions. ELISA results for LAB samples subjected to enhancement can be observed on Table 30. 185 Texas Tech University, Alejandro Echeverry, December 2007 Table 30. ELISA Results For Salmonella in Enhanced LAB Samples (Purveyor). Controlling for ELISA=1 intervention=LAB Day Frequency 14 21 Day Frequency 14 21 TOP 2 2 TOP 1 1 Location A 0 0 Location A 3 3 B 3 3 Total 8 8 B 0 0 Total 1 1 Controlling for ELISA=0 intervention=LAB Enhanced Samples ASC (PU) After processing and microbial analysis, the pathogen was detected once on one of the samples on day 14 (top subsection). There was no detection of the pathogen on either location A or B within the subprimal on day 14. On day 21 there was no detection of the pathogen on any of the sampled locations after treatment with ASC. ELISA results for ASC samples subjected to needle tenderization can be observed on Table 31. Enhanced Samples LA (PU) After processing and microbial analysis there was no detection of the pathogen on any of the sampled locations within the subprimal on day 14. Similar results were obtained for LA samples after 21 days of aging under refrigerated 186 Texas Tech University, Alejandro Echeverry, December 2007 conditions. ELISA results for LA samples subjected to needle tenderization can be observed on Table 32. Table 31. ELISA Results For Salmonella in Enhanced ASC Samples (Purveyor). Controlling for ELISA=1 intervention=ASC Day Frequency 14 21 Day Frequency 14 21 TOP 2 3 TOP 1 0 Location A 0 0 Location A 3 3 B 3 3 Total 6 9 B 0 0 Total 1 0 Controlling for ELISA=0 intervention=ASC Table 32. ELISA Results For Salmonella in Enhanced LA Samples (Purveyor). Controlling for ELISA=1 intervention=LA Day Frequency 14 21 Day Frequency 14 21 TOP 3 3 TOP 0 0 Location A 0 0 Location A 3 3 B 3 3 Total 9 9 B 0 0 Total 0 0 Controlling for ELISA=0 intervention=LA 187 Texas Tech University, Alejandro Echeverry, December 2007 Discussion Needle tenderization, deep marination, brine enhancement and moisture injection are some of the various processes commonly used in the industry to increase the tenderness, juiciness and quality attributes of certain meat cuts; unfortunately, contaminated non-intact meat products have been implicated in several foodborne outbreaks and product recalls, prompting the FSIS to address the safety of these types of products (6, 14). Even though the pathogen implicated on these unfortunate events was Escherichia coli O157:H7, Salmonella must also be considered and addressed in this type of products due to the number of infections and deaths that it causes every year. As reported by Johnston and others in 1969 (12) injected meat products have been implicated with outbreaks of salmonellosis; however, they have occurred due to the combination of various factors, including 1) contaminated water and spices used in the brine formulation and 2) undercooking of the meat product. Nevertheless, translocation of Salmonella from contaminated surfaces into the internal muscle of meat can occur and lead to potentially similar outbreaks and recalls, as they are often perceived as whole muscle cuts and are, therefore, prepared to customer specifications,(23). Previous studies have characterized the levels of contamination that can be translocated from the surfaces of meat into the internal muscle with either tenderization (22) or moisture enhancement process (1). Additional studies have analyzed samples that have been tenderized at the processing plant (11) or at 188 Texas Tech University, Alejandro Echeverry, December 2007 the retail level (10, 11); however, they have focused on total aerobic counts, coliform counts, and other native meat microflora. In this study we characterized the translocation levels of Salmonella that occur from contaminated meat surfaces of strip loins into the interior of the muscle after processing with either needle tenderization (NT) or moisture enhancement (EN). Two different settings where the interventions can be applied were analyzed. In the packer setting (PA) application of interventions to the meats surface occur after the animal has been slaughtered, split and fabricated. These cuts are held under refrigerated conditions for an aging period (that varies according to producer and needed requirements) before the subprimals are subjected to a process to increase the tenderness/juiciness of the final product. In the second setting, purveyor application (PU), the establishments receive the different meat cuts and proceed to process them in-house with either NT or EN (or both). We analyzed this situation as a potential intervention point for the subprimals. High Inoculation Results Packer Results At high inoculation levels effectiveness of the interventions varied according to the process (NT or EN) or the day of application. At day 0, 14 and 21 no differences were observed among interventions; however, all sampling days presented statistical significant differences between the treatments and the 189 Texas Tech University, Alejandro Echeverry, December 2007 control. Based solely on the results obtained from the surface, the subprimals treated with the interventions and then subjected to enhancement yielded a reduction between 1.59 (LAB) to almost 2.0 log10 units (ASC) by day 14. At day 21 a significant reduction in the levels of the pathogen was obtained with the application of LAB and LA to the surfaced of inoculated meat (Figure 56). 7.00 6.00 a b b b b a a a b b b b a ab b INOC CTRL LAB ASC LA Log 10 cfu/cm2 5.00 4.00 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 56. Summary of surface Salmonella counts on enhanced subprimals over time (Packer). ab Means for each day with different superscripts differ (P < 0.05) Based solely on the results obtained from the surface, the subprimals treated with the interventions and followed by needle tenderization yielded approximately between 1.1 to 2.0 log10 units less than control samples (day 14). Numerically, the largest reduction was obtained with the application of ASC followed by LA and LAB; however, no differences between the treatments were 190 Texas Tech University, Alejandro Echeverry, December 2007 observed. On day 21 the largest reduction was achieved with LAB and LA, with a reduction of approximately 1.2 log10 units when compared to the control (Figure 57). 7.00 6.00 5.00 a b b b b a a b b b b a a b b INOC CTRL LAB ASC LA Log 10 cfu/g 4.00 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 57. Summary of surface Salmonella counts on needle tenderized subprimals over time (Packer). ab Means for each day with different superscripts differ (P < 0.05). Based solely on the results obtained from the surface, the subprimals treated with the interventions followed by enhancement did not present differences due to the treatments on day 0. By day 14 only those samples treated with LAB were significantly lower when compared to the control (Figure 58); however, no differences were observed between the interventions at this 191 Texas Tech University, Alejandro Echeverry, December 2007 time. On day 21 no significant differences were observed between interventions and control, but samples treated with the interventions showed surface counts that were between 0.6 and 1.0 log10 units less than those obtained on day 0. 6.00 5.00 a a a CTRL a a b ab ab a a a a LAB ASC LA Log 10 cfu/cm2 4.00 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 58. Summary of surface Salmonella counts on enhanced subprimals over time (Purveyor). ab Means for each day with different superscripts differ (P < 0.05). Based solely on the results obtained from the surface, the subprimals treated with the interventions followed by needle tenderization did not present differences due to the treatments on day 0. By day 14 only those samples treated with LA were significantly lower when compared to the controls; however, no differences were found among interventions at this time. On day 21 no significant differences between interventions and control were found. Still, 192 Texas Tech University, Alejandro Echeverry, December 2007 microbial levels on day 21 were approximately 1.0 log10 cfu/cm2 lower than those reported on day 0 (Figure 59). 6.00 a 5.00 a CTRL a a a ab ab a b a a LAB ASC a LA Log 10 cfu /cm2 4.00 3.00 2.00 1.00 0.00 Day 0 Day 14 Day 21 Figure 59. Summary of surface Salmonella counts on needle tenderized subprimals over time (Purveyor). ab Means for each day with different superscripts differ (P < 0.05) Top results also presented significant differences among treatments for each one of the processes. In the packer setting on day 14, enhanced samples that were treated with LAB were significantly lower than the control by approximately 0.8 log10 cfu/g units. On day 21 enhanced samples presented a similar trend and only the LAB treatment presented a significant reduction on the levels of the pathogen (approximately 0.8 log10 cfu/g units). Needle tenderized samples presented a similar trend to those results obtained in the 193 Texas Tech University, Alejandro Echeverry, December 2007 enhancement application: Only those samples treated with LAB were significantly lower than the control with 1.2 log10 cfu/g units and approximately 0.8 log10 cfu/g cycle reduction on day 14 and 21, respectively (Figure 60). 6.00 a 5.00 a b ab a a a a a b a a ab ab a a b bc c INOC CTRL LAB ASC LA 4.00 b Log 10 cfu/g 3.00 2.00 1.00 0.00 EN 14 EN 21 NT 14 NT 21 Figure 60. Summary of TOP Salmonella counts on EN and NT subprimals over time (Packer). ab Means for each day with different superscripts differ (P < 0.05). Analysis of Top subsamples in the purveyor setting yielded the following results: On day 14 no statistical differences between treatments and control were observed, even though samples treated with LAB were approximately 0.5 log10 cfu/g lower than the control. On day 21 a similar trend was obtained for all samples and no differences between interventions and control are reported. For 194 Texas Tech University, Alejandro Echeverry, December 2007 the top subsection, samples subjected to the needle tenderization process presented a similar trend to the enhanced samples: No significant differences between interventions and control are reported at day 14 or 21 (Figure 61). 6.00 a 5.00 a a a a a a a a a a a a a a a CTRL LAB ASC LA 4.00 log 10 cfu /g 3.00 2.00 1.00 0.00 EN 14 EN 21 NT 14 NT 21 Figure 61. Summary of TOP Salmonella counts on EN and NT subprimals over time (Purveyor). a Means for each day with different superscripts differ (P < 0.05). Salmonella counts in subsections A and B resulted in mixed results in the degree of effectiveness of the interventions. In the packer application, subsection A, the levels of contamination with Salmonella varied from 1.0 logs (LAB, LA) to 195 Texas Tech University, Alejandro Echeverry, December 2007 >2.70 log (CTRL, ASC) in enhanced samples on day 14. On day 21, enhanced samples showed significant differences in this subsection, with the lowest counts (1.0 log10 cfu/g) on those samples treated with LA. Subsection A from needle tenderized samples also presented statistical lower counts on subsection A when treated with LA (day 14 and 21). In the purveyor application, subsection A also presented statistical lower counts when treated with the interventions: Enhanced samples treated with LAB and ASC were approximately 3.0 and 2.8 log10 cfu/g lower than the controls on day 14 and 21, respectively. Needle tenderize samples treated with ASC and LA were approximately 2.7 and 2.4 log10 cfu/g lower than the controls on day 14 and 21, respectively. In subsection B of the packer application, enhanced samples treated with ASC and LA presented the lowest pathogen recovery on day 14. On day 21, the lowest recovery were found on samples treated with ASC and CTRL; with levels of contaminations being approximately 1.7 log10 cfu/g lower than the control. Needle tenderize samples treated with LAB and with LA were approximately 2.6 and 2.0 log10 cfu/g lower than the controls on day 14 and 21, respectively. In subsection B of the purveyor application the recovery levels between interventions were more constant: No differences among treatments were found in EN samples on day 14 or 21, with the recovery levels being between 1.0 and 1.89 log10 cfu/g on day 14 and 21, respectively. The overall level of Salmonella recovery on needle tenderize samples were higher in this process when compared to enhanced samples, with counts ranging from 1.97 to 3.01 log10 196 Texas Tech University, Alejandro Echeverry, December 2007 cfu/g on day 14 to 1.0 to 2.1 log10 cfu/g on day 21, respectively. The lowest recoveries were obtained in those samples treated with LAB and LA on day 14 and 21, respectively. Low Inoculation Results Packer Result At low inoculation levels the interventions used to spray the subprimals resulted in mixed results in eliminating Salmonella from the surfaces and internal subsections that were analyzed. For the packer application (EN) positive Salmonella results were obtained on both moisture enhanced and needle tenderized meat: all the treatments (day 14) showed at least one positive obtained from the top subsections; however, no positives were found on subsections A and B. On day 21, subsections top, A and B obtained from control samples yielded at least a positive result. Except for one positive on the top section of ASC samples on day 21, no other positives were found at this sampling time. These results might be an indication that at this level of surface contamination the interventions are effective in controlling and reducing the levels of Salmonella. For the packer application (NT) all the treatments (day 14) showed at least one positive obtained from the top subsections; only CTRL samples showed positive top subsections on day 21. For all treatments none of the A or B subsections tested positive for the pathogen at this specific process. 197 Texas Tech University, Alejandro Echeverry, December 2007 Purveyor Results At low inoculation levels, positive ELISA results for Salmonella were obtained once in top enhanced CTRL, LAB, and ASC samples (day 14), but LA samples were all negative. No positives were obtained on subsections A or B for any given treatment or the control. On day 21 only the top section of enhanced CTRL and LAB samples yielded positive ELISA results. Subsections A and B for all the interventions and the control were negative on day 21. Positive ELISA results were obtained once in top needle tenderize CTRL, ASC, and LA samples on day 14; however, LAB samples were negative in this subsection in all of the replications. On day 14, only A and B subsections from CTRL and LA samples were positive. It is worth noting here that when LA tested positive in subsections A and B the top subsection of the same steak (containing the inoculated surface) tested negative for the pathogen (data by specific replications are not shown). In general terms, the use of interventions to control translocation of Salmonella into the internal muscle of the steaks was very effective: For both processes (EN and NT) only the CTRL samples yielded positive A and B results while subsections A and B from LAB and ASC were all negative. The LA samples tested positive once on subsection A and B, but when this occurred the top was negative, indicating that even though the interventions are effective in controlling the surface contamination some areas might not be exposed fully to the antimicrobial and microorganisms might survive and be internalized later on in the process. We also have to be cautious in what we inferred from these results: 198 Texas Tech University, Alejandro Echeverry, December 2007 Just because subsections A and B were negative for the pathogen it doesnt necessarily mean that the microorganism was absent of the interior of the muscle: As a reminder, only 25 grams were collected and analyzed for each subsection with the ELISA test, leaving plenty of muscle material out of the analysis where the pathogen (if present) could have been internalized with the process. There was a trend to higher Salmonella counts in subsections A and B on those samples processed with needle tenderization. We hypothesize that the variation on the contamination levels for each subsection was a function of how deep inside the muscle the needles and the blades cut throughout the meat. Microbial contamination is internalized in the muscle, but this doesnt occur at a constant rate. We observed higher microbial counts on the top subsection as expected, but in some cases the numbers on subsection A were not higher than those on subsection B, which was located closer to the conveyor belt. It could happen that when the blades and the needles pierce the meat all the way to the bottom (another external surface contaminated with the pathogen) they might carry some bacteria back in into subsections B and A (in that order). Other possible causes include carriage of the pathogens inside the meat using the brine as a vehicle through the pierced locations of the meat: as the subprimals are in the conveyor belt and they are transported to the injectors they are subjected to a brine shower right before they are puncture with the needles, 199 Texas Tech University, Alejandro Echeverry, December 2007 which in turn, can drain and carry contaminated brine through the punctures in the meat. From the results obtained in this study it was found that the use of interventions such as LAB, ASC, and LA in inoculated subprimals was very effective in reducing the levels of Salmonella contamination in the surface and internal muscle of meat. Reduction of approximately 1.1 and 1.2 log10 cfu/cm2 units were obtained by day 14 and 21 with the application of ASC and LA, respectively (Packer application). In the purveyor setting, needle tenderized samples treated with LA on day 14 yielded the lowest reduction (0.6 log 10 units) when compared to the control; LAB was the most effective in reducing the levels by day 14 on enhanced samples (approximately by 0.7 log10 cfu/cm2). Processors should take notice of this study and must try to adapt their process to implement interventions in subprimals intended for needle tenderization/ enhancement steaks. Acknowledgements This study was Funded by the Beef Checkoff. 200 Texas Tech University, Alejandro Echeverry, December 2007 References 1. Bohaychuk, V. M., and G. G. Greer. 2003. Bacteriology and storage life of moisture-enhanced pork. J Food Prot. 66:293-9. 2. Brashears, M. M., A. Amezquita, and J. Stratton. 2001. Validation of methods used to recover Escherichia coli O157:H7 and Salmonella spp. subjected to stress conditions. J Food Prot. 64:1466-71. 3. Carr, M. A., K. L. Crockett, C. B. Ramsey, and M. F. Miller. 2004. Consumer acceptance of calcium chloride-marinated top loin steaks. J Anim Sci. 82:1471-4. 4. Davis, K. A., D. L. Huffman, and J. C. Cordray. 1975. Effect Of Mechanical Tenderization, Aging And Pressing On Beef Quality. Journal of Food Science. 40:1222-1224. 5. FSIS-USDA. 2006. Salmonella verification sample result reporting: agency policy and use in public health protection. Federal Register Docket No. 04 026N. p. 9772-7. In W. Department of Agriculture, D.C. (ed.), vol. 71. 6. FSIS. 2005. HACCP plan reassessment for mechanically tenderized beef products. p. 30331-30334. United States Department of Agriculture. (USDA) (ed.), vol. 70, No 101. Federal Register. 7. FSIS. February 1, 1998. Directive 10,010.1, Microbiological Testing Program for Escherichia coli O157:H7 in Raw Ground Beef. In W. Department of Agriculture, D.C. (ed.). 8. Fung, D. Y. C., R. K. Phebus, D. H. Kang, and C. L. Kastner. 1995. Effect Of Alcohol-Flaming On Meat Cutting Knives. Journal of Rapid Methods and Automation in Microbiology. 3:237-243. 9. Gill, C. O., and J. C. McGinnis. 2004. Microbiological conditions of mechanically tenderized beef cuts prepared at four retail stores. International Journal of Food Microbiology. 95:95-102. 10. Gill, C. O., and J. C. McGinnis. 2005. Factors affecting the microbiological condition of the deep tissues of mechanically tenderized beef. J Food Prot. 68:796-800. 201 Texas Tech University, Alejandro Echeverry, December 2007 11. Gill, C. O., J. C. McGinnis, K. Rahn, D. Young, N. Lee, and S. Barbut. 2005. Microbiological condition of beef mechanically tenderized at a packing plant. Meat Science. 69:811-816. 12. Johnston, R. W., M. E. Harris, and A. B. Moran. 1978. The Effect Of Mechanical Tenderization On Beef Rounds Inoculated With Salmonellae. Journal of Food Safety. 1:201-209. 13. Kang, D.-H., and D. Y. C. Fung. 1999. Thin Agar Layer Method for Recovery of Heat-Injured Listeria monocytogenes. Journal of Food Protection. 62:1346-1349. 14. Laine, E. S., J. M. Scheftel, D. J. Boxrud, K. J. Vought, R. N. Danila, K. M. Elfering, and K. E. Smith. 2005. Outbreak of Escherichia coli O157:H7 infections associated with nonintact blade-tenderized frozen steaks sold by door-to-door vendors. J Food Prot. 68:1198-202. 15. Mandigo, R. W., and D. G. Olson. 1982. Effect of Blade Size for Mechanically Tenderizing Beef Rounds. Journal of Food Science. 47:2095-2096. 16. MMWR. 2006. Preliminary FoodNet Data on the Incidence of Infection with Pathogens Transmitted Commonly Through Food --- 10 States, 2006. MMWR CDC Surveill Summ. . p. 56(14); 336-339. Center for Disease Control and Prevention (CDC). 17. Petersohn, R. A., D. G. Topel, H. W. Walker, A. A. Kraft, and R. E. Rust. 1979. Storage Stability, Palatability And Histological Characteristics Of Mechanically Tenderized Beef Steaks. Journal of Food Science. 44:1606-1614. 18. PS, M., S. L, D. V, M. LF, B. JS, S. C, G. PM, and T. RV. 1999. - Foodrelated illness and death in the United States. Emerg Infect Dis. 5:607-25. 19. Raccach, M., and R. L. Henrickson. 1979. Microbial aspects of mechanical tenderization of beef. Journal of Food Protection. 42:971-973. 20. SAS Institute, I. 2004. Version 9.1.3 for Windows, Cary, North Carolina. 21. Schwartz, W. C., and R. W. Mandigo. 1977. Effect on Conveyor Speed on Mechanical Tenderization of Beef inside Rounds. J. Anim Sci. 44:581-584. 22. Sporing, S. 1999. Escherichia coli O157:H7 risk assessment for production and cooking of blade tenderized beef steaks. p. vii, 102 leaves : ill. ; 28 cm. In, Food Science, Master Thesis...

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