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DATE DUE I DATE DUE I DATE DUE W'izzoagggfil D§j41139290ijlt t JAN '3 l 2006 11/00 clam-.9659.“ COMPETITION OF THERMALLY INJURED LISTERIA MONOCYTOGENES WITH A MESOPHILIC LACTIC ACID STARTER CULTURE DURING MILK FERMENTATION By Finny P. Mathew A THESIS Submitted to Michigan State University In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 2000 ABSTRACT COMPETITION OF THERMALLY INJURED LISTERIA MONOCYTOGENES WITH A MESOPHILIC LACTIC ACID STARTER CULTURE DURING MILK FERMENTATION By Finny P. Mathew The relationship between heat treatment of milk and the ability of sublethally injured Listeria monocytogenes to survive mesophilic fermentation in milk was investigated. A three-strain cocktail of L. monocytogenes, suspended in 200 ml of tryptose phosphate broth, was heated at 56°C/20 min and 64°C/2 min to obtain low heat-injured (LHI) and high heat-injured (HHI) cells, respectively, showing >99% injury. Flasks containing 200 ml of raw, low heat-treated (56°C/20 min), high heat-treated (64°C/2min), pasteurized or UHT milk were tempered to 31 .1°C, inoculated to contain 104-106 LHI, HHI or healthy L. monocytogenes cells and Lactococcus lactis subsp. lactis/Lactococcus lactis subsp. cremoris starter culture at 0.5, 1.0 or 2.0% levels. Numbers of healthy and injured L. monocytogenes cells were determined using tryptose phosphate agar w/o 4.0% NaCl at selected intervals during the 24h fermentation period along with the numbers of starter organisms. In starter-free controls, ~76-81% and 59-69% of LHI and HHI cells, respectively, were repaired after 8 hours of incubation, with lowest repair in raw milk. Increased injury was observed for healthy L. monocytogenes cells at 1.0 and 2.0% starter levels, with less injury seen for LHI and HHI cells. The extent of sublethal injury for all L. monocytogenes was inversely related to severity of the milk heat treatment. Dedicated to my family in India and East Lansing iii ACKNOWLEDGMENTS Firstly, I would like to thank God for giving me the opportunity and wisdom to get my MS degree in Food Science at Michigan State University. I want to extend my heartfelt thanks to my adviser, Dr. Elliot T. Ryser, without whose immense help, guidance and cooperation in academics as well as in research I would not have been able to finish my degree. I am very thankful to him for providing me the opportunity to tap into his expertise on Listeria monocytogenes. I want to thank my committee members, Dr. Zeynep Ustunol and Dr. James Pestka, for their kind cooperation and good advice at all times. I would like to thank my lab mates for their cooperation during my research work and for making my time in the lab enjoyable. I would also like to thank Dr. John Partridge, Mr. John Engstrom and his dairy plant team for making my experience in the dairy plant a learning (and fun) experience during the first year of my degree as well as for all help extended towards my research. I thank the National Food Safety and Toxicology Center and the Department of Food Science and Human Nutrition, Michigan State University, for funding my research and providing me with graduate assistantships. Last but not the least, I would like to confer my deepest thanks to my family in India as well as in East Lansing (Alociljas) who constantly supported, helped and guided me in every possible way and prayed for my success at Michigan State University. iv TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS AND ABBREVIATIONS INTRODUCTION LITERATURE REVIEW Raw milk cheese regulations Cheesebome epidemics Listeria monocytogenes Surveillance for L. monocytogenes in cheese Behavior of L. monocytogenes in different products Milk fermentation Behavior in cheese Feasibility of raw milk cheese Political climate surrounding raw milk cheese Raw milk versus pasteurized milk cheese Beneficial effects of milk heat treatment Detrimental effects of milk heat-treatment Effect of heat-treatment on L. monocytogenes Effect of acid/acidity Sublethal thermal/acid injury Goals of the study RESEARCH PAPER FOR PUBLICATION Title page Abstract Introduction (no title) Materials and Methods Culture preparation Sublethal injury Experimental design Milk inoculation Microbiological analysis Statistical analysis Results Sublethal injury Indigenous microflora in milk Growth of L. monocytogenes without starter culture Growth of L. monocytogenes in the presence of starter culture vii xiii xiv Discussion Acknowledgments Tables Figure legends Figures BIBLIOGRAPHY Appendix A — Competitive Inhibition Of Acid-Injured Listeria monocytogenes Introduction Materials and Methods Culture preparation Sublethal injury Results APPENDIX B - Research Data Used for Manuscript vi 69 73 74 85 86 89 106 106 106 106 106 107 108 LIST OF TABLES Table no. Title/Caption Page 1. Foodbome Illness Associated with cows’ Milk Cheese in the 5 United States, Canada, and Europe 2. Chronological List of Class I Recalls in the United States for 18 Domestic Cheese Contaminated with L. monocytogenes 3. Chronological List of Class I Recalls in the United States for 26 Imported Cheese Contaminated with L. monocytogenes 4. Behavior of L. monocytogenes During Cheese Ripening as 34‘ Affected by Cheese Composition 5. Growth and Inactivation of L. monocytogenes in Surface- 35 Inoculated Retail Cheeses During Storage at 4-30°C 6. Percent Heat-injury of L. monocytogenes in TPB and UHT Milk 74 7. Mixed Covariance Procedure Table for Growth of Healthy L. 74 monocytogenes Cells 8. Repair of L. monocytogenes in Different Types of Milk Without 75 Starter Culture 9. Mixed Covariance Procedure Table for Repair of Sublethally 76 Injured L. monocytogenes Cells Afier 24 h 10. Mixed Covariance Procedure Table for Log Increase of Total L. 76 monocytogenes after 24 h 11. Mixed Covariance Procedure Table for Percent Increase in Injured 76 L. monocytogenes Cells 12. Percent Increase in Injured L. monocytogenes Cells in Raw Milk at 77 Different Starter Culture Levels 13. Percent Increase in Injured L. monocytogenes Cells in Low Heat- 78 Treated Milk at Different Starter Culture Levels 14. Percent Increase in Injured L. monocytogenes Cells in High Heat- 79 Treated Milk at Different Starter Culture Levels vii Table no. Title/Caption Page 15. 16. 17. l8. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Percent Increase in Injured L. monocytogenes Cells in Pasteurized Milk at Different Starter Culture Levels Percent Increase in Injured L. monocytogenes Cells in UHT— Pasteurized Milk at Different Starter Culture Levels Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 0.5% LLLC Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 1.0% LLLC Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 2.0% LLLC Heat Injury of L. monocytogenes in Tryptose Phosphate Broth and UHT Milk at 56°C Heat Injury of L. monocytogenes in Tryptose Phosphate Broth and UHT Milk at 64°C Fate of Uninjured L. monocytogenes in Raw Milk without a Starter Culture Fate of LHI L. monocytogenes in Raw Milk without a Starter Culture Fate of HHI L. monocytogenes in Raw Milk without a Starter Culture Fate of Uninjured L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Fate of LHI L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Fate of HHI L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Fate of Uninjured L. monocytogenes in Raw Milk at a 1.0% Starter Inoculum Fate of LHI L. monocytogenes in Raw Milk at a 1.0% Starter Inoculum viii 80 81 82 82 84 108 108 109 109 109 110 110 110 111 111 Table no. Title/Caption Page 30. Fate of HHI L. monocytogenes in Raw Milk at a 1.0% Starter 111 Inoculum 31. Fate of Uninjured L. monocytogenes in Raw Milk at a 2.0% Starter 112 Inoculum 32. Fate of LHI L. monocytogenes in Raw Milk at a 2.0% Starter 112 Inoculum 33. Fate of HHI L. monocytogenes in Raw Milk at a 2.0% Starter 112 Inoculum 34. Fate of Uninj ured L. monocytogenes in LHT Milk without a 113 Starter Culture 35. Fate of LHI L. monocytogenes in LHT Milk without a Starter 113 Culture 36. Fate of HHI L. monocytogenes in LHT Milk without a Starter 113 Culture 37. Fate of Uninjured L. monocytogenes in LHT Milk at a 0.5% 114 Starter Inoculum 38. Fate of LHI L. monocytogenes in LHT Milk at a 0.5% Starter 114 Inoculum 39. Fate of HHI L. monocytogenes in LHT Milk at a 0.5% Starter 114 Inoculum 40. Fate of Uninjured L. monocytogenes in LHT Milk at a 1.0% 115 Starter Inoculum 41. Fate of LHI L. monocytogenes in LHT Milk at a 1.0% Starter 115 Inoculum 42. Fate of HHI L. monocytogenes in LHT Milk at a 1.0% Starter 115 Inoculum 43. Fate of Uninjured L. monocytogenes in LHT Milk at a 2.0% 116 Starter Inoculum 44. Fate of LHI L. monocytogenes in LHT Milk at a 2.0% Starter 116 Inoculum Table no. Title/Caption Page 45. Fate of HHI L. monocytogenes in LHT Milk at a 2.0% Starter 116 Inoculum 46. Fate of Uninjured L. monocytogenes in HHT Milk without a 117 Starter Culture 47. Fate of LHI L. monocytogenes in HHT Milk without a Starter 117 Culture 48. Fate of HHI L. monocytogenes in HHT Milk without a Starter 117 Culture 49. Fate of Uninjured L. monocytogenes in HHT Milk at a 0.5% 118 Starter Inoculum 50. Fate of LHI L. monocytogenes in HHT Milk at a 0.5% Starter 118 Inoculum 51. Fate of HHI L. monocytogenes in HHT Milk at a 0.5% Starter 118 Inoculum 52. Fate of Uninjured L. monocytogenes in HHT Milk at a 1.0% 119 Starter Inoculum 53. Fate of LHI L. monocytogenes in HHT Milk at a 1.0% Starter 119 Inoculum 54. Fate of HHI L. monocytogenes in HHT Milk at a 1.0% Starter 119 Inoculum 55. Fate of Uninjured L. monocytogenes in HHT Milk at a 2.0% 120 Starter Inoculum 56. Fate of LHI L. monocytogenes in HHT Milk at a 2.0% Starter 120 Inoculum 57. Fate of HHI L. monocytogenes in HHT Milk at a 2.0% Starter 120 Inoculum 58. Fate of Uninjured L. monocytogenes in Pasteurized Milk without a 121 Starter Culture 59. Fate of LHI L. monocytogenes in Pasteurized Milk without a 121 Starter Culture Table no. Title/Caption Fag: 60. Fate of HHI L. monocytogenes in Pasteurized Milk without a 121 Starter Culture 61. Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 0.5% 122 Starter Inoculum 62. Fate of LHI L. monocytogenes in Pasteurized Milk at a 0.5% 122 Starter Inoculum 63. Fate of HHI L. monocytogenes in Pasteurized Milk at a 0.5% 122 Starter Inoculum 64. Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 1.0% 123 Starter Inoculum 65. Fate of LHI L. monocytogenes in Pasteurized Milk at a 1.0% 123 Starter Inoculum 66. Fate of HHI L. monocytogenes in Pasteurized Milk at a 1.0% 123 Starter Inoculum 67. Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 2.0% 124 Starter Inoculum 68. Fate of LHI L. monocytogenes in Pasteurized Milk at a 2.0% 124 Starter Inoculum 69. Fate of HHI L. monocytogenes in Pasteurized Milk at a 2.0% 124 Starter Inoculum 70. Fate of Uninjured L. monocytogenes in UHT Milk without a 125 Starter Culture 71. Fate of LHI L. monocytogenes in UHT Milk without a Starter 125 Culture 72. Fate of HHI L. monocytogenes in UHT Milk without a Starter 125 Culture 73. Fate of Uninjured L. monocytogenes in UHT Milk at a 0.5% 126 Starter Inoculum 74. Fate of LHI L. monocytogenes in UHT Milk at a 0.5% Starter 126 Inoculum xi Table no. Title/Caption Page 75. Fate of HHI L. monocytogenes in UHT Milk at a 0.5% Starter 126 Inoculum 76. Fate of Uninjured L. monocytogenes in UHT Milk at a 1.0% 127 Starter Inoculum 77. Fate of LHI L. monocytogenes in UHT Milk at a 1.0% Starter 127 Inoculum 78. Fate of HHI L. monocytogenes in UHT Milk at a 1.0% Starter 127 Inoculum 79. Fate of Uninjured L. monocytogenes in UHT Milk at a 2.0% 128 Starter Inoculum 80. Fate of LHI L. monocytogenes in UHT Milk at a 2.0% Starter 128 Inoculum 81. Fate of HHI L. monocytogenes in UHT Milk at a 2.0% Starter 128 Inoculum 82. Growth of Starter Culture at 0.5% Inoculum Level without L. 129 monocytogenes 83. Growth of Starter Culture at 1.0% Inoculum Level without L. 129 monocytogenes 84. Growth of Starter Culture at 2.0% Inoculum Level without L. 129 monocytogenes 85. Acid-Injury of L. monocytogenes in Tryptose Phosphate Broth (pH 129 3.5) xii LIST OF FIGURES Figure Title/Caption Page No. l. Surveillance programs for Listeria spp. in domestic and imported 22 cheese 2. Preparation of heat-inj ured L. monocytogenes 62 3. Fermentation of milk at 31 .1°C 63 4. Sublethal Heat injury of L. monocytogenes at 56°C in UHT milk 86 5. Sublethal Heat injury of L. monocytogenes at 64°C in UHT milk 87 xiii 10 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. LIST OF SYMBOLS OR ABBREVIATIONS . CF U, colony-forming units diameter, diam gram, 8 high heat-injured, HHI high heat-treated, HHT hour(s), h Lactococcus lactis subsp. lactis/Lactococcus lactis subsp. cremoris, LLLC low heat-injured, LHI low heat-treated, LHT . minute(s), min modified tryptose phosphate agar, MTPA modified tryptose phosphate agar with 4% salt, MTPNA parts per million, ppm pound, lb revolutions per minute, rpm second, 5 specie, sp. species, spp. tryptose phosphate agar, TPA tryptose phosphate agar with 4% NaCl, TPNA ultra high temperature, UHT xiv INTRODUCTION Most United States “Standards of Identity” for cheese and cheese related products (1948-49) provide cheese manufacturers with the option of pasteurizing [716°C (161°F )/ l 5 see] the milk or holding the cheese for a minimum of 60 days at 317°C (35°F) to eliminate pathogenic microorganisms. Thus, any cheese prepared from raw or heat- treated milk has to be held at least 60 days. Since 1948, at least 10 foodbome outbreaks have been linked to domestically produced cheese. Reports have shown that three important foodbome pathogens, namely, Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli OlS7:H7 can respectively survive up to 434 days, 210 days and 138 days in Cheddar cheese produced from pasteurized milk inoculated with the pathogen. Consequently, the adequacy of the 60 day hold at 2 1.7°C still remains very much in question. Based on available data, the United States Food and Drug Administration (FDA) is re-examining current regulations. However, given the superior flavor characteristics of raw milk Cheddar Cheese that result from non-starter lactic acid bacteria and enzymes naturally present in the milk, cheese manufacturers as well as certain consumer groups are reluctant to any change in the current aging policy. Listeria monocytogenes is the hardiest of the three aforementioned foodbome pathogens in terms of heat/acid resistance, temperature, aw and pH ranges at which it can survive and grow. It can cause abortion in pregnant women and meningitis in immunocompromised individuals. The disease, listeriosis, has a very high mortality rate among susceptible individuals (~20%). Consequently, United States has a "zero tolerance" policy for L. monocytogenes in ready-to-eat foods. Dairy cows that appear healthy can serve as reservoirs for L. monocytogenes with this pathogen reportedly present in l.6-12.0%, l.3-5.4%, and 2.5-6.0% of the raw milk produced in the United States, Canada and Western Europe, respectively. In the United States, this pathogen has been responsible for at least 46 class I recalls involving domestically produced cheese, 3 of which were prepared from raw milk. Thus, the current "zero tolerance" policy for L. monocytogenes has extracted a particularly heavy toll on the dairy industry. Fermentation is an age-old food preservation method used to inhibit the growth and survival of pathogenic bacteria. Studies of survival and growth of healthy L. monocytogenes in Cheddar, Colby and Cottage cheese indicate that Listeria numbers slowly decrease during ripening of the cheese. Incomplete pasteurization can lead to the survival and recovery of sublethally injured cells. Such repair requires an optimal pH near 7.0. Given the low pH of Cheddar cheese (~pH 5.0) combined with high levels of salt in the moisture phase, survival of sublethally injured should be far less than that for healthy cells. The purpose of the study was to assess the ability of healthy and sublethally injured cells of L. monocytogenes to compete with different levels of a mesophilic lactic acid starter culture in milks that have undergone various degrees of thermal processing. The underlying hypothesis was that a sub-pasteurization heat treatment can be identified which will sufficiently injure L. monocytogenes to prevent its survival in Cheddar cheese beyond 60 days of ripening and thereby preserve the raw milk cheese industry. 1.- ‘1 1' 1‘ a m. .... TIC R01 LITERATURE REVIEW RAW MILK CHEESE REGULATIONS Present-day laws regarding use of pasteurized, heat-treated (sub-pasteurized), and raw milk for cheesemaking date back to World War II. These standards were established more as a safety measure than from any documented scientific evidence. Most U.S. Standards of Identity for cheese and cheese related products (Anon. 1949) specify three safety options: (a) milk pasteurization - min. 71.6°C (161°F)/15 see, (b) holding finished cheese for a minimum of 60 days at a temperature of 1.7°C (3 5°F) or greater or (c) neither milk pasteurization nor a 60 days holding period for cheeses used as ingredients in further manufacture. Thus, the holding option is required when cheese is prepared from raw or heat-treated milk. Cheeses that can be made from raw milk with a 60-day hold at 335°F include Asiago (soft and fresh, medium, old), blue, Nuworld, Parmesan/Regiano, Roquefort, Swiss/Emmentaler, brick, Cheddar, Colby, cold pack/club cold pack cheese food, cold pack cheese food with meat, fruits and vegetables, Edam, Gouda, Granular and stirred curd, grated American cheese food, Limburger, Provolone, sofi ripened cheeses, Samsoe, Caciocavallo siciliano, Gorgonzola, Gruyere, hard grating cheese and Romano. CHEESEBORNE EPIDEMICS Since institution of the Federal Standards of Identity for cheese in 1948, some foodbome pathogens have survived longer than 60 days in cheese made from raw or heat- treated milk (i.e. less than legal pasteurization) and caused major outbreaks of illness and/or recalls (Anon. 1999d). Post processing contamination of cheese prepared from pasteurized milk is also a problem (Kornacki 1982, Marier et. a1 1973). Epidemiological T.) surveys in the United States (Bryan 1983, 1988, Sharp 198 7), Canada (D 'Aoust et. al 1985b, Sharp 198 7), England and Wales (Barrett 1986, Galbraith et. al 1982, Sharp 1987) and several Western European countries (Sharp 1985, 1987) have verified that dairy products are a relatively safe class of foods. In the United States, dairy products have been vehicles in only 1-3% of all reported foodbome outbreaks (Barza 1985, Bryan 1983, 1988, CDC 1985, Finch and Blake 1985, Kaplan et a1. 1962, Kleeburg 1975, Parry 1966, Potter 1984, Sharp 1 98 7). Commercially produced cheeses have been sporadically linked to foodbome illness (Table 1). Since 1948, 10 confirmed outbreaks in the United States were traced to domestically produced Cheese (Table 1). Several pathogens including Brucella melitensis, Clostridium botulinum, Staphylococcus aureus, Streptococcus zooepidemicus and Shigella sonnei have caused cheese-related outbreaks (Table 1). S. aureus grth and enterotoxin production during cheesemaking is a potential problem only if there is subnormal acid development by the starter bacteria (Stadhouders et. al 1978, T atini et. al 1971, 1973, Tuckey et. al 1964, Zehren and Zehran 1968). From 1950 to 1965, a series of food poisoning outbreaks caused by Staphylococcus aureus enterotoxins occurred in raw milk Cheddar and some other cheeses in the US. (Table 1). Attention will be given to outbreaks caused by L. monocytogenes, Salmonella, E. coli OlS7:H7 since these pathogens can reportedly survive the mandatory 60-day ripening period in various cheeses made from inoculated pasteurized milk (Goepfert et al. 1968, Hargrove et. al 1969, Park et al. 1970, Reitsma and Henning 1996, Ryser and Marth 1987a). :00: SEE 28m mm 88: SNEOEBM 080:0 00m «03 000388 8:33.889 3:8..— oonNA 320853: 828:0 33— 8% baa 3am 9352: £882.30. 88 Sea .588 $2 058.: 82 me 808: M. 880 080:0 owa 008:3 0302 3858380 2 0.308: .m. 005m :2 .3880 :08 DEE 38 E 320858.: em £2: HQ Eatsafigh .m. 080:0 :8 2.00-8382 32 0:8 38 8 38853: on :2: HQ Esta—Egg. 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D E 008880 .0080": gm :8 .0 0880588803: 08:80:80 : 3: ..§§o.0 Snagam 3880M 880 88:8“: b08> 080:0 80 > .6280 _ 23 Salmonella was responsible for five cow-milk cheese-related outbreaks in the United States, three in Canada, and at least four in Western Europe. One United States outbreak in 1976 involving seven lots of Cheddar prepared from pasteurized milk caused 339 cases of salmonellosis (Fontaine et. a1 1980). The contaminated cheese was traced to a Kansas manufacturer. Low numbers of Salmonella heidelberg were isolated from all seven cheese lots and from three vats of cheese at the Kansas factory. Examination of the plant revealed no environmental or employee contamination. Raw milk for cheesemaking was stored unrefiigerated for 1 to 3 days in insulated holding tanks before being pasteurized at 71 .7°C (161 .5°F)/15 s. The pasteurized milk was not examined for bacterial count or alkaline phosphatase activity. The average pH of contaminated cheese was 5.6 vs. 5.4 for the uncontaminated cheese. Slow acid production leading to an abnormally high cheese pH likely facilitated survival and growth of S. heidelberg (Fontaine et. a1 1980). This outbreak can thus be attributed to poor manufacturing practices and inadequate control programs in the cheese plant. Salmonella enterica serotype Typhimurium definitive type 104 (S. Typhimurium DT104) has emerged as the most common multidrug-resistant Salmonella strain in the United States and is resistant to 5 different antibiotics (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline). During spring of 1997, two cheese-related outbreaks involving S. Typhimurium DT104 were investigated in a matched case-control study The first outbreak peaked in February 1997; 31 patients were culture-positive for a strain of S. Typhimurium var. Copenhagen that exhibited the same pulsed-field gel electrOphoresis (PFGE) pattern. This strain was identified as phage type DT104. In a subsequent case-control study, 15 of 16 S. Typhimurium var. Copenhagen cases compared with 14 of 24 matched controls reported eating unpasteurized Mexican-style cheese. Enhanced surveillance uncovered a second outbreak, which peaked in April 1997 and was caused by a non-Copenhagen variant of S. Typhimurium. During this second outbreak, S. Typhimurium was isolated from 79 people who ate fresh Mexican-style cheese from street vendors, as well as from some cheese samples and raw milk used in cheesemaking. The PFGE pattern of the milk isolate matched 1 of 3 patient strains with all isolates identified as phage type DT104b (Cody et. al 1999). In early 1997, a 5-fold increase in salmonellosis among Hispanics was observed in Yakima County, Washington. Bacterial strains and risk factors for infection with S. Typhimurium in Yakima County were investigated in laboratory, case-control and environmental studies. Between January and May 1997, 54 culture-confirmed cases of S. Typhimurium were reported. The median age of patients was 4 years and 91% were Hispanic. Overall, 77% of the cases reported eating unpasteurized Mexican-style sofi cheese in the 7-day period preceding onset of illness, compared to 28% of the controls. All case isolates were phage type DT104 or DT104b. Cheese consumed by two unrelated patients was made from raw milk, which was traced back to the same local farm. Milk samples fi'om nearby dairies yielded S. Typhimurium DT104 (Villar et. al 1999). During 1980 to 1982, several outbreaks of salmonellosis were traced to raw milk Cheddar that was produced in Ontario, Canada (Wood et. al 1984). The milk used for cheesemaking came from a farm where one cow was shedding Salmonella muenster in her milk. This naturally contaminated raw milk was then used to determine survival of S. muenster during commercial preparation of raw milk cheese. Curd from 11 of 181 vats tested positive with two of these lots still positive after pressing. During curing at 41°F, one lot was negative after 30 days, but one lot was positive after 125 days. No significant compositional differences were observed between the lots of contaminated cheese. Another large Canadian outbreak of salmonellosis involving Cheddar cheese occurred during March-July 1984 in the four Maritime Provinces (D'Aoust 1985a, D'Aoust et. al 1985b, Ratnam and March 1986). Over 2700 people were infected with S. Typhimurium. Epidemiological evidence implicated Cheddar cheese that was manufactured by a single plant on Prince Edward Island. Salmonella Typhimurium was sporadically detected in Cheddar cheese that was manufactured at this facility from either pasteurized milk [73.8°C (165°F)/16 s] or heat-treated milk [66.7°C (152°F)/ 16 s]. Salmonella was first confirmed in a cheese trim bucket. One of the employees who used their hands to transfer curds to a forming machine also tested positive for S. Typhimurium. Testing of the raw milk supply ultimately identified two cows in separate herds, one shedding S. Typhimurium from one quarter of her udder, the other shedding S. heidelberg. A thorough evaluation of the pasteurization process revealed that the pasteurizer operator manually over-rode the electronic controller, thereby shutting down the pasteurizer while milk continued to flow through the unit and into a vat, leading to Salmonella-positive vats. This only occurred when raw milk from the infected cow shedding S. Typhimurium was used. D'Aoust et al. (D ’Aoust et. al 1985b) investigated survival of S. Typhimurium in the contaminated cheese lots. Salmonella Typhimurium was present in mild Cheddar made from either heat-treated or pasteurized milk. Analysis of six contaminated cheese lots indicated that the cheeses contained 0.39 to 9.3 Salmonella CFU/ 100 g. Salmonella Typhimurium was detected in 1 of 6 cheese lots cured for eight months at 5°C (41°F). OCC “11 However, some cheeses also showed heavy mold growth, which may have contributed to survival of S. Typhimurium (D ’Aoust et. al I985b). Four Salmonella outbreaks in Europe have been traced to cheese (Table 1). The latest community-wide outbreak of salmonellosis was reported in France during 1997. A total of 113 cases were identified in a case control study with one batch of Morbier cheese (soft raw milk cheese) from one processing plant identified as the source of S. Typhimurium (Valk et al. 2000). These studies demonstrate that Salmonella can survive past the 60 day holding requirement at 335°F. The outbreaks also indicate that soft cheese made from unpasteurized milk is an important vehicle for S. Typhimurium transmission. The need for good manufacturing practices and adequate process control programs in the cheese factory is also underscored. A major outbreak of gastrointestinal illness caused by enteropathogenic E. coli occurred in the United States in 1971 (Kornacki 1982, Marier et. al 1973). This outbreak which included at least 387 cases was traced to Camembert cheese prepared by a single manufacturer in France. All the contaminated cheese was manufactured during a 2-day period at one plant and contained 105 to 107 E. coli 0:124 per gram. The same serotype was found in stool specimens. The attack rate was >94% for people who consumed the cheese. Although the source of contamination was never confirmed, the filtration system for river water used in washing equipment was not working properly when the contaminated cheese was manufactured. Enteric pathogens were not isolated from the water or from those employees that were available for examination. While the epidemic strain was never isolated from the starter culture, salt, or the equipment, this organism was recovered from the curdling tank which suggests post-pasteurization contamination. 10 In 1983, three outbreaks of enteropathogenic Escherichia coli affected 45 persons who attended office parties in Washington DC. Additional cases were later identified in Illinois (75 cases), Wisconsin (35 cases), Georgia (10 cases), and Colorado (4 cases). Brie cheese imported from France was identified as the vehicle by epidemiological and laboratory investigations. Stools of the victims contained enterotoxigenic E. coli serotype 027:H20. Cultures of cheese did not yield E. coli 027:H20 although coliform counts ranged from 102 to 108 CFU/g (MacDonald et. al 1985). Isolation from stools but not from cheese suggests that other foods or mishandling of cheese during distribution may have contributed to the outbreak. Listeria monocytogenes was not identified as a serious foodbome pathogen until 1981 when 41 cases of listeriosis in Canada, including 17 deaths, were linked to consumption of contaminated coleslaw (Gravani 1999). Despite further evidence 2 years later suggesting possible involvement of pasteurized milk in an outbreak of listeriosis in Massachusetts, the presence of L. monocytogenes in food was not yet regarded as a major threat to public health. However, this situation changed dramatically in June of 1985 when a major listeriosis epidemic occurred in California (Linnan et. al 1988). As many as 300 cases, including 85 fatalities were reported (Gravani 1999). In 1988, Linnan and his team published their findings concerning 142 cases in Los Angeles County that were linked to this outbreak. Ninety-three (65.5%) cases involved pregnant women or their off- spring with the remaining 49 (34.5%) cases involving non-pregnant adults. F orty-eight of the 142 listeriosis victims died giving a mortality rate of 33.8%. L. monocytogenes serotype 4b comprised over 80% of the patient isolates (Linnan et. al 1988). Listeria- contaminated Mexican-style cheese fi'om a single factory was confirmed as the vehicle of 11 (I transmission, since the serotypes and phage types of isolates from cheese and the clinical cases were identical. Listeria was not detected in raw milk samples from dairy herds that produced milk for the factory. However, these samples were taken after the cheese factory closed. The factory environment and equipment were grossly contaminated with L. monocytogenes, including a vat pasteurizer that yielded the organism after clean up. This pasteurizer, used to process a milk-vegetable fat premix used in cheese, had neither controls nor a head space heater. Final reports indicated that L. monocytogenes most likely entered the cheese during manufacture through direct addition of raw milk. Since the plant received 10% more raw milk than could be pasteurized by their pasteurizer, unpasteurized milk was possibly mixed deliberately with pasteurized milk for cheesemaking (Linnan et. al 1988). Considerable evidence indicates that L. monocytogenes and Listeria innocua are primarily introduced into cheese during curing. Such contamination has occurred in cheeses produced from either pasteurized or raw milk (Bradshaw et. al 1 98 7, Mossel I 98 7, Prentice and Neaves I 98 7). Outbreaks of listeriosis associated with soft and semi- sofi ripened cheeses prepared from either pasteurized or raw milk have been reported in France (Anon. 1988b), Switzerland (including a large listeriosis outbreak traced to Vacherin Mont d'Or soft-ripened cheese) (Breer 198 7, Bula et al. 1988, Malinverni et. al 1985, Mossel 198 7), and the United Kingdom (Azadian et. al 1989, Bannister 1987). Investigations in the UK. have shown that listeriosis can be transmitted via soft cheese to immunocompetent, healthy individuals (Azadian et. al 1989, Bannister 198 7). In 1987, a woman was hospitalized with symptoms of fever, back pain, aching legs, and neck stiffness which led to severe meningitis (Bannister 198 7). Listeria monocytogenes 12 serotype 4b was isolated from cerebrospinal liquid (CSF) and the remaining portions of some French soft cheese from her refrigerator. However, L. monocytogenes was not recovered from unopened packages of commercial cheese, which were prepared from pasteurized milk. In another case, a 40-year-old immunocompetent woman was hospitalized with a 4-day history of headache, fever, and one episode of vomiting. She had consumed most of a 4 oz (114 g) package of goat's milk whey cheese about 24 h before developing symptoms. Listeria monocytogenes serotype 4b was isolated from the patient's CSF and from four packages of cheese (30 to 50 million organisms/g). Listeria populations reportedly increased while the cheese was stored in a display cabinet at 8°C (46.4°F) (Azadian et. al 1989). These incidents emphasize that post-pasteurization contamination and growth of L. monocytogenes are important risk factors in cheese-bome listeriosis. Standard sanitation operating procedures for the factory, good manufacturing practices, use of active starter cultures, good personal hygiene, and careful cheese handling until consumption must be followed, especially when cheese is prepared from raw and subpasteurized milk. LISTERIA MONOC YT OGENES Listeria monocytogenes, one of six species of Listeria, is generally hardier than the aforementioned foodbome pathogens including S. Typhimurium, E. coli 01 57:H7 and S. aureus, in terms of heat/acid resistance, temperature, aw and pH ranges for survival and growth. Listeria monocytogenes is a small (0.5um x 1-2um), gram-positive, non- spore forming rod with rounded ends. Cells are usually found singly, or in short chains, or may be arranged in V and Y forms. Listeria is motile by peritrichous flagella when cultured at 20-25°C, but not motile when grown at 37°C. Listeria grows well on most 13 (I commonly used bacteriological media. The growth rate is increased by the presence of fermentable sugars, particularly glucose. Normal temperature limits for growth are +1- 2°C to 45°C (Gray and Killinger 1966) with an optimum range of 30-37°C (Petran and Zottola 1989, Seeliger 1961). Growth is slow at refrigeration temperatures, with generation times of 30-40 h at 4°C. Listeria is one of the few foodbome pathogens that can grow at an aW value of 0.93 (Rocourt 1999). Listeria monocytogenes is ubiquitous in nature, being commonly found in soil and water and on plant material, particularly that which has undergone decay. The organism can survive longer under adverse environmental conditions than many other non spore-forming foodbome pathogens. This resistance, together with the ability to colonize, multiply, and persist on processing equipment makes L. monocytogenes a major threat to the food industry (F enlon 1999). Listeriosis, the human disease caused by L. monocytogenes usually occurs in certain well-defmed high-risk groups including pregnant women, neonates and immunocompromised adults (elderly people and those suffering from diseases like AIDS) but may occasionally occur in people who have no predisposing underlying condition. Unlike infection with other common foodbome pathogens, listeriosis has a mortality rate of ~20% (Gellin and Broome 1989). Manifestations include septicemia, meningitis (or meningoencephalitis), encephalitis, and bacteremia in immunocompromised individuals; sepsis or meningitis in neonatal infection (depending on onset time) (Gray and Killinger 1966, Seeliger 1961) and intrauterine or cervical infections in pregnant women, which may result in spontaneous abortion (2nd/3rd trimester) or stillbirth. Overall mortality may be as high as 70, 80 and 50% in cases of meningitis, septicemia and perinatal/neonatal infections, respectively (F DA/CFSAN). The onset of the aforementioned disorders is 14 97‘. ,-4 its Kl r; usually preceded by influenza-like symptoms including persistent fever. Gastrointestinal symptoms such as nausea, vomiting, and diarrhea may precede more serious forms of listeriosis or may be the only symptoms expressed in normal hosts that consume foods contaminated with L. monocytogenes (Dalton et al. 1997, Slutsker and Schuchat 1999). The onset time for the most serious forms of listeriosis is unknown but may range from a 3 to 70 days. The onset time for the gastrointestinal form of listeriosis is far shorter, ranging from 12 hours to a few days (FDA/CFSAN). This uncertainty in onset time leads to obvious difficulties in identifying cases of foodbome listeriosis. According to the Food, Drug and Cosmetic Act of 1938, a food may be considered adulterated and therefore unfit for human consumption if the product contains harmful substances (e. g., pathogenic organisms). While still unknown, the oral infective dose of L. monocytogenes varies widely with the strain and susceptibility of the individual. Evidence from cases contracted through raw or supposedly pasteurized milk as well as the California cheese outbreak suggests that the number of L. monocytogenes cells needed to induce listeriosis may be quite low — perhaps as few as several hundred to a few thousand cells in certain high-risk segments of the population. Consequently, because of the moral obligation to the public, the FDA has adopted and continues to uphold the current policy of “zero tolerance” for the presence of L. monocytogenes in ready-to-eat foods (Ryser I999c). L. monocytogenes may invade the gastrointestinal epithelium. Once the bacterium enters the host's monocytes, macrophages, or polymorphonuclear leukocytes, it is bloodbome (septicemic) and can grow. Its presence intracellularly in phagocytic cells also permits access to the brain and probably transplacental migration to the fetus in 15 pregnant women. The pathogenesis of L. monocytogenes centers on its ability to survive and multiply in phagocytic host cells (FDA/CFSAN). Sporadic cases of listeriosis in dairy cows (symptoms include encephalitis, abortion and septicemia) in which L. monocytogenes was intermittently shed in milk over several lactation periods have been recorded in the literature for more than 50 years. The apparently normal appearance of milk and consumption of raw milk on farms could be important factors 1n the transmission and epidemiology of milkbome listerial infection (Wesley 1999). Dairy cows that appear healthy can serve as reservoirs for L. monocytogenes and secrete the organism in milk (Ryser 1999b). Milk and milk products have been linked to cases of foodbome listeriosis for over 17 years. Following the pasteurized milk outbreak in Massachusetts and the California cheese outbreak, FDA officials in cooperation with state governments and the dairy industry intensified their surveillance programs under the Dairy Safety Initiative Program, which began April 1, 1986 (Kozak 1986). FDA surveys in 1986 indicated that an average of 2.5 % of all dairy products manufactured from pasteurized milk contained L. monocytogenes (Anon. 1986). A subsequent report in February 1987 indicated generally similar contamination rates with 2.6% of dairy processing facilities manufacturing finished products containing L. monocytogenes (Anon. 1987c). Listeria monocytogenes is reportedly present in 1.6 to 12.0% of all raw milk produced in the United States (4% average) (Donnelly et al. 1988, Hayes et. al 1986, Liewen and Plantz 1988, Lovett 1987). Incidence rates outside the United States are generally similar with 1.3 to 5.4% of Canadian and 2.5 to 6.0% of Western European raw milk yielding L. monocytogenes (Ryser 1999a). While some early reports indicated that L. monocytogenes could survive pasteurization (Beams and Girard l6 1958), others proved these findings to be false (Bradshaw et. al 1985, Farber 1989, Mackey and Bratchell 1989). As mentioned before, this pathogen has thus far been responsible for four major soft cheese-related outbreaks that included over 100 deaths (Ryser 1999a). Owing to the current "zero tolerance" policy, L. monocytogenes has extracted a particularly heavy toll on the dairy industry in terms of Class I recalls. SURVEILLANCE FOR L. MONOCYTOGENES IN CHEESE As a result of the 1985 listeriosis outbreak in California, FDA officials added L. monocytogenes to their list of bacterial pathogens that should be of concern to cheesemakers and began surveying various sofi domestic cheeses for listeriae. Less than one month after the first nationwide Class I Listeria-associated recall (Table 2) was issued for 22 varieties of Mexican-style cheese (~500,000 lb) contaminated with L. monocytogenes, the FDA developed a series of programs designed to prevent the reoccurrence of such an outbreak (Skinner 1989) (Figure 1). The Domestic Soft Cheese Surveillance Program-the first of the Dairy Initiative Programs-was instituted by the FDA in July of 1985 and involved on-site inspection of firms manufacturing soft cheese (Anon. 1985). Priority was given to manufacturers of Mexican-style soft cheese, followed by firms producing other ethnic-type soft cheeses such as Edam, Gouda, Liederkranz, Limburger, Monterey Jack, Muenster, and Port du Salut from raw, heat-treated [<71.7°C (161°F )/ 15 sec] or pasteurized [>71.7°C (161°F)/15 sec] milk. Between June 1985 and October 1988, FDA inspectors collected cheese samples to be analyzed for L. monocytogenes using the original FDA procedure (Ryser 1 999a). l7 1.. Alt .1 03.? W, 1 AA .1!) Vv. “c ”'1 (I) Less than 2 months into this program, FDA officials isolated a pathogenic strain of L. monocytogenes from one sample of domestically produced Liederkranz cheese. In general, FDA inspections of other soft cheese factories uncovered problems similar to those encOuntered during inspections of Grade A fluid milk factories: (a) potential bypasses of the pasteurizer, (b) post pasteurization blending of product, and (c) a general lack of education and/or training of factory personnel (McBean 1988). Items of particular concern to cheesemakers that were not generally found during the above visits included defects in the pasteurization process, discrepancies in pasteurization/production records, and a higher incidence (than in Grade A milk factories) of pathogenic microorganisms (including L. monocytogenes) on environmental surfaces in production and storage areas (Ryser 1999a). Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988 under four separate programs (Figure 1) with FDA officials reaching nearly half of the 400 soft cheese factories in the United States by April of 1986 and the remaining factories (including follow-up inspections of problem factories) by late 1987 (Anon. 198 7b). According to FDA records (Archer 1988), L. monocytogenes was identified in 12 of 586 (1.82%) domestic cheese samples analyzed during 1986. During these inspection programs, six Class I recalls were issued for various ethnic-type soft and semi-soft cheeses contaminated with L. monocytogenes (Table 2). Given the ability of L. monocytogenes to grow in these soft cheeses during refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a significant public threat, with these varieties thus far accounting for 13 of 38 recalls issued (Table 2), including one large recall in 1990 involving approximately 500,000 lb of product. 18 008m 00%03003 -00- ooodom .00w05 .000>0Z 0:03 ”0000.300 0000.2 0:008:00 oQQ : 800:0 08 008000082 0:00.. 3000002 000 060—. 30m + m00000> 008m 00:00 w 000 .0805 8000 -00- 030503 00%0203 0000:. .0002": 00:00:00 .0083 00:03:00 30m: .0300 ”0:30-003082 00m -8- 8:2 28 0305083 008000 58803 5:8 02020 32... 009 300 00000 -00- coma :2 000500002 303000: SEE 000 _0:00m “000000-08 080000 -00- cm: 0000380000 .030 0020000 :002 £002: 000:: 2:2? 05 ”000000-08 00:3 -8- 80.2 00 0900233 .0505 0505 00: 00 2088908 08008 0000fi0> 00:00 m 000 080$ -00- 30.02 00x00. .00w80 0000300 00005 0:008:00 0w\m\m 8000 ”030-003082 00m 90:80:00 -00- 000.32 002 00000 600500002 0EC 332w “.006 500000005 008m 08902003 005 805,—. .0080m .0003 0005— .00w05 080:030 .0000: 302 00082 302 £800.. 30 Z .000>0Z 0080:0083): £0003 =0:E02 000880.: 0000000, 0050 cm 000 333 88000: .$002: 0:03 .0030: .8000 .0mw0000 .0800“: 8000 .0300 ”008 .0835 000.com... 0000200 0:008:00 88000—2 000004 0:008:00 .3026 0000002 a8 000.5 0820—. 30 8003 00% 500000 00:05:85 0&8 =008 00D 800:0 «0 09C. 00=0M00A00=0£ {N 505 000050000000 800:0 0:80.03: 00.: 00005 00:03 05 0: 020.00% _ 80.0 .00 83 00%200030 ”N 030:. 19 00:3 -00- 0300003 0008-:- m0x0-_- 0QQO 08 0300-0000002 00:3 -00- 0300003 0000:- 00x0:- 00: NR 08 0008-0000002 00:3 -00- 0300000 0000-:- 00000- onQm 08 038000002 -00- om 0080:0800: 000000 0000000000 0080:8802 00: _ \m 08: 000 800:0 00000 -00- am: 00000803 $800—- 302 .0002”: 00000803 00% : \0 00800.00 8000 00000803 68000000. 000000 500m 000—0000 5.50 500 050200 582 000.502 008.0502 -00- 3.3 .030: .000000: 0000:: 0&0000 .0002”: 00000000 00000803 35:5: 800:0 80000 0800000.:- 50- 20.: 0.00502 00350 .0505 .020: 05000.. 8020.0- 3.00 002% 0820 -00- . cow _ 0800803 00000803 N00: :3 0080:0000 -00- 0300003 008m 00%02003 .00m05 00%03003 NQE 5: 0800 8000 00000803 0000?; J00000> .0000:- .0800000-_- 00002000000 60:0 08> 302 .3885: .5022: 05.32 .0505 .020: -00- 030000: 003000 .0002: 6000200 0000-000 00000-2. 00000803 NQotm 000-: 800:0 0000-0—00 -8- 88.2 08803 08053 .032 58803 3002 0.20 -8- 8: 00> 302 .0280 00> >52 5: E 00800 00800803 4000?; 803 .0000:- .0000>_>m000m 00:0 -8- 80.0% 08> 52 .5022: .080: .0080 080888 58803 530 £230.02 000000 .030 08 T 808 0300 E88580 800: E00 002% 885 30 8003 00.0 000000 0000:0000 003.00 :0000 0000 800:0 00 00>:- 03080 N 0300- 20 A39“ N mammmouu S35 555:5 oursaocmz £83804 ma: \m Hum—mm 88:0 2.5 fimqi Swag N .233 8.3 85232 588m; 32% 3.8% 02m -8- 598%: «852 .8828 aéoaau «E025 35m saga: «335 638500‘ £5330 -0? 850533 £50m rag—850 5.52 dmwuooo 62.82 6.5an 260809 meQm 88a 80:0 -ow- wmadvm $35232 £9835, maxim 83c 08:0 EoEB> “Ea—mm 209E .x~o> BoZ Samba 302 -oc- :25:ch .EEmQEmE >62 .mtomanommmmg .0522 4:282:80 3085:0832 3:1: 0 88:0 :820 Eo:5> .mEmmeEBA .982 393 Jr; 302 £36.. 302 838%? 65, -ov- ovfin day—mafia: 262 $535832 6532 .3082800 30950332 35 Q2 38:0 :320 588mm? 63% :Swaflmma .oommozcoh dig—35$ 65630 5.82 1.8% 302 inflow 302 508552 -ou- 83 £25: dmwuooO £3.85 63.8.00 d€8§0 58083? onN SoNaowSO -ov- onmm mafiixflfig oEO vaQS mmrsm v08 08vo -ov- S m 030 .953va Ema—comm? $2 302 £082 30 Z 2080—. 302 00: B 0085 08:0 008m 00098003 .000880H 080320802 05—080 582 28000:): 002080 002: .2200 000 .5022: 8500 .0288 £232 000%? 0:802 00: :0 88 88 >30 :05 6008800. 0:20:00 £20m 0802202002 .80> 302 80000:): 8003002 3:00 83:0 @003 .202 3 mm: .0802: 08800 8008—00 0:88:00 .085 820082 onQm 0:8 30M 0:00.. 2080002 082 .2200 88 80¢ 033 332m 8808 0::080 00025 Q32 .202 3 N: m 0:20:00 :Som 05—0800 5.82 :082 00R: 080:0 00008:: 080:0 02:3 :802 383 .202 3 030583 80> 302 80> 302 8ka 020m 000882 080:0 A0000: .2023 mo— 80> B02 80> 302 woBQm 02:3 880% 30 8008 00% 280000 0000:8005 2880 :82 0000 080:0 .8 09¢ 8,8003 N 0::0H 22 .0000 N 200.05 80:0 0088000 008cm 030:0 008008: 0:0 0800800 8 .000 0.2050 00.: 080.&0:0 00:0: 50: 00:80000 8&0: I 0880 00.00800 8 080:0 8008: 00 008m a $2 :3 503 I 80&0:0 0080:0080 800:0 :000: a $2 :5. 503 I 80&0:0 800000000 5:000 + 50: 0:3. I 800:0 0:080”: 80:0: 800.: 00300: 0020M0~A002022 d =0>8m A 0.:&E 30: I 80.&0:0 0080:0080 :0&0500 80:0& 000:: 030:0 A £2 :00. 203 I 00080 0503 300:0 00:00:08 :0:0.£\0>8m H 52 0002 I 3:3 080:0 £00 .«0 8:080:00 : 0&0: E0083. 8&0: 0080 080:0 00:00:: 0:0 00w< 0&0: :0002.8&0: I 80&0:0 f 0805080 800:0 .000 008008: 90.50002: £0: E083. I 30800600 080:0 08m 5:000 800.: 080:8: 0020M000002022 IN £0: fidm:<-.¢0=:0m ”0:000:80 80800: 00800000 a 30: 00:. 8&0: I 80.&80 08050200 800:0 000 0000800 03mmEOQ 23 MI.” 3" 1' an. PIS 1H. 1 J in in] While all products contaminated with L. monocytogenes must be retrieved from the marketplace, formal Class I recalls do not have to be issued for contaminated products that have not yet left the factory. Since such situations typically lead to nonpublished "internal recalls" issued by the manufacturer, more cheese was likely destroyed during this 12-year period than has actually been reported (Ryser I999a). Following a report by Ryser and Marth (Ryser and Marth 1987a) that L. monocytogenes can survive more than one year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period for Cheddar cheese manufactured from raw milk), the FDA modified its Domestic Cheese Program in August of 1987 to include cheese prepared from unpasteurized milk (Anon. 1987b). Between April and October of 1987, 181 samples of domestic aged [held a minimum of 60 days at 217°C (35°F)] natural cheese manufactured fiom raw milk, as well as similar imported cheeses in domestic status, were collected from retail stores by FDA field personnel and analyzed for L. monocytogenes. These efforts uncovered one positive sample-a sharp Cheddar cheese manufactured in Wisconsin, which was subsequently recalled from the market in July of 1987 (Table 2). Isolation of L. monocytogenes from several imported Brie cheeses between 1986 and 1988 led to the eventual recall of approximately 300,000 tons of Brie cheese imported from France (Table 3) which prompted a real concern about the incidence of this pathogen in other European cheeses. Recall of the Brie cheese led to two corrective measures: (a) adoption of a cheese certification program by the United States and France to prevent importation of Listeria-contaminated cheese and (b) initiation of numerous large-scale surveys to determine the extent of Listeria contamination in virtually all types 24 of cheese manufactured in the United States, Canada, and Western Europe. After first isolating listeriae from a hard cheese (Italian Pecorino Romano cheese prepared from goat's milk) in June of 1987, (Figure 1) (Skinner 1989), the previous import alert was extended to include both soft and hard varieties of Italian cheese (Anon. 1987a). Subsequently, the FDA ordered intensified sampling of soft and hard cheese for the next two months as part of the ongoing imported cheese surveillance program (Figure 1) (Anon. 1988b). This action prompted the recall of several Danish cheeses in early 1988, four separate Class I recalls for Listeria-contaminated soft cheeses manufactured in Cyprus (Table 3) as well as an import alert for contaminated soft and hard cheeses produced by two Italian firms in the latter half of 1989 (Farber et al. 1988). The overall situation regarding presence of L. monocytogenes in imported cheese has greatly improved since 1986 (Anon. 1990) with only four additional recalls of imported cheese since 1990 (Table 3). Sporadic detection of listeriae in domestic and imported cheeses suggests that surveillance of such products is still necessary to safeguard public health. Moreover, as mentioned earlier, class I recalls lead to heavy economic loss in terms of product retrieval, product disposal and consumer lawsuits as well as possible loss of market share for the company’s products, lost productivity and related medical expenses. 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MONOC YT OGENES IN DIFFERENT PRODUCTS Cheesebome listeriosis outbreaks prompted scientists on both sides of the Atlantic Ocean to determine the incidence of Listeria spp. in various cheeses and examine the behavior of L. monocytogenes during manufacture and storage of fermented dairy products (Ryser 1999a). While results from two Yugoslavian studies concerned with behavior of L. monocytogenes in various fermented dairy products were published in 1964 (Ikonomov and Todorov 1964) and 1981 (Stajner et al. 1979), neither surveys dealing with the incidence of listeriae in fermented dairy products nor research on behavior of L. monocytogenes in cheese was conducted before contaminated Mexican- style cheese was linked to the California listeriosis outbreak in June of 1985. Milk Fermentation Schaack and Marth (1988a) investigated the fate of L. monocytogenes in sterile skim milk that was fermented with Lactococcus lactis subsp. lactis (LL) and Lactococcus lactis subsp. cremoris (LC) in sterile skim milk. Milk samples containing different levels of LL or LC (5.0, 1.0, 0.5 or 0.1%) was inoculated to contain one of two L. monocytogenes strains at a level of 103 CFU/ml. Inoculated milks were fermented for 15 h at 21 or 30°C, followed by refrigeration at 4°C. Listeria monocytogenes survived all fermentations and grew to some extent. Incubation at 30°C with 5.0% LL was most inhibitory to L. monocytogenes. At 30°C, LC was less inhibitory to L. monocytogenes than LL at inoculum levels of 0.1 and 5.0%. Growth of L. monocytogenes generally ceased when the pH dropped below 4.75. In a similar study by El-Gazzar et al. (1992), L. monocytogenes was inhibited by a four strain mixture of LC in sterile skim milk but survived the 36-h fermentation at 30°C. 28 When this milk was stored at 4°C, L. monocytogenes survived 4 to 6 weeks, with the length of survival dependant on the Listeria strain. Both of these studies show that L. monocytogenes can survive in milk fermented by mesophilic lactic acid bacteria used in cheesemaking, thus, suggesting potential public health problems if post-processing contamination of cheesemilk occurs. Behavior in cheese Ryser and Marth (198 7a) studied the fate of L. monocytogenes during Cheddar cheesemaking and ripening. Pasteurized whole milk inoculated to contain ~2.5 logs of L. monocytogenes CFU/ml was made into stirred-curd Cheddar cheese. Cheese was ripened at 6 or 13°C. During cheese manufacture, Listeria counts remained relatively constant. After overnight pressing, numbers of L. monocytogenes increased to about 3 logs/g of curd. Highest numbers of Listeria, ~3.5 logs/g, were detected in cheese after 14 days of ripening. The three different L. monocytogenes strains studied survived as long as 224, 154 and at least 434 days in Cheddar cheese of normal composition with greatest survival generally seen in cheese ripened at 6 rather than 13°C. Additional studies conducted with Salmonella Typhimurium and Escherichia coli OlS7:H7 showed that these pathogens could survive up to 210 days (Goepfert et al. 1968, Hargrove et. al 1969, Park et al. 1970) and 138 days (Reitsma and Henning 1996), respectively, in Cheddar cheese produced from artificially contaminated pasteurized milk. Yousef and Marth (I988) prepared Colby cheese from pasteurized milk that was inoculated to contain 102-103 L. monocytogenes CFU/ml. Up to 3.2% of the Listeria population was recovered in the whey and the mean count in the curd was 1.27 log higher . than in the milk. The cheese was ripened at 4°C for 140 days. Listeria populations 29 remained fairly constant during the first 3 to 5 weeks of ripening. Thereafter, numbers of Listeria decreased almost linearly. The D-values were 143 and 105 days in 2 cheeses having >40% moisture and 51-67 days in 4 cheeses with <40% moisture. After 140 days, survival differences were observed between the two strains with higher initial numbers of Listeria in milk leading to greater survival. Hence, both of these studies indicate the ability of L. monocytogenes to survive beyond the mandatory 60-day ripening for Cheddar and Colby cheese. Parmesan cheese, a hard cheese with a low moisture content, was prepared by Yousef and Marth (1989) from pasteurized milk inoculated to contain ~104-105 L. monocytogenes CFU/ml (2 strains studied). Unlike the previous cheeses, a lipolytic enzyme (lipase) is ofien added to cheesemilk to produce the characteristic flavor of fully ripened Parmesan cheese. The coagulum was cut into very small particles and cooked at ~52°C (125°F) for 45 min until the pH decreased to 6.1, producing a dry, rice-like curd which was pressed to form a very dense, low-moisture cheese. Following manufacture, the cheese was brine salted (22% NaCl) for 7 days at 13°C, dried 4-6 weeks in a humidity-controlled chamber at 13°C, vacuum-packaged, and ripened at 13°C for a minimum of 9 months. During the first 2 h of cheesemaking, populations of both Listeria strains increased approximately 6- to 10-fold. Although Listeria counts remained relatively stable during cooking, populations decreased appreciably during pressing of the curd. During brining, drying and ripening at 13°C, numbers of both Listeria strains decreased almost linearly, with estimated D-values ranging between 8 and 36 days. Using direct plating, the 2 strains were no longer detected in cheese after ~14-112 days of ripening at 13°C. Despite large differences in survival of L. monocytogenes between 30 different batches of cheese, both Listeria strains decreased at a faster rate in Parmesan than in Colby or Cheddar cheese (Ryser and Marth 1987a, Yousef and Marth 1988) during ripening. Decreased viability of L. monocytogenes in Parmesan cheese is probably related to a combination of factors, including action of lipase added to the milk, cooking of the curd during cheesemaking, and low water activity of the fully ripened cheese. To decrease the moisture content and develop proper flavor, the present regulation in the United States requires that Parmesan cheese be aged a minimum of 10 months regardless of whether or not the cheese is prepared from raw or pasteurized milk thus ensuring its safety. Buazzi et al. (1992a) examined the fate of L. monocytogenes during manufacture and ripening of Swiss cheese, which involves cooking of the curd at 50-53°C and ripening the finished cheese at an elevated temperature for "eye" development. When rindless Swiss cheese was prepared from pasteurized milk inoculated to contain 104-105 L. monocytogenes (l of 3 strain) CFU/ml, the pathogen was generally unable to grow during cheesemaking, with populations increasing 43% during the early stages of cooking owing to physical concentration and curd shrinkage. Thereafter, about 57% of the population in the curd was inactivated after 30-40 min of cooking at 50°C. After pressing, the curd contained 50% fewer listeriae, with this population decreasing most sharply after 30 h of brining at 7°C. Storing the finished cheese (pH 5.2-5.4) for 10 days at 7°C reduced the Listeria population to very low numbers. Complete inactivation of the pathogen occurred after 66-80 days of ripening at 24°C, with production of propionate by eye-forming bacteria likely contributing to the death of listeriae. Two studies conducted 31 in Switzerland (Bachmann and Spahr 1995, Kaufmann 1990) demonstrated that the environments within Emmentaler and Gruyere cheese (other varieties of Swiss cheese) also are not conducive to Listeria survival, with the pathogen no longer being present in 24-h-old cheeses (pH 5.2-5.4) prepared from raw milk inoculated to contain 104 L. monocytogenes CPU/ml. These studies indicate that the manufacturing steps involved in Swiss cheesemaking should ensure the safety of cheese made from raw, heat-treated or pasteurized milk. In another study, Brick cheese was made from pasteurized whole milk inoculated to contain ~102-103 L. monocytogenes CPU/ml (4 strains) (Ryser and Marth 1989). Cheeses were ripened at 15°C and 95% RH with a surface smear for 2, 3 or 4 weeks to simulate production of mild, ripened and Limburger-like Brick cheese, respectively. Cheeses were then stored an additional 20-22 weeks at 10°C. Populations of the four Listeria strains increased 1-2 orders of magnitude following completion of brining ~32 h after the start of cheesemaking. All 4 strains leached from cheese into the 22% brine solution during 24 h and survived in the brine at 10°C for at least 5 days after cheese removal. During initial smear development, two strains grew rapidly to different levels depending on the type of sample and the pH - i.e. 6.6 and 6.2 logs/g in 4-week-old slice sample (pH 6.0-6.5); 7.0 and 6.9 logs/g in the surface (pH 6.5-6.9) samples; and 5.6 and 5.1 logs/g in the interior (pH 5.6-6.2) samples. Numbers of these two strains generally decreased l-to 7-fold during 20-22 weeks at 10°C. The two remaining strains failed to grow appreciably in any cheese during or after smear development, despite a pH of 6.8- 7.4 in fully-ripened cheese, and were not isolated from 2- and 3-week-old cheeses. Using direct plating, both strains were detected sporadically at ~4 log CFU/ g in 4-week old 32 cheese. Cold enrichment of 4—week old slice, surface and interior samples generally yielded positive results for L. monocytogenes. Inhibition of these two strains could have been due to smear-ripening organisms, which can reportedly produce bacteriocin—like substances active against listeriae (Ennahar et al. 1996, Ryser et al. 1994), or heightened sensitivity of these L. monocytogenes strains to the inhibitory effects of certain listeriocidal fatty acids (i.e., linoleic) and monoglycerides (Wang and Johnson 1992) produced during cheese ripening. In 1995, Bachmann and Spahr (1995) manufactured Tilsiter cheese, a semi-firm, slightly yellow, smear-ripened variety similar to brick cheese from milk inoculated to contain 104 L. monocytogenes CFU/ml. Overall, their findings were similar to those observed for two of the L. monocytogenes strains in brick cheese (Ryser and Marth 1989), with Listeria populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of ripening at 10-13°C. The above studies show that L. monocytogenes can survive during manufacture and ripening of smear-ripened cheeses due to the increase in pH to that occurs as a result of bacterial growth on the cheese surface. Similar studies have been done on other cheese varieties (Margolles et al. 1997, Papageorgiou and Marth 1989a, 1989b, Razavilar 1997, Rodriguez et al. 1998, Ryser and Marth 1987b, Stecchini et al. 1995); the results of which are summarized in Table 4. Growth and survival of L. monocytogenes also was investigated in market cheeses that were purchased, inoculated and then stored at 4 to 30°C (Genigorgis et al. 1991). Results from this study are summarized in Table 5. All of the aforementioned studies except those for Swiss and Parmesan show that L. monocytogenes can persist beyond the mandatory 60-day ripening period for cheeses 33 that can be legally prepared from raw or heat-treated milk. These studies point out the need to re-evaluate the safety of current cheesemaking practices. If found inadequate, appropriate changes in current regulations or alternative technologies should be introduced so that safety of these cheeses can be reassured. FEASIBILITY OF RAW MILK CHEESE According to current FDA regulations (Anon. 1949), milk pasteurization or use of a similar heat treatment during cheesemaking is required for the manufacture of 16 cheese varieties including Brie, cottage, cream, Neufchatel, Monterey, mozzarella, Scamorza, Muenster, Gammelost, Koch Kaese, and Sapsago (Johnson et al. 1990b). Seven varieties of manufacturing cheese (i.e., for use in pasteurized processed cheese, cheese foods, cheese spreads) require neither pasteurization of the cheese-milk nor a 60- day minimum ripening period at >1.7°C (35°F); whereas the 34 remaining cheese varieties (mentioned previously) recognized under current standards of identity must either be manufactured from pasteurized milk or held a minimum of 60 days at >1.7°C (35°F) to eliminate pathogenic microorganisms. Although statistics on milk pasteurization for cheesemaking are scarce, available evidence indicates that ~10% of all cheese produced in the United States (~646 million lb/month for 1999) is prepared from raw or heat-treated (subpasteurized) milk (~65 million lb/month) (Dairy Marketing andamentals, Groves 2000b). 34 332 $35 88m Ham-aux ”850m omega 883 new 27% E :3 mo owfiaoobm u detach 8 Z u .35? wfibmxom .8 3332805 5233 @8395 a mac—«Someone mo 9% 05 Saw : vm memeoam’x a n woo-H 38:0 2: 3-: 3-3 3-3 v 3 3 one 33 E... 88.28 2 2 3.3.3. 3.3 v 3 3 3.8 33 .mz 5:533: SEN 2-3 3-3 3-3 2 3 3 33 and cam 3855 82: 21mm 3.33 233 a. 3 3 33 mad 2:. £8 «mm-E o 3-3 4.33 2 3 3 63 33 NR 3325 :1: 2-0 3-3 3-2 3 3 3 8+ 33 NR .885 8 3.32 3-3 3-3 $2 3. 3 $3 $3 33 see 32 3-3 3-3. 31: oz: 2 3 a: 83 on. firm o2 3-3 3-3. 3-3. «a; 3 3. No: 83 3m 02m 3 3-3 2-3 33-3 35 2 3. N3 323 3.3 203825 GOV Axflsfi was an; sages: .. 3:5 nee 3E . Ea: an; 3 3°C 38.6 325m 358 Egg mm 5 as. Bantam name: wmzmmgnuozos 4 mo Ewe-H 388me ”dogma—:00 83:0 3 cogent/x mm chan 88:0 warn—Q umzmmgnuozofi 4N .3 Hoggom ”v 03mg. 35 Table 5: Growth and Inactivation of L. monocytogenes in Surface-Inoculated Retail Cheeses During Storage at 4—30°C: Cheese category and type pH % NaCl in Growth moisture phase Sofi mold ripened Brie 6.0-7.7 2.5-3.6 + Camembert 7.3 2.5 + Blue 5.1 6.1 - Bacterial surface ripened Limburger 7.2 4.8 - Muenster 5.5 3.8 - Soft Italian Provolone 5.6 4.6 - String cheese 5.5 4.4 - Semisofi and hard ripened Monterey Jack 5.0-5.2 1.0-3.0 - Colby 5.5 4.9 - Cheddar 4.9-5.6 2.6-5.4 - Swiss 5.5 2.7 - Hispanic Queso Fresco 6.5-6.6 4.5-6.6 +/- Queso Ranchero 6.2 4.1 + Queso Panelia 6.2-6.7 2.5-3.9 + Cotija 5.5-5.6 9.6-12.5 - Pickled cheese Feta 4.2-4.3 2.2-7.5 - Ewe '3 milk cheese Kasseri 4.8-5.3 5.5-5.8 - Soft unripened Cottage Cheese 4.9-5.1 1.0-1.2 +/- Cream Cheese 4.8 <0.9 - Whey cheeses Ricotta 5 .9-6.1 <0.7 + Processed cheese American 5.7 2.1 - Monterey Jack 5.7 4.4 - Piedmont 6.4 5.1 - Source: Adapted from (Genigorgis et al. 1991, Ryser I999a). 36 Research on the use of pasteurized milk for cheesemaking began in 1907 as a joint effort between the United States Department of Agriculture and the University of Wisconsin Agriculture Experiment Station. Although the primary goal was improved quality, product safety was also a concern. During the World War 11, many cheesemakers were called into service. Those who took their place were less experienced and had to meet government demands for huge amounts of cheese to fuel the war effort. Thus, quality and safety of the cheese were sometimes compromised. By 1949, pasteurization of milk and dairy products was adopted nationwide (American Cheese Society). The mandatory 60-day holding period at >1 .7°C (3 5°F) for cheeses manufactured from raw or heat-treated milk was also adopted at that time (Anon. 1949, Johnson et al. 1990b) afier researchers demonstrated that Brucella abortus, the causative agent of brucellosis, was eliminated from cheese by such an aging process. Based on epidemiological evidence and outbreak information, the current 60-day holding period generally has been deemed adequate to eliminate most foodbome pathogens. However, considering the results from the aforementioned challenge studies, it appears that organisms such as Listeria and Salmonella can survive well beyond the 60- day ripening process (Goepfert et al. 1968, Hargrove et. al 1969, Park et al. 1970, Ryser and Marth I 98 7a). Consequently, the adequacy of the 60 day hold at > 1.7°C (3 5°F) still remains very much in question with safety concerns regarding such cheeses recently voiced by the FDA as well as the Australian Dairy Industry, the Government of Canada (F arber et al. 1996) and the International Dairy Federation. In keeping with the grave nature of listeriosis as compared to most other foodbome illnesses, the FDA has continued to maintain a policy of “zero tolerance” for L. monocytogenes in all ready-to- 37 eat foods. Thus far, no well-documented cases of listeriosis have been associated with consumption of cheeses that were legally prepared from raw milk and held a minimum of 60 days at a temperature of 317°C (35°F) before sale. At FDA's request, the Cheese Subcommittee of the National Advisory Committee for the Microbiological Criteria of Foods reviewed the current data and concluded that the 60 day holding period at _>_ 1.7°C may be insufficient to eliminate all foodbome pathogens (Anon. 1997). The Cheese Subcommittee also recommended that the FDA re-exarnine its current policy regarding the 60-day aging period for hard cheeses prepared from raw and heat-treated milk. Since ~4% of the raw milk supply can be expected to contain L. monocytogenes, it would be prudent to manufacture cheeses from pasteurized milk whenever possible. Although Yousef and Marth (1989) demonstrated that ripening Parmesan cheese for 10 months, as legally required, is sufficient to produce a high-quality, Listeria-free product, desirable flavor and texture characteristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteurized milk. Hence, alternative means should be developed to enhance the safety of these products. Methods used could include cold sterilization of the milk via microfiltration, sublethal heat treatment (short of pasteurization) or addition of various flavor- and texture-enhancing enzymes (or microorganisms) to pasteurized milk, which would allow the cheesemaker to obtain a higher quality product (Johnson et al. 1990c). POLITICAL CLIMATE SURROUNDING RAW MILK CHEESE On the international front, the Codex Committee on Food Hygiene, at its meeting in October 2000, considered a proposed code of hygienic practices for milk and milk products that stops short of requiring mandatory pasteurization of milk. The proposal will 38 be considered at Step 3 in the eight-step Codex process. Several years may be needed until this policy is officially adopted by Codex. Codex is not in favor of mandating pasteurization, but will leave the decision to individual countries. These countries also will be able to determine their own level of public health protection concerning imported dairy products (Groves 20000). On the domestic front, current participants include government officials along with various dairy industry representatives and consumer groups, all of which have diverse opinions. In 1999, FDA announced that it was rethinking the 60-day aging rule. On July 28, 2000, the front page headline in the Cheese Reporter (Anon. 2000b) read: “60-Day aging may be inadequate to eliminate E. coli in cheese: Research.” This article discussed some studies being conducted at the Illinois Institute of Technology and FDA’s National Center for Food Safety and Technology in Summit-Argo, IL to confirm prior work suggesting that 60-day aging of hard cheese made from unpasteurized milk is inadequate to protect public health. Based on preliminary findings, E. coli OlS7:H7 decreased 1 log in raw milk cheese (initial inoculum of 105 cells/ml in raw milk) after 60 days of ripening with E. coli still detected after 360 days (Anon. 2000b). This decision by FDA to review the 60-day aging rule is part of the Food Safety Initiative Program developed by President Clinton. Whether his successor shares the same zeal for the safety of raw milk remains to be seen. On the industry front, there is certainly nothing resembling unanimity on this issue. The National Cheese lnstitute’s proposed general standard, which is somewhat misunderstood, calls for pasteurization or an equivalent process for dairy ingredients used in cheese. "Equivalent process" is not defined. The Cheese of Choice Coalition was 39 formed recently by the American Cheese Society and Old-Ways Preservation Trust to advocate continued use of raw milk in cheesemaking. The American Dairy Products Institute's Cheese Division also supports traditional "curing" methods for cheeses made from unpasteurized milk, including the 60-day aging period (Groves 2000a). Consumer groups also are divided on this issue. The Center for Science in the Public Interest seems likely to support mandatory pasteurization. However, Consumer Alert prefers consumer choice in this matter. On July let, 2000, Digby Anderson, director of the Social Affairs Unit, decried FDA’s possible ban on unpasteurized cheeses in an editorial column of the Wall Street Journal. His column prompted three letters to the editor of that paper, one from Consumer Alert's executive director, all supporting his views (Groves 20000). Based on the preliminary E. coli findings and earlier work with Listeria, FDA may eventually propose mandatory pasteurization, or an equivalent, which would force opponents of mandatory pasteurization to come up with an acceptable alternative. The annexes in Codex's proposed milk hygiene code could play a key role since alternatives to pasteurization are outlined that can help ensure the same level of public health protection. Barring an outcome that satisfies all parties, Congress might eventually ask to "referee" this issue, making it more political than it should be (Groves 2000a). RAW MILK VERSUS PASTEURIZED MILK CHEESE Beneficial effects of milk heat treatment The proportion of casein and milk fat converted to cheese primarily dictates potential cheese yield. Casein (in micellar form) is in colloidal suspension while fat (triglycerides) is in an oil-in-water emulsion. Enzymatic degradation can increase the 40 solubility of casein and milk fat. Casein can become more water soluble via chemical changes that do not require enzymatic catalysis. Researchers (Ali et. al 1980a, 1980b, Pierre and Brule 1981) have reported that cold storage of raw milk causes solubilization of colloidal calcium phosphate (casein bound) and a concomitant shift in caseins from the micellar to soluble state. Ali et al. (1980a) showed that these events caused an increase in rennet clotting time, reduction in firmness of rennet clot and reduced cheese yield. They also demonstrated that solubilization during cold storage could be reversed by heating at 60°C (140°F) for 30 min or 72°C (161.6°F) for 30 to 60 sec, although the milk equilibrium system never fully regained its initial state. Qvist (Qvist 1979) reported that pasteurization at 72°C (161.6°F) for 15 sec after cold storage at 5°C (41°F) caused the dissociated casein micelle components to return to micellar form. Additionally, pasteurization shortened the secondary (ionic) phase of coagulation to the level of , uncooled milk, but did not reestablish the original rennet clotting time because the primary (enzymatic) phase was not shortened. In some cases, pasteurization further prolonged the primary phase during cold storage. Johnston et al. (1981) provided evidence that pasteurization afier cold storage resulted in the recovery of soluble casein and calcium, but showed that both pasteurization at 72°C (161.6°F) for 15 sec and heat- treatrnent at 60, 65 or 70°C (140, 149 or 158°F), for 10 sec and repeated for 15 sec, resulted in significantly prolonged primary and secondary stages of coagulation relative to an unstored unpasteurized control. However, heat-treatment did not cause serious changes in cheesemaking performance. Dzurec and Zall (1985) showed that soluble 13- casein decreased with severity of the heat-treatment and subsequent cold storage of milk. 41 81.: pr 1? . lu 1.\ Several investigators (Banks et. al 1986, Price 192 7, Price and Call 1969, Wilson et. a1 1945) demonstrated that cheese made from pasteurized milk exhibited better overall quality and fewer flavor defects than raw milk cheese. Proliferation of psychrotrophic bacteria in either raw or pasteurized milk before cheesemaking, can lead to development of off flavors, gassiness and poor cheese quality (Cousin 1982, Law 1979). These proteolytic enzymes produced by psychrotrophic bacteria are not destroyed by pasteurization or thermization (Stadhouders 1982). Studies reported that on-the-farm thermization kept the bacterial numbers low and improved the quality of cheese when compared to cheese made from unthermized milk (Banks et. al 1986, Coghill et. al 1982). Wilster (Wilster 1980) stated that pasteurizing milk for cheesemaking afforded much easier control of the cheesemaking process, especially in regard to control of acid development, which is almost solely due to the starter culture with little influence from microorganisms present in raw milk. The cheesemaking process, and consequently the cheese, would be more uniform from day to day using pasteurized milk. Detrimental effects of milk heat-treatment When milk is heated sufficiently, B—lactoglobulin reacts with K-casein on the casein micelles resulting in denaturation. Depending on the severity of the heat-treatment given to milk and consequent denaturation of B—lactoglobulin, heated milk may show poor rennetability (increased clotting and hardening time, reduction in firmness of the coagulum) and less spontaneous whey drainage from the coagulum (syneresis) compared to untreated milk (Hermier and Cerf 1986, Hooydonk et. al 198 7). These effects could result in lost yield, high moisture, and body/textural defects. Ustunol and Brown (1985) stated that milk used for cheesemaking should not be heated more than required to meet 42 current pasteurization requirements since it could impair the enzyme-catalyzed clotting of milk. However, other researchers suggested that pasteurization [72°C (161.6°F)/l6 3] would not have any appreciable effect on enzymatic clotting of milk (Morr 198 7, Wilson and Wheelock 1972). Marshall (Marshall 1986) was able to make Cheshire cheese from milk heated at 97°C (206.6°F) for 15 sec, but Cheddar cheese from similarly treated milk was excessively crumbly. Even by changing the manufacturing steps, a satisfactory Cheddar cheese could not be produced. Amantea et al. (1986) showed that cheese made from heat-treated milk [63°C (145.4°F)/16 s] was firmer than cheese produced from pasteurized milk, although the cheeses were similar in moisture, salt, pH, and age. The difference in firmness reportedly resulted from irreversible protein denaturation. Over- pasteurization can also lead to a cheese with a "short" or "brittle" body (O'Keefle et. a1 1982, Price and Call 1969). According to Reinhold (Reinhold 1972), Swiss cheese can be routinely made from fully pasteurized milk without harmful effects on eye development. However, the impact of pasteurization on flavor development during curing was not described. Ginzinger et al. (1999) manufactured Bergkaese, a Swiss-type hard cheese, to examine the effect of raw milk flora on cheese quality. Milk pasteurization had no significant effect on physical properties of the cheese. However, pasteurization adversely affected aroma intensity and bitterness with cheese produced from pasteurized milk having lower flavor intensity and increased bitterness compared to raw milk cheese. They concluded that it would be inappropriate to pasteurize milk intended for making Bergkaese, even for elimination of indigenous milk microflora, due to adverse effects on sensory quality. 43 Several researchers reported that flavor develops slower in pasteurized as compared to raw milk cheeses (Banks et. al 1986, Franklin and Sharpe I 962, Hanrahan et. a1 1963, Kristofirersen 1985, Melachouris and T uckey 1966, Price 192 7, Scarpallino and Kosikowski 1962, Wilson et. al 1945). Price and Call (1969), and Melachouris and Tuckey (1966) observed that cheese made from excessively heated milk was of inferior quality compared to that made from pasteurized milk. Among the enzymes in milk thought to function in cheese curing are plasmin and lipase. Alichanidis et al. (1986) indicated that plasmin is largely unaffected by pasteurization. A 30-40% increase in milk protease activity was reported in pasteurized milk compared to raw milk, with this change possibly due to inactivation of a protease inhibitor (Noomen 1975). In contrast, milk lipase, is heat sensitive but not completely destroyed by pasteurization. Pasteurization at 72°C (161.6°F)/15 s will decrease milk lipase activity greater than 90%, while heating at 60-67°C (140-152.6°F) for 15 sec results in more than a 60% loss in activity (Johnson 1974). Loss of milk lipase and other enzyme activity may adversely affect typical flavor development in Swiss and hard Italian cheeses such as Romano, Parmesan, and Asiago. The contribution of other enzymes present in milk such as acid phosphatase, lactoperoxidase and xanthine oxidase, all of which are not appreciably inactivated by standard pasteurization, to the curing of cheese is unknown (Andrews 1974, Johnson 1974). Some lactobacilli and pediococci remaining after pasteurization increased the rate and extent of flavor development (Law 1984). Franklin and Sharpe (1962) observed a decrease in flavor development in Cheddar cheese made from milk heat-treated at 628°C (145°F) for 17 s. As a result of pasteurization, flavor scores also decreased as the number of lactobacilli in cheese milk decreased. 44 :hetsemi Prism raw mill conrol a of Wilt) problem as food and pro hr: ma cheese Italian nati \‘e for ch m {Olin Slain Ihat 1 In conclusion, heat treatment or pasteurization does not adversely affect the cheesemaking process or the resulting physical properties of the cheese to a great extent. Pasteurized milk will yield a cheese of more consistent quality than cheese made from raw milk. Pasteurization and other heat-treatments enable improved uniform process control and quality during cheesemaking. However, heating results in some denaturation of whey protein (with pasteurization) as well as some body/texture and moisture control problems. Whey proteins can also lose fimctionality which could affect their usefulness as food ingredients. Moreover, cheeses made fiom pasteurized milk ripen more slowly and probably not to the same flavor intensity as do cheeses prepared from raw milk. This has major adverse implications for manufacturers of processed cheese which require cheese with accelerated body breakdown and intense, sharp flavors. Swiss and hard Italian type cheeses, the traditional flavor of which is strongly related to the activity of native milk enzymes and microflora, also would be adversely affected if pasteurized milk for cheesemaking became mandatory. EFFECT OF HEAT-TREATMENT ON L. MONOC YT OGENES The established association of L. monocytogenes with raw milk in the 19508 gave rise to several early studies dealing with the possible resistance of this organism to pasteurization. In 1983, interest in this topic was revived as a result of a listeriosis outbreak in Massachusetts that was epidemiologically linked to consumption of pasteurized milk. Reports of unusual heat resistance of L. monocytogenes in milk can be found in the early literature (Ikonomov and T odorov 1 96 7, Ozgen 1952, Potel 1951, Stajner et al. 1979, Stenberg and Hammainen 1955). In 1951, Potel (1951) demonstrated that L. monocytogenes died rapidly in milk held at 80°C. However, the following year, 45 Ozgen (1952) reported that L. monocytogenes survived 15 s at 100°C. These early findings indicated that L. monocytogenes could survive HTST pasteurization at 71 .6°C/ 1 5 3, including a study by Beams and Girard (1958) using the open-tube method. However, later studies proved that these early studies were flawed. Donnelly et al. (1987) showed that the open-tube method used by Beams and Girard (1958) was unreliable to determine thermal death time. Using a "sealed-tube" method, they demonstrated that L. monocytogenes was rapidly inactivated in milk at 62°C. Thermal-inactivation profiles obtained by the sealed-tube method were linear for three strains of L. monocytogenes during the entire inactivation period and gave rise to D62oc values between 0.1 and 0.4 min depending on the strain of bacteria. The capillary tube method (a standard method now widely accepted) was used by several investigators to determine thermal resistance of L. monocytogenes in liquid media and foods (El-Shenawy et al. 1989, Lou and Yousef 1997a). Thermal inactivation rates for L. monocytogenes ‘ were linear throughout the entire course of heating in the range of 50-75°C. All these studies were conducted using suspended cells. Results from investigations on resistance of intracellular L. monocytogenes (cells present in leukocytes) are in conflict as some have shown increased heat resistance of internalized cells (Banning et al. 1988, Doyle et. a1 1987, Knabel et al. 1990). Knabel et al. (1990) compared heat resistance data of L. monocytogenes when the heat-treated cells were recovered from sterile, whole, and homogenized milk by incubation under aerobic and anaerobic conditions. When grown at 43°C and recovered by anaerobic incubation after heating, L. monocytogenes had D6234; of 243 3 compared to 36 s for Listeria grown at 37°C and plated aerobically after thermal inactivation at 628°C. The FDA (Bradshaw et. al 198 7, Banning et al. 1992, Lovett et al. 1990), Centers 46 for Disease Control and Prevention (CDC) (Anon. 1988a, 1988c, 1989), and the World Health Organization (WHO) (WHO Working Group 1988) support HTST pasteurization as a safe process. In their review, Lou and Yousef (Lou and Yousef1999) also concluded that "pasteurization is a safe process which reduces the number of L. monocytogenes occurring in raw milk to levels that do not pose an appreciable risk to human health." EFFECT OF ACID/ACIDITY Although HTST pasteurization is sufficient to destroy L. monocytogenes in milk, a growing concern in thermal inactivation is the survival of sublethally injured cells. Garazyabal et al. (1987) reported that L. monocytogenes was not recoverable from raw milk immediately after heating at 60 to 73°C but grew in the product during extended incubation. Such repair requires an optimal pH near 7.0. According to Bergey's Manual of Systematic Bacteriology (1986) (Seeliger and Jones 1986), L. monocytogenes can only grow at pH values from 5.6 to 9.6, with optimal growth occurring at neutral to slightly alkaline values; the latter was verified by Petran and Zottola (1989). The minimum pH value for growth was based on the work of Seeliger (Seeliger 1961), who, in 1961, reported that L. monocytogenes failed to grow in dextrose (glucose) broth at pH <5.6 after 2-3 days of incubation at 37°C. In addition, subcultures fiom the medium were no longer routinely successful. Subsequent investigations have shown that L. monocytogenes can proliferate in laboratory media adjusted to far lower pH values. Results from these studies (Borovian 1989, George et a1. 1988, Parish and Higgins 1989, Sorrells et al. 1989) confirm the ability of L. monocytogenes to multiply in similar laboratory media adjusted to pH 4.4-4.6 with hydrochloric, citric, or malic acid. Farber et al. (1989) observed growth of L. monocytogenes at 30°C in double-strength brain heart infusion (BHI) broth 47 acidified with hydrochloric acid to a pH value as low as 4.3. Furthermore, L. innocua, L. seeligeri, and L. ivanovii also were reported to grow in BHI broth acidified with hydrochloric acid to pH values as low as 4.53, 4.88, and 5.16, respectively. Thus, the minimum pH at which L. monocytogenes and most other Listeria spp. can grow is well below pH 5.0 provided that these organisms are incubated at near-optimum temperatures and allowed sufficient time to overcome an extended lag phase. Fermentation is an age-old method of food preservation which has an inhibitory effect on the growth and survival of pathogenic bacteria. However, proper acid development is critical to the safety and quality of fermented foods. Behavior of L. monocytogenes in these foods depends on numerous extrinsic and intrinsic factors, including pH. Camembert (Ryser and Marth 1987b) (a mold-ripened cheese), Brick cheese (Ryser and Marth 1989), and white pickled cheese (Abdalla et al. 1993) supported growth of L. monocytogenes, with the pH of these cheeses being 5.9-7.2, 6.9-7.3, and >60, respectively. In contrast, the bacterium was inactivated rapidly in cottage (Ryser and Marth 1985), Parmesan (Yousef and Marth 1989), mozzarella (Buazzi et al. 1992b), and water-buffalo mozzarella cheese (Villani et al. 1996), having final pH values of 5.0- 5.1, 5.2-5.3, and 4.0, respectively. Various degrees of survival have been reported in most other cheeses. L. monocytogenes persisted 70 to _>_434 days in Cheddar cheese at pH 5.0- 5.15 (Ryser and Marth 198 7a), _>_ 115 days in Colby cheese at pH 5.0-5.18 (Yousef and Marth 1988), 270 days in semihard Manchego-type cheese at pH 5.10-5.80 (Dominguez et al. 1987), ~90 days in Trappist cheese at pH 4.70-5.42 (Kovincic et al. 1991) and feta cheese at pH 4.6 (Papageorgiou and Marth 1989a), <66-80 days in Swiss cheese (Buazzi 48 et al. 1992a), >50 days in blue cheese (Papageorgiou and Marth 1989b), and ~ 180 days in cold-pack cheese food without preservatives at pH 5.21-5.45 (Ryser and Marth 1988). SUBLETHAL THERMAL/ACID INJURY In nature, L. monocytogenes may be subjected to various environmental stresses, such as high/low temperature, acidic/alkaline conditions and starvation (Foster and Spector 1995, Miller 1992). Environmental stresses can induce stress-adaptive or stress- protective responses e.g., incubating a microorganism such as L. monocytogenes at a high but sublethal temperature will induce a heat-shock response. Resistance of L. monocytogenes to heat or other lethal factors can be greatly increased by heat-shock or adaptation to other stresses. Bacteria respond to heat shock by synthesizing new proteins, termed heat-shock proteins (HSP) (Agard 1993, Craig et al. 1993). Induction of the heat- shock response or HSP usually increases the thermotolerance of microorganisms. As opposed to the intrinsic thermotolerance of microorganisms, heat-shock-induced thermotolerance is transient and non-inheritable and therefore an acquired or adaptive response (Watson 1990). Temperatures at which microorganisms are heat-shocked affect the magnitude of the acquired thermotolerance. Optimal heat-shock temperatures for maximal thermotolerance are ~10-15°C above the microbe's optimal growth temperature. Listeria monocytogenes has optimal heat-shock temperatures in this range (F arber and Brown 1990, Lou and Yousef l 999). The magnitude of heat-shock-related thermotolerance is also affected by the length of exposure to heat, the heating menstruum, heating rates, physiological state of the cells, and the method used to recover injured cells. 49 pTOQCSS‘r'.‘ thermal establish} hmdling I mlrlmir'." m3) OCCU Cheese cu; time and increased, 01111112“ 3 tesrsram 11118311; Writ ' acidic a1 pH Conditions similar to heat-shock can develop in some foods during thermal processing or hot-holding. Slow heating or cooking, preheating, hot water washing, mild thermal processes, and holding food in warm trays (as occurs in food service establishments) are examples of heat-shock that may occur during food processing and handling. Farber and Brown (1990) suggested that heat-shock may result when foods are minimally processed or when the food is too bulky to allow rapid heating. Heat-shock may occur during vat pasteurization of dairy products (Linton et al. 1990) or cooking of cheese curds during the make process (e. g. Swiss cheese), which involves a long come-up time and low-temperature heating/cooking. Therrnotolerance of L. monocytogenes is increased by low heating rates (Quintavalla and Campanini 1991, Stephens et al. 1994). Quintavalla and Campanini (1991) found that L. monocytogenes became more heat resistant during slow (0.5°C/min) rather than fast heating. Stephens et al. (1994) investigated thermal inactivation of a l7-h-old culture of L. monocytogenes (Scott A) in tryptic phosphate broth at 50-64°C by both instantaneous heating and slow heating and found that slow heating significantly increased heat resistance of L. monocytogenes. Besides heat shock, adaptation to other environmental stresses may also increase the thermotolerance of pathogens. F arber and Pagotto (1992) found that exposing a stationary-phase culture of L. monocytogenes to a laboratory broth at pH 4.0 for 1 h increased the Dsgoc-value in sterile whole milk fi'om 2.75 to 3.90 min. A gradual decrease to pH 4.0 during 4 or 24 h also significantly increased heat resistance (acid adaptation). Acid adaptation can enhance survival of L. monocytogenes when exposed to lethal acidic conditions. Kroll and Patchett (1992) found that acid shocking L. monocytogenes at pH 3.0 for 20 min prolonged the lag-phase when the organism was subsequently grown 50 at pH 7.0. Prior incubation at pH 5.0 rather than pH 7.0 increased survival of L. monocytogenes by 3 logs during acid shock at pH 3.0 for 40 min. Synthesis of "acid stress proteins" is presumably required for induction of the acid-tolerance response (O'Driscoll et a1. 1996). Lou and Yousef (1997b) found that acid resistance in L. monocytogenes was significantly greater after adaptation to mild acidic conditions or after a stepwise increase to high acid-conditions. They suggested that food fermentations, which involve a gradual lowering of pH, could lead to acid adaptation in L. monocytogenes. Acid adaptation also cross protects L. monocytogenes against a variety of deleterious factors such as lethal doses of hydrogen peroxide, heat, NaCl, ethanol, and certain surface active hydrophobic compounds (Lou and Yousef 1999). Since acid adaptation increases general resistance, including acid tolerance, acid-adapted cells of L. monocytogenes may survive better in both acidic and fermented foods (e. g. cheese) than unadapted cells (Guhan et al. 1996). When present in a sublethally injured state in food, L. monocytogenes cannot be enumerated directly since the recovery media contains various Listeria selective agents, some of which are inhibitory to the repair process while others are toxic and cause death of these injured cells. In order to successfully detect and accurately enumerate sublethally injured cells, an environment favorable for repair of sublethally injured cells must be provided. Current detection procedures for L. monocytogenes (FDA, USDA-FSIS, IDF), with the exception of cold enrichment (which is very time consuming and laborious) rely on highly selective enrichment and/or plating media. Therefore, these methods frequently underestimate the true incidence of Listeria. Busch and Donnelly (1992) developed 51 31 1C) II: Listeria Repair Broth (LRB) which permits complete repair of injured Listeria within 5 h at 37°C after which various selective agents can be added to inhibit the growth of competing microflora upon incubation. Considerable research has been conducted to evaluate the efficacy of LRB, University of Vermont Broth as well as LRB modified by adding certain components, to resuscitate heat-, acid-, sanitizer- and freeze-injured L. monocytogenes cells (Donnelly 1999). Based on the earlier study by Knabel et al. (1990), Teo and Knabel (2000) developed modified Penn State University (mPSU) Broth for anaerobic recovery of heat-injured L. monocytogenes from pasteurized milk. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteurized whole milk were detected using mPSU broth. Use of a suitable recovery-enrichment medium is necessary if all L. monocytogenes (healthy and injured) cells are to be detected in foods. To summarize, survival and growth of healthy L. monocytogenes, S. Typhimurium and E. coli OlS7:H7 in Cheddar, Colby and most other aged cheeses generally decreases during storage (Hargrove et. al 1969, Park et al. 1970, Reitsma and Henning 1996, Ryser and Marth 198 7a, 1985). Although milk pasteurization is sufficient to destroy pathogens, a growing concern is the survival and recovery of sublethally injured cells (Garayzabal et. al 198 7). Since such repair requires an optimal pH near 7.0, the harsh nature of the cheese environment (acid + salt) should limit survival of sublethally injured cells in a product such as Cheddar cheese. GOALS OF THE STUDY The goal of this study was to investigate the relationship between the heat treatment milk receives prior to cheesemaking and the ability of L. monocytogenes to 52 inure cheese suhletl cheese mom/e euhun Chedd 1163161 pesteL caulk Cherie lllClUSl survive similar conditions to those encountered during the early stages of a Cheddar cheese fermentation. The potential for L. monocytogenes to become inactivated and/or sublethally heat/acid injured during sub-pasteurization heating of the milk before cheesemaking as well as during a simulated Cheddar cheese fermentation was investigated. Procedures were developed for obtaining heat-injured cells of L. monocytogenes based on an earlier study by Busch and Donnelly (1992). These injured cultures were used to study the influence of a lactic starter culture typical of those used in Cheddar cheese manufacture on growth and survival of the pathogen in raw, low heat- treated (LHT), high heat-treated (HHT), pasteurized and ultra high temperature (U HT) pasteurized milk. The underlying hypothesis was that a sub-pasteurization heat treatment can be identified which will sufficiently injure L. monocytogenes to prevent its survival in Cheddar cheese beyond 60 days of ripening and thereby preserve the raw milk cheese industry. 53 RU‘. m0. C0 Ac Running Head: Competitive Inhibition of Sublethally Injured Listeria monocytogenes Competition of Thermally Injured Listeria monocytogenes with a Mesophilic Lactic Acid Starter Culture during Milk Fermentation Finny P. Mathew, and Elliot T. Ryser. Department of Food Science and Human Nutrition Michigan State University, East Lansing, MI 48824-1224, USA. Running Head/Keywords: Listeria monocytogenes, thermal injury, competitive inhibition, mesophilic milk fermentation. *Corresponding Author: Elliot T. Ryser Phone: (517) 353 9734 Fax: (517) 353 1676 E-mail: aser@msu.edu 54 ABSTRACT The relationship between heat treatment of milk and the ability of sublethally injured Listeria monocytogenes cells to survive mesophilic fermentation in milk was investigated. Overnight tryptose broth cultures of three L. monocytogenes strains were centrifuged, suspended in 200 ml of tryptose phosphate broth and heated at 56°C/20 min and 64°C/2 min to obtain low heat-injured (LHI) and high heat-injured (HHI) cells, respectively, showing >99 % injury. Flasks containing 200 ml of raw, low heat-treated (56°C/20 min), high heat-treated (64°C/2min), pasteurized or UHT milk were tempered to 31.1°C, inoculated to contain 104-106 LHI, HHI or healthy L. monocytogenes cells and a Lactococcus lactis subsp. lactis/Lactococcus lactis subsp. cremoris starter culture at levels of 0.5, 1.0 or 2.0%. Numbers of healthy and injured L. monocytogenes cells were determined using tryptose phosphate agar with or without 4.0% NaCl at selected intervals during the 24h fermentation period, numbers of starter organisms were also measured. Presence of L. monocytogenes did not adversely affect the growth of the starter culture at any inoculation level. Overall, L. monocytogenes survived the 24 h fermentation process and grew to some extent. In starter-free controls, ~76-81% and 59-69% of LHI and HHI cells, respectively, were repaired after 8 hours of incubation, with lowest repair rates observed in raw rather than heat-treated or pasteurized milk. Increased injury was observed for healthy L. monocytogenes cells at 1.0 and 2.0% starter levels, with less injury seen for LHI and HHI cells. The extent of sublethal injury for all L. monocytogenes was inversely related to severity of the milk heat treatment. 55 Present-day laws regarding use of pasteurized, heat-treated (sub-pasteurized), and raw milk for cheesemaking date back to World War II (Anon. 1949). Options provided to cheese manufacturers were to either (a) pasteurize the milk [71.6°C (161°F)/15 sec] or (b) hold the cheese for a minimum of 60 days at >1.7°C (35°F). Thus, any cheese prepared from heat-treated milk was required to be held at least 60 days. Subsequent reports have shown that three important foodbome pathogens, namely, Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli OlS7:H7 can survive up to 434 days (Ryser and Marth 1 98 7a), 210 days (Goepfert et al. 1968, Hargrove et. al 1969, Park et al. 1970) and 138 days (Reitsma and Henning 1996), respectively, in Cheddar cheese produced from pasteurized milk inoculated with these pathogens. Consequently, the adequacy of the 60 day hold at Z 1.7°C still remains very much in question. The United States Food and Drug Administration (FDA) as well as the Australian Dairy Industry, the Government of Canada (F arber et al. 1996) and the International Dairy Federation recently voiced concerns regarding safety of cheeses made from raw and heat-treated milk. At FDA's request, the Cheese Subcommittee of the National Advisory Committee for the Microbiological Criteria of Foods reviewed the data and concluded that the 60 day holding period at 2 1.7°C may be insufficient to eliminate all foodbome pathogens; the Subcommittee recommended that the FDA re-examine its current policy (Anon. 1997). The Codex Committee on Food Hygiene is considering a proposed drafl code of hygienic practice for milk and milk products that stops short of requiring mandatory pasteurization of milk (Groves 2000a). However, given the superior flavor characteristics of raw milk Cheddar Cheese that result from non-starter lactic acid bacteria and enzymes naturally present in the milk (Kristofiersen 1985, Melachouris and 56 Tuckey 1966, Price 192 7, Scarpallino and Kosikowski 1962), cheese manufacturers are reluctant to any change in the current aging policy. The American Cheese Society and the American Dairy Products Institute's Cheese Division support traditional "curing" methods for cheeses made from unpasteurized milk, including the 60-day aging requirement (Groves 2000a). Listeria monocytogenes is one foodbome pathogen of particular concern because it can cause abortion in pregnant women and meningitis in immunocompromised adults (Gray and Killinger I966, Seeliger 1961). Sporadic cases of bovine mastitis and abortion in which L. monocytogenes was intermittently shed in milk over several lactation periods have been recorded for more than 50 years. Dairy cows that appear healthy also can serve as reservoirs for L. monocytogenes (Ryser 1999b) with this pathogen reportedly present in 1.6-12.0%, 1.3-5.4%, and 2.5-6.0% of the raw milk produced in the United States, Canada and Western Europe, respectively (Donnelly et al. 1988, Hayes et. al 1986, Liewen and Plantz 1988, Lovett and Hunt 198 7, Ryser 1999a). In 1983, pasteurized milk was epidemiologically implicated as the vehicle of infection in a listeriosis outbreak in Massachusetts that resulted in the death of 14 of 49 individuals (Fleming et. a1 1985). After 85 fatal cases of listeriosis were traced to consumption of Jalisco-brand Mexican-style cheese in 1985, surveillance efforts were intensified under the Dairy Safety Initiative Program (Kozak 1986). FDA reports in 1986 indicated that an average of 2.5 % of all dairy products manufactured from pasteurized milk was contaminated with L. monocytogenes (Anon. 1986). A subsequent report in February 1987 indicated that 2.6% of all dairy-processing facilities contained L. monocytogenes (Anon. 198 7c). In the United States, this pathogen has been responsible 57 for at least 46 class I recalls involving domestically produced cheese, 3 of which were prepared from raw milk (Ryser I999a). Thus, the current "zero tolerance" policy for L. monocytogenes has extracted a particularly heavy toll on the dairy industry. Although high temperature-short time pasteurization is sufficient to destroy L. monocytogenes in fluid milk (Bradshaw et. al 198 7, Farber 1989, Mackey and Bratchell 1989), incomplete pasteurization can lead to the survival and recovery of sublethally injured cells. Garazyabol et al. (I 98 7) reported that L. monocytogenes was not recoverable from raw milk immediately after heating at 60 to 73°C but grew in the product during extended incubation. Such repair requires an optimal pH near 7.0 and is reportedly enhanced under anaerobic conditions (Knabel et al. 1990). Fermentation is an age-old food preservation method used to inhibit the growth and survival of pathogenic bacteria. Studies showed that healthy L. monocytogenes cells survived and grew to some extent in samples of sterile skim milk that were fermented with mesophilic and thermophilic starter cultures (Schaack and Marth 1988a, 1988b). Studies on survival and growth of healthy L. monocytogenes, S. Typhimurium and E. coli OlS7:H7 cells in Cheddar, Colby and other aged cheese indicate that their numbers slowly decrease during cheese ripening (Hargrove et. a1 1969, Park et a1. 1970, Reitsma and Henning 1996, Ryser and Marth 198 7a). Demise of these pathogens during aging is . principally due to acid development by the starter culture. Given the low pH of Cheddar cheese (~pH 5.0) combined with high levels of salt in the moisture phase, survival of sublethally injured should be far less than that for healthy cells. The purpose of the study was to assess the ability of low heat-injured (LHI), high heat-injured (HHI) and healthy cells of L. monocytogenes to compete with different 58 levels of a mesophilic lactic acid starter culture in milks that have undergone various degrees of thermal processing. MATERIALS AND METHODS Culture preparation: Three strains of L. monocytogenes (CWD 95 and CWD 246 from silage, and CWD 17 from raw milk) were obtained from C. W. Donnelly (Dept. of Nutrition and Food Sciences, University of Vermont, Burlington, VT). The cultures were maintained at -70°C in trypticase soy broth (Becton Dickinson and Co., Cockeysville, MD) containing 10% (v/v) glycerol (J. T. Baker, Phillipsburg, NJ) and subjected to two consecutive overnight transfers (18-24 h/35°C) in 9 m1 of tryptose phosphate broth (TPB) (Difco Laboratories, Detroit, MI) containing 0.6% (w/v) yeast extract (Difco). A 3-strain cocktail suitable for sublethal injury work was then prepared by combining equal volumes of these cultures in a sterile 50 ml centrifuge tube (Clear Propylene, Plug Seal Cap, Coming Inc., Corning, NY), centrifuging at 10,000 rpm at 4°C/ 15 min (Super T21, Sorvall® Products, Newtown, CT), and resuspending the pellet in 9 ml of phosphate buffered saline (PBS) to obtain a suitable culture for injury. Sublethal Injury: Heat-injured cells were obtained using the procedure of Busch and Donnelly (1992) (Figure 2). In this method, 200 ml of TPB [in a 2800 ml wide bottom Fembach flask] was tempered to 56°C/64°C in a shaking water bath [50 rpm] (Reciprocal Shaking Bath, Precision Scientific, Winchester, VA), inoculated to contain 108-109 L. monocytogenes CFU/ml and heated at 56°C/up to 30 min and 64°C/up to 5 min to obtain LHI and HHI cells, respectively, showing >99.0%. Samples were appropriately diluted in 59 PBS and spiral plated (Autoplate® 4000, Spiral Biotech, Inc., Bethesda, MD) on tryptose phosphate agar (Difco) + 0.6 % (w/v) Yeast Extract (non-selective medium, TPA) and TPA + 4.0% (w/v) NaCl (selective medium, TPNA) and incubated at 35°C/48 h to determine numbers of healthy and injured L. monocytogenes cells, respectively. Percent injury was determined from the following equation: % Injury: {l—Count on selective medium } X100 Count on non-selective medium These heat-injured cultures were then centrifirged at 10,000 rpm at 4°C/15 min, resuspended in PBS and appropriately diluted for inoculation into milk. The heat-injury trials also were repeated in UHT milk to investigate the influence of the heating medium on sublethal injury. Experimental Design: A 5 x 3 x 4 factorial design was used to assess the effect of milk type [raw, low heat-treated (LHT), high heat-treated (HHT), pasteurized, and ultra high temperature (UHT) pasteurized] on the ability of L. monocytogenes cells in different physiological states [healthy, LHI, and HHI] to compete with different inoculum levels (0%, 0.5%, 1.0% and 2.0%) of a Lactococcus lactis subsp. lactis/L. lactis subsp. cremoris (LLLC) starter culture normally used to manufacture Cheddar cheese. Each trial was carried out in triplicate. Fresh raw milk (chilled ~4°C) was obtained from the Michigan State University (MSU) Dairy Farm in sterile 2-liter flasks (autoclaved 121°C/15 min), divided into 200 ml aliquots and heated to 56°C and 64°C in a shaking water bath (Precision Scientific) for the same times in the sublethal injury trials (to obtain 99.0% injury) to obtain LHT and HHT milk, respectively. Freshly pasteurized (72°C/25 3) milk was obtained from the 60 MSU Dairy Plant in sterile 2-liter flasks (autoclaved 121°C/ 15 min). UHT pasteurized milk (Parmalat whole milk, Parmalat USA, Teaneck, NJ) was purchased locally. Cans of frozen (-70°C) LLLC starter culture (Blue Label, Direct Vat Set, Chr. Hansen, Milwaukee, WI) were thawed by submersion in deionized water containing 100 ppm available chlorine for 30 min after which 2-ml aliquots were transferred to sterile freezer vials and frozen at —70°C. Working LLLC cultures were prepared by thawing a vial of culture and transferring 0.5m] of the contents to a flask containing 100 ml of sterile (autoclaved at 121°C/15 min) skim milk. Following 4-6 h of incubation at 30°C, the working LLLC starter culture was ready for use in trials. Milk Inoculation: Three sets of flasks containing 250 ml of raw, LHT, HHT, pasteurized and UHT pasteurized milk were tempered to 31 .1°C in a water bath (Microprocessor Controlled 280 Series water Bath, Precision Scientific, Winchester, VA) (Figure 3). A l-ml sample was withdrawn to determine the numbers of indigenous bacteria in the milk. One set each was inoculated with healthy, LHI (56°C/10-30 min), or HHI (64°C/1-5 min) cells of L. monocytogenes at a level of 104-~106 CFU/ml. Thereafter, a working LLLC starter culture was added at a level of 0.5%, 1.0% or 2.0%. Additional flasks containing the LLLC starter culture alone, and the pathogen alone served as controls for assessing the impact of starter on the pathogen and pathogen on the starter, respectively. 61 lnoculate Inoculate 10 ml of Tryptose Phosphate Broth with L. monocytogenes 35°C 18h Inoculate 200 ml of Tryptose Phosphate Broth to contain ~107-109 CF U/ml 1 Heat to obtain >99.0% injury 56°C - low heat-injured cells (LHI) 64°C - high heat-injured cells (HHI) l Spiral-plated on TPA and TPNA l Centrifuge heat-injured cultures (10,000 rpm at 4°C/15 min) Resuspend in Phosphate Buffer Saline Figure 2: Preparation of heat-injured L. monocytogenes 62 250 ml raw, LHT, HHT, pasteurized or UHT milk 1 Determine numbers of native (contaminating) bacteria 1 Add 104-106 CFU/ml Healthy, LHI or HHI cells of L. monocvtogenes 1 Add 0, 0.5, 1.0 or 2.0% starter culture Analyze at 0, 2, 4, 6 and 8 h for L. monocytogenes, Starter and pH Refrigerate and sample after 24 hrs Figure 3: Fermentation of Milk at 31.1°C 63 Numbers of both healthy and injured cells of L. monocytogenes as well as the starter culture were determined from l-ml samples, which were taken initially and thereafter at 2-h intervals during a fermentation period of 8 h. The pH was also monitored at the time of sampling using a pH meter (ORION model 620, Thermo Orion, Beverly, MA) equipped with a standard combination electrode (ORION model 6157 Solid State pHuture Probe, Thermo Orion). One additional sample was taken after 24 h for analysis. Microbiological Analysis: Numbers of indigenous microflora and total (healthy + sublethally injured) L. monocytogenes cells in UHT milk were determined by spiral plating samples appropriately diluted in PBS on TPA, while populations of healthy L. monocytogenes cells were determined by spiral plating samples on TPNA followed by 48 h incubation at 35°C. Modified tryptose phosphate agar (MTPA) containing esculin (0.1% w/v) (Sigma Chemical Co., St. Louis, MO) and ferric ammonium citrate (0.05% w/v) (Sigma) [non- selective] and MTPA + 4% NaCl [selective] (MTPNA) were used to examine other milk types. Numbers of LLLC starter culture were determined by pour plating appropriately diluted samples in MRS agar. These plates were counted after 48 h of incubation at 35°C. Statistical Analysis: Two-way Analysis of Variance (ANOVA) was performed on the data using the Statistical Analysis System (Proc Anova, SAS© Version 8, SAS Institute, Inc., Cary, NC). Arithmetic means were compared using the Duncan grouping test at 95% confidence level (p=0.05). Interactive effects were analyzed using the Autoregressive 64 Mixed Covariance Model (Proc Mixed Covtest) with the Satterthwaite Degrees of Freedom Method. RESULTS Sublethal Iniuflz Heating the 3-strain cocktail of L. monocytogenes in UHT milk at 56°C/20 min (Figure 4) and 64°C/2 min (Figure 5) produced >99.0% injury. No significant (p<0.05) differences were obtained in % injury between trials conducted in TPB and UHT milk at 56°C/20 min as well as 64°C/2 min (Table 6). Indigerm; microflora in milk: Fresh raw milk samples used for competitive inhibition trials had bacterial populations in the range of 3.0x101- 4.1x102 CFU/ml. Except for one sample (9.99x100 CF U/ml — LHT milk), no detectable counts were observed when raw milk was subjected to heating at 56°C/20 min (LHT) and 64°C/2 min (HHT). Pasteurized and UHT milks did not yield any detectable bacterial counts. Black colonies of L. monocytogenes on the non- selective medium (MTPA) could be easily differentiated from the naturally contaminating bacteria. The catalase test was also used for confirmation. Growth of L. monocytogenes without starter culture: When healthy L. monocytogenes cells were grown in different types of milk at 31 .1°C (typical milk ripening temperature for Cheddar cheesemaking), steady growth was observed during 24 h of incubation. The heat treatment that the milk received before inoculation did not have a significant effect (p<0.05) on the growth rate of L. monocytogenes during incubation (Table 7). 65 When LHI and HHI cells of L. monocytogenes were grown in different types of milk, populations continually increased in all samples. As the incubation period increased, 78.95-87.74% of the injured cells were repaired after 24 h (Table 8). Repair for LHI cells in raw, LHT and HHT milk was significantly (p<0.05) lower than in pasteurized and UHT milk with HHI L. monocytogenes cells showing significantly greater (p<0.05) repair in UHT milk compared to the other milk types. Maximum repair occurred in pasteurized and UHT milk for LHI (87.57%) and HHI (87.74%) cells, respectively. The extent of repair was generally greater for LHI rather than HHI cells for all time periods up to 8 h, e.g., ~47 % of the LHI cells repaired after 6 h of incubation in raw milk compared to 32% for HHI cells. In UHT milk, ~55% of the LHI cells repaired after 6 h of incubation compared to 40% for HHI cells. However, after 24 h of incubation, differences in the % repair were not significant (p<0.05) (Table 9). Growth of L. monocytogenes in the Presence of Starter Cm: Initial LLLC populations of 4.6x106 to 5x107 CFU/ml increased to about 109 CFU/ml after the 24 h fermentation period in all types of milk. Final pH values ranged from 3.85 to 4.4 after fermentation depending on the level of LLLC. Populations of LLLC as well as the pH drop in control (inoculated only with LLLC) and competitive inhibition samples (inoculated with both LLLC starter culture and L. monocytogenes) were comparable (raw data in Appendix); therefore, most attention will be given to the behavior of L. monocytogenes in competition with LLLC. When healthy, LHI or HHI L. monocytogenes cells (initial level of ~104-5x106 CF U/ml, representing moderate to severe contamination of the milk) were grown in different types of milk in competition with 0.5, 1.0 and 2.0% starter culture, a steady 66 increase in the total population of Listeria was observed in all cases irrespective of the starter inoculum, type of milk or physiological state of L. monocytogenes. Listeria attained final populations of ~108 to 5x109 CFU/ml with growth affected by the physiological state of Listeria and LLLC level (Table 10). Overall, growth of sublethally injured L. monocytogenes was greater than that of healthy cells at all LLLC levels, e.g. 3.09 and 3.46 log increase (significant, p<0.05) for healthy and HHI cells, respectively, in pasteurized milk containing 0.5% LLLC after 24 h A greater increase in total populations of HHI was observed as compared to LHI cells at each LLLC inoculum level, e.g., 3.21 log increase versus 3.12 log for HHI and LHI cells, respectively, after 24 h of incubation in LHT milk containing 1% LLLC. Growth of healthy as well as sublethally injured L. monocytogenes cells was inhibited as the LLLC inoculum level increased, e.g., 3.17 and 3.45 log increase for LHI and HHI cells, respectively, in pasteurized milk containing 0.5% LLLC compared to 2.82 and 2.93 log in the same milk containing 2.0% LLLC after 24 h (p<0.05). Injury of healthy L. monocytogenes cells increased as the fermentation process progressed (counts on selective MTPNA decreased steadily compared to non-selective MTPA). At the end of the 24-h fermentation period, >90% of the healthy L. monocytogenes cells were injured, with slightly higher injury observed at higher LLLC inoculum levels of 1.0% and 2.0%. For LHI and HHI L. monocytogenes cells, >99.0% of the initial population was injured, and no repair or significant change was observed in percent injury. The primary interest of this study was to assess the behavior of sublethally injured cells during fermentation. Analysis of the percent increase in the number of injured cells 67 showed a significant effect of the type of milk, LLLC inoculrun level as well as physiological state of Listeria, interactive effects of these factors were also found to be significant (Table 11). A significant increase (p<0.05) in the percentage of healthy L. monocytogenes cells that became injured was generally observed as the LLLC inoculum increased from 0.5% to 2.0% (Table 1216), e.g., injured cells increased by 51.63% and 64.93% in UHT milk at LLLC inoculum levels of 0.5% and 2.0%, respectively, after 8 h of fermentation. For LHI and HHI L. monocytogenes cells, a reverse trend was observed for the increase in the population of injured as compared to healthy cells. Where significant differences were observed (p<0.05) for different LLLC levels (Tables 12-16), the numbers were generally greater for 0.5% than for 2.0% starter culture. In LHT milk containing 0.5% starter culture, the increase observed for LHI and HHI cells after 6 h of incubation was 47.44% and 54.18%, respectively, while at 1.0%, these numbers increased to 46.32% and 50.00%, respectively (Table 13). The increase in percentage of injured cells also was greater for HHI as compared to LHI L. monocytogenes cells, although the trend was not always significant (p<0.05), e.g. in LHT milk containing 0.5% starter culture, the increase observed for LHI and HHI cells after 6 h of incubation was 47.44% and 54.18%, respectively, while at 1.0%, these numbers increased to 46.32% and 50.00%, respectively (Table 13). The extent of increase in the number of injured cells was dependent on the type of milk in which L. monocytogenes was grown, e.g., in the case of healthy cells, significantly greater percentages of cells became injured in raw milk than in heat treated milks (LHT, HHT, pasteurized and UHT) for all fermentation periods (Tables 17-19). 68 If Id If Similar trends were observed for LHI and HHI cells at all LLLC inoculum levels (Tables 17-19). Analysis of the raw data to investigate the interactive effects of LLLC inoculum level and milk type confrrmed the results obtained from the individual analyses. In general, repair of sublethally injured L. monocytogenes cells (in absence of LLLC) increased as the milk heat treatment became more severe. After 6 h of incubation, 32.16% and 40.00% of HHI cells repaired in raw and UHT milk, respectively. The increase in the number of healthy cells that became injured was greater for less severely heated milk and higher LLLC inoculum levels, e.g. 74.03, 69.36, 68.07, 65.89, and 62.48% in raw, LHT, HHT, pasteurized, and UHT milk, respectively, containing 2.0% LLLC after 6 h compared to 59.49, 57.72, 59.62, 53.42, and 47.26% respectively, using an LLLC inoculum level of 0.5%. Conversely, the increase in percent injury for LHI and HHI L. monocytogenes cells was greater for less severely heat-treated milk containing lower levels of LLLC, e.g., 56.63, 54.18, and 54.11% for raw, LHI and HHT milk, respectively, containing 0.5% LLLC compared to 52.52, 50.00, and 48.09% for a starter inoculum of 1.0% (for HHI cells). DISCUSSION Listeria monocytogenes was sublethally injured (>99.0%) in both UHT milk and TPB when heated at 56°C/20 min and 64°C/2 min. This shows that the heating medium did not have a significant effect on sublethal injury of L. monocytogenes at the temperatures studied. These findings were similar to those observed by others (Busch and Donnelly 1992, Meyer and Donnelly 1992). 69 ol In general, L. monocytogenes (in all physiological states) grew steadily in the absence of starter culture in all types of milk at 31.1°C (typical milk ripening temperature for Cheddar cheesemaking) during 24 h of incubation. Similar growth trends were also observed in control samples from other studies (El-Gazzar et al. 1992, Schaack and Marth 1988a, 1 988b, Wenzel and Marth 1991). Greater repair for LHI and HHI L. monocytogenes cells in more severely heat-treated milk (e. g. UHT milk) as compared to raw milk could be explained by the presence of native enzymes and microflora in raw milk that inhibit repair by providing a more hostile environment to the pathogen than heat-treated milk. The extent of repair was generally greater for LHI rather than HHI cells. The more severe heat treatment received by HHI cells compared to LHI cells could be responsible for slower repair. At the end of the 24-h fermentation period, the repair was similar. Meyer and Donnelly (1992) observed that the lag time for heat-injured (at 55°C) cells was inversely proportional to the incubation temperature between 4°C (8 days) and 37°C (none detectable). In our trials, L. monocytogenes in all physiological states showed some growth within the first 2 h of incubation. Thus, our results concur with their study since no detectable lag phase was observed at an incubation temperature of 31 .1°C. Steady growth was observed for healthy, LHI and HHI L. monocytogenes cells in all types of milk (as in controls) when grown in competition with 0.5, 1.0 and 2.0% starter culture. Sublethally injured L. monocytogenes cells grew to a greater extent than healthy cells at all LLLC levels. This may be due to greater susceptibility of healthy cells to acid injury from acid produced by the starter culture, while stress-adaptive responses (e.g. production of heat shock proteins) induced by sublethal heating, resulting in cross 70 protection against other lethal factors such as acid production, could be responsible for enhanced growth of sublethally injured cells (Craig et al. 1993). Several studies were conducted to assess competitive inhibition of healthy L. monocytogenes in sterile skim milk. El-Gazzar et al. (1992) observed that L. monocytogenes survived during a mesophilic fermentation process (starter culture containing a 4 strain mixture of Lactococcus lactis subsp. cremoris) in skim milk as well as further storage for 4-6 weeks at 4°C. Schaack and Marth (1988a) reported variable growth of L. monocytogenes in the presence of Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris depending on inoculum level, with highest populations observed in starter-free controls. Greatest inhibition was observed using a 5.0% starter culture inoculum and an incubation temperature of 30°C. While these results agree with our findings, neither of these studies examined sublethal injury of healthy L. monocytogenes cells that may result from competitive inhibition and/or acid production. In our study, more than 90% of the healthy L. monocytogenes cells were injured after 24 h fermentation period in all types of milk. The extent of increase in the number of injured cells was inversely related to severity of the heat-treatment that the milk received. As mentioned previously, this could again be due to the more hostile environment of raw as compared to heat-treated milk. For LHI and HHI L. monocytogenes cells, increasing LLLC inoculum levels from 0.5% to 2.0% resulted in a lesser increase in the number of injured cells. This is likely due to increased inhibition of LHI and HHI L. monocytogenes cells by higher inoculum levels of LLLC, causing the total population and consequently the percentage of injured L. monocytogenes cells to decrease, thus explaining the reverse trend. Schaack and Marth 71 (1988a) also showed that inhibition of healthy L. monocytogenes cells increased with increasing levels of starter culture. Our results show the same trend for healthy as well as sublethally injured L. monocytogenes cells. A greater increase in the percentage of injured cells was observed for HHI L. monocytogenes as compared to LHI cells. Increased growth (total population) of HHI cells during the 24 h fermentation period as compared to LHI cells (resulting in concomitant increase in injured cells) could possibly explain this trend. Williams and Golden (1998) observed that acid injury of L. monocytogenes was enhanced by prior heat stress. The extent of sublethal injury could also influence recovery on selective media. Thus, the above results show that L. monocytogenes (irrespective of initial physiological state) can survive the 24-h fermentation period at 31.1°C. In all instances, the pathogen exhibited some growth in the presence of LLLC, albeit less than in the controls. Some inhibition was observed at higher LLLC inoculum levels for L. monocytogenes in all physiological states. Higher levels of LLLC increased acid production, resulting in a concomitant increase in the number of healthy L. monocytogenes cells that became injured. Growth and activity of the starter culture was not affected by the presence of L. monocytogenes as observed from comparable values obtained for the LLLC population as well as the pH drop in controls and test samples. Most studies investigating the behavior of L. monocytogenes during cheesemaking and curing have used pasteurized milk inoculated with the pathogen (Ryser and Marth 1 98 7a, 1989, Yousef and Marth I 988). Factors affecting the fate of pathogens during cheesemaking and subsequent aging include the characteristics of the pathogen (heat, acid and salt tolerance, physiological state), temperature/time profile of the milk 72 from silo storage to completion of cheesemaking, pH profile, generation of metabolites (volatile compounds, inhibitors and bacteriocins produced by the starter culture), native milk enzymes and added enzymes. In addition, raw milk contains various antibacterial factors including antibodies, complement and non-antibody proteins such as lysozyme, lactoferrin and lactoperoxidase as well as macrophages, polymorphonuclear leukocytes, and lymphocytes (Johnson et al. 1990a). Their presence will influence the survival of intracellular pathogens such as L. monocytogenes in fermented dairy products. In our study, raw and subpasteurized milk allowed less repair of sublethally injured cells and also showed higher numbers of injured cells compared to pasteurized milk. Given the low pH and high salt content of cheese, complete inactivation of sublethally injured L. monocytogenes in cheese (even if it survives the cheesemaking/fermentation process) during the 60-day storage period may be possible. The various heat treatments given to milk for cheesemaking should be investigated to better define conditions that will minimize pathogen survival in cheeses that are subject to the mandatory 60-day ripening rule. ACKNOWLEDGMENTS We would like to thank the National Food Safety and Toxicology Center, Michigan State University, East Lansing, M1 for funding this research. We would also like to thank Mr. Kadir Kizilkaya and Ms. Arzu Cagri for helping with programming in SAS. 73 TABLES Table 6: Percent Heat-injury of L. monocytogenes in TPB and UHT Milk Heating Medium 56°C/20 min 64°C/2 min Tryptose Phosphate Broth 9971:2036a 9974212018a UHT Milk 99.44i0.31a 99.413:O.36a Meansistandard deviations (n=3). Means in the same column with different superscript are significantly different (p<0.05). Table 7: Mixed Covariance Procedure Table for Growth of Healthy L. monocytogenes Cells Effect Num DF Den DF F Value Pr > F Milk 4 12.5 17 <.0001 Time 5 48.7 554.44 <.0001 Milk * Time 20 46.9 1.59 0.0974 Note: Pr > F value less than 0.05 indicates significant effect of the particular interaction 74 Table 8: Repair of L. monocytogenes in Different Types of Milk without Starter Culture Incubation Type of Milk Low Heat Injured High Heat injured period (h) % % 2 Raw 5.1843.548 0.2440.138 LHT 1.754039" 0.1840088 HHT 0.394012" 0.3240138 Pasteurized 2.6840748" 02540.1 18 UHT 22541058" 25642888 4 Raw 172543.678" 26040.72c LHT 153743.14" 3.7141 .77"8 HHT 208246.488" 6.8840268 Pasteurized 213242.948" 4.4241 .03"8 UHT 251542.918 5.6241438" 6 Raw 47.124123c 321641.53c LHT 51.194154" 33.724124"8 HHT 512740.92" 33.794153"c Pasteurized 541541.488 35.744093" UHT 553641.428 40.0041268 8 Raw 76.434187" 595440.928I LHT 785841.428" 682941.478" HHT 81.3242248 65.014272“ Pasteurized 799143.048" 63.034266cd UHT 801741.328" 690341688 24 Raw 78.954074" 80.934157" LHT 842844.608" 82.594271" HHT 836343.988" 820442.67" Pasteurized 875741.628 801741.10" UHT 872943.308 877443.888 Meansistandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 75 Table 9: Mixed Covariance Procedure Table for Repair of Sublethally Injured L. monocytogenes Cells After 24 h Effect Num DF Den DF F Value Pr > F Milk 4 14.3 5.64 0.0062 Phys. State 1 6.52 1.99 0.2046 Milk * Phys. State 4 13.4 2.12 0.1352 Note: Pr > F value less than 0.05 indicates significant effect of the particular interaction Table 10: Mixed Covariance Procedure Table for Log Increase of total L. monocytogenes after 24 h Effect Num DF Den DF F Value Pr > F Milk 4 60.2000 6.3300 0.0003 Starter 3 91.3000 35.2800 <.0001 Milk * Starter 12 88.1000 3.2400 0.0007 Phys. State 2 97.7000 10.2000 <.0001 Milk“ Phys. State 8 93.3000 0.9100 0.5101 Starter "' Phys. State 6 63.2000 4.2600 0.0011 Milk“ Starter "‘ Phys. State 24 71.7000 0.7500 0.7849 Note: Pr > F value less than 0.05 indicates significant effect of the particular interaction Table 11: Mixed Covariance Procedure Table for Percent Increase in Injured L. monocytogenes Cells Effect Num DF Den DF F value Pr >F Milk 4 84 4.48 0.0025 Starter 3 67.4 1 1.38 <.0001 Milk "' Starter 12 83 0.94 0.5129 Phys. State 2 258 259.70 <.0001 Milk * Phys. State 8 183 0.80 0.6035 Starter "‘ Phys. State 6 238 28.84 <.0001 Milk * Starter * Phys. State 24 185 3.43 <.0001 Note: Pr > F value less than 0.05 indicates significant effect of the particular interaction 76 Table 12: Percent Increase in Injured L. monocytogenes Cells in Raw Milk at Different Starter Culture Levels Fermentation Starter Healthy Low Heat- High Heat- Period (h) Culture (%) (%) Injured (%) Injured (%) 2 0.5 323041.98" 232944.478 369246.488 1.0 391746.868" 224846.338 252046.57" 2.0 480945.068 218343.178 18.104346" 4 0.5 482643.22" 38.0042558 491844.058 1.0 482445.89" 360342.088 402041.56" 2.0 628143.788 36.804425" 33.044272" 6 0.5 595941.78" 486040.658 566341.548 1.0 60.584133" 467641.398" 525240.66" 2.0 740341.628 447140.95" 456140.478 8 0.5 642342.88" 496340.488 586340.388 1.0 64.164246" 494544.148 558740.40" 2.0 744242.268 470440.858 47.5141 .718 24 0.5 662745.328 489943.088 605841.878 1.0 672843.598 515347.088 605140.948 2.0 743344.278 474141.628 47.974224" Meansistandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 77 Table 13: Percent Increase in Injured L. monocytogenes Cells in Low Heat-Treated Milk at Different Starter Culture Levels Fermentation Starter Healthy Low Heat- High Heat- Period (h) Culture (%) (%) Injured (%) Injured (%) 2 0.5 246943.18" 221941.278 27.1842588 1.0 246542.59" 21 .8442868 23.4344488 2.0 396243.598 230443.288 224446.968 4 0.5 43.804146" 38.9641888 42.6841208 1.0 397244.16" 345641.588" 396144.888" 2.0 545947678 33.684343" 347745.16" 6 0.5 57.724088" 474441.308 541840.638 1.0 563840.62" 463240.988 500041.04" 2.0 693340588 44.134041" 448240848 8 0.5 653441.70" 517341.138 555041.758 1.0 586241478 49.034131" 507741.03" 2.0 706640.448 45.9641 .008 479841.178 24 0.5 678643.72" 53.4741388 57.1342508 1.0 615842638" 50.1942488 52.004226" 2.0 743542.788 495441.738 505040.51" Meansistandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 78 Table 14: Percent Increase in Injured L. monocytogenes Cells in High Heat-Treated Milk at Different Starter Culture Levels Fermentation Starter Healthy Low Heat- High Heat- Period (h) Culture (%) (%) Injured (%) Injured (%) 2 0.5 24.5742148 259540.968 26.6540998 1.0 308942.638 245641.158 21.1145378 2.0 348345.568 19.174425" 222642.658 4 0.5 4477418388 41 .0441348 430540908 1.0 428744.27" 354941.328" 34.294340" 2.0 533647.938 314242.17" 36.034085" 6 0.5 59.624138" 475040.558 541140.728 1.0 553141.15" 46.104080" 48.094060" 2.0 68.0840738 43.3640278 44.9140608 8 0.5 656240.698" 522541.348 56.7340898 1.0 61.154150" 48.914091" 48.944043" 2.0 712641.188 448240.468 501441.00" 24 0.5 705643.638" 538141.138 577241.688 1.0 65.644202" 514840.748 503340.64" 2.0 742442858 46.6340608 51.864084" Meansistandard deviations (n=3). Means in the same column and fermentation period With different superscript are significantly different (p<0.05). 79 Table 15: Percent Increase in Injured L. monocytogenes Cells in Pasteurized Milk at Different Starter Culture Levels Fermentation Starter Healthy Low Heat- High Heat- Period (h) Culture (%) (%) Injured (%) Injured (%) 2 0.5 227243.478 243142.848 295943.048 1.0 27.67451 18 22.6842668" 220745.658" 2.0 315040538 18.874267" 202945.34" 4 0.5 411043.378 402841.688 442742.068 1.0 428244.078 368942.008 36.164277" 2.0 451944.768 305740.58" 36.084212" 6 0.5 534240.52" 474341.388 53.7640888 1.0 538241.95" 459241.498 47.394137" 2.0 658941468 43.014163" 43.1841288 8 0.5 640945.108" 51.4142178 581641.918 1.0 599841.59" 475941.688" 49.054077" 2.0 695142.198 45.084134" 472443.26" 24 0.5 674946.458 532943.788 608942.998 1.0 627140.808 496542.558" 504341.14" 2.0 705543.598 45.904186" 48.534460" Meansdzstandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 80 Table 16: Percent Increase in Injured L. monocytogenes Cells in UHT—Pasteurized Milk at Different Starter Culture Levels Fermentation Starter Healthy Low Heat- High Heat- Period (h) Culture (%) (%) Injured (%) Injured (%) 2 0.5 180047.02" 257640.678 319541.158 1.0 249444.79" 228346.098 14.874233" 2.0 413744.218 310746.618 189142.83" 4 0.5 349442.00" 337942.998 454341.528 1.0 36.164545" 326745.798" 32.344235" 2.0 57.1945378 35.8444928 31.524868" 6 0.5 472640.58" 474140.828 534640.938 1.0 48.004147" 450141.00" 45.724081" 2.0 624841.278 426741.148 42.6440888 8 0.5 516343.71" 494042.748 548941.948 1.0 53.924201" 44.784114" 472641.14" 2.0 649343.518 460941.708" 480340648 24 0.5 51.684422" 505244.678 538944.358 1.0 568343.03" 42.774329" 494141.188" 2.0 714343.068 497343.198 45.644164" Meansistandard deviations (n=3). Means in the same column and fermentation period With different superscript are significantly different (p<0.05). 81 Table 17: Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 0.5% LLLC Fermentation Type of Milk Healthy Low Heat- High Heat- Period (h) (%) Injured (%) Injured (%) 2 Raw 323041988 23.2944478 36.9246488 LHT 246943.18" 22.1941278 27.184258" HHT 24.574214" 259541.278 266540.99" Pasteurized 22.724347" 243142.848 295943.04" UHT 180047.01" 257640.678 319541.158" 4 Raw 48.2643228 380042.558 491744.05a LHT 438041.468" 389641.888 42.684120" HHT 447741.838" 41 .0441348 43.054090" Pasteurized 41.104337" 402841.688 44.274206" UHT 349442008 33.794299" 45.4241 .528" 6 Raw 59.4941968 486040.658 566341.548 LHT 577240.888 474441.308 54.184063" HHT 596241.388 475040.558 54.1 140.73" Pasteurized 534240.52" 474341.388 53.764088" UHT 472640.588 47.4140828 534640.93" 8 Raw 642342888 496340.488 586340.388 LHT 653441.708 517341.138 55.5041.75"8 HHT 65.6240698 52.2541348 56.7340.89°°° Pasteurized 640945.118 514742.178 581641.918" UHT 516343.71" 494042.748 548941.948 24 Raw 66.2745338 489943.088 605841.878 LHT 678643.728 53.4741388 571342.508" HHT 705543.638 538141.138 577241.688" Pasteurized 67.49fl:6.45'l 532943.78a 60.894299a UHT 51.684422" 505244.678 53.894435b Meansistandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 82 Table 18: Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 1.0% LLLC Fermentation Type of Milk Healthy Low Heat- High Heat- Period (h) (%) Injured (%) Injured (%) 2 Raw 391746.868 224746.338 252046.578 LHT 24.654259" 21 .8542868 234344.488" HHT 308942.638" 243641.148 21.1145378" Pasteurized 27.67451 1" 226842.668 220745.658" UHT 249444.79" 228346.098 14.874233" 4 Raw 48.2445898 36.0342088 402041.598 LHT 397244.168" 345641.588 39.6044888 HHT 428744.278" 35.4941328 342943.408" Pasteurized 428244.078" 368842.008 36. 1642.778" UHT 36.164545" 326645.798 32.344235" 6 Raw 605841.338 46.7641398 525240.668 LHT 563840.63" 463240.988 500041.04" HHT 553141.15" 46.1040808 480940.608 Pasteurized 538241.95" 459241.498 473941.378 UHT 480041.478 456141.008 45.384026d 8 Raw 64. 1642.468 494544.148 558740.408 LHT 58.624147" 49.0341318" 507741.03" HHT 61.1441 .508" 489140.918" 48.9440438 Pasteurized 599841.59" 475941.688" 490540.778 UHT 53.9242018 44.784114" 472641.14d 24 Raw 672843.598 515347.088 585140.948 LHT 61.584263" 50.1942478 52.004226" HHT 656342.028" 514840.748 503340.64" Pasteurized 627140.808" 49.6542548" 504341.14" UHT 568343.038 427743.29" 494141.18" Vensfltandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 83 Table 19: Percent Increase in Injured L. monocytogenes Cells in Different Types of Milk Fermented with 2.0% LLLC Fermentation Type of Milk Healthy Low Heat- High Heat- Period (h) (%) Injured (%) Injured (%) 2 Raw 480945.068 218343.17" 18.1043468 LHT 396243.59" 23.044328" 224446.968 HHT 348345.56" 19.174425" 222642.658 Pasteurized 315040.538 188742.67" 202945.348 UHT 41 .3744218" 316746618 189042838 4 Raw 628143.788 368044258 380442728 LHT 545947.678" 336843.438 347745.168 HHT 533647.938" 314242.178 360340.858 Pasteurized 45.194476" 305740.588 36.0842128 UHT 57.1945378 358444.928 315248688 6 Raw 740341.628 447140.958 45.6140468 LHT 693640.55" 441340.418" 44.8240848 HHT 680740.73" 433640.278" 449140.568 Pasteurized 658941.468 430141.638" 43.184128" UHT 62.4841 .278 426741.14" 42.644088" 8 Raw 74.4242268 47.0440858 475141.718 LHT 70.6640448" 459641.008 479841.178 HHT 712641.188" 448240.468 501441.008 Pasteurized 69.514219" 45.0841348 47.2443268 UHT 649343.518 460941.708 43.034064" 24 Raw 74.3344278 474141.628 47.9742368" LHT 743542.788 495441.738 505040.518 HHT 742442.848 46.6340608 518640.848 Pasteurized 705543.598 45.9041868 485344.608" UHT 714343.068 497343.198 45.644164" Meansistandard deviations (n=3). Means in the same column and fermentation period with different superscript are significantly different (p<0.05). 84 FIGURE LEGENDS Figure 4: Sublethal heat injury of L. monocytogenes in UHT milk at 56°C. Key: TPA represents the total population of both healthy as well as sublethally injured cells, TPNA represents the population of healthy cells. Figure 5: Sublethal heat injury of L. monocytogenes in UHT milk at 64°C. Key: same as that for Figure 1. 85 Mnlurv. 8 8 9 a 0 9:. av .erm 3 via Hg 5 amzmmSAogeE 4 mo 49%: :33 6 233m egg: mm om a 2 m. o WagsigfihrtEPr-L 4.. I 1 11...- l? #1 .6 .6 4 N 0 Minds 601 O T. 86 .0640 an 058 FED E ”mauMSAoonoE 4 mo 405.45 :33 um 2:me €5.65: m mN N we F me o o . . 4 1 4 o om -- N 1.” -. _I m ow ' 1.4 .m m. of. ' w. m ’11 O m 8 -. w cow + if T #1 lHrl 11 m biz. 4 IT .420th 0 2 4 6 8 24 MTPA 5.814010 6.804002 7.654025 8.614002 8.884004 8.99401 1 MTPNA 3.054005 5.464031 6.884017 8.294001 8.774003 8.894011 Injured 5.814010 6.784003 7.574027 8.334003 8.254007 8.314010 % Injury 99.82401 94.64436 82.57437 52.70412 23.39419 20.87407 (*Native contaminating bacterial count in milk: 2.054048) Table 24: Fate of HHI L. monocytogenes in Raw Milk without Starter Culture Time ——_> 0 (h) 2 4 6 8 24 MTPA 5.604024 6.794024 7.804029 8.394021 8.934008 9.044007 MTPNA 2.794025 4.374032 6.244020 7.904019 8.704007 8.944006 Injured 5.604024 6.794024 7.794021 8.224021 8.534009 8.314010 % Injury 99.82401 99.58402 97.22407 67.6641.4 40.28409 18.89415 (*Native contaminating bacterial count in milk: 1.284068) 109 1 Table 25: Fate of Uninjured L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Time —» 0 2 4 6 8 24 (h) MTPA 5.864030 7.144046 7.934060 8.414046 8.624044 8.714032 MTPNA 5.744028 6.644017 7.364037 7.504031 7.384033 7.324035 Injured 5.234037 6.934058 7.774069 8.354048 8.594045 8.694032 Starter 6.834064 7.574066 8.334033 8.844019 9.014017 8.884021 pH 6.414003 6.254004 5.984015 5.024042 4.604034 3.754023 (*Native contaminating bacterial count in milk: 2.494021) Table 26: Fate of LHI L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Time —pp 0 2 4 6 8 24 (111 MTPA 6.024043 7.414030 8.304060 8.944068 9.004063 8.954045 MTPNA 2.854035 4.184050 5.274026 5.114035 5.284045 5.1 140.10 Injured 6.024043 7.414030 8.304060 8.944068 9.004063 8.954045 Starter 6.874021 7.674059 8.384021 8.774013 8.954017 9.034009 pH 6.434002 6.224005 5.994002 5.254018 4.704022 4.164013 (*Native contaminating bacterial count in milk: 1.694044) Table 27: Fate of HHI L. monocytogenes in Raw Milk at a 0.5% Starter Inoculum Time ——> 0 2 4 6 8 24 (h) MTPA 5.604024 7.674051 8.354047 8.774030 8.884039 8.994033 MTPNA 2.924046 3.794020 4.724034 5.214008 5.244015 5.154007 Injured 5.604024 7.674051 8.354047 8.774030 8.884039 8.994033 Starter 6.844031 7.834026 8.554010 8.904015 9.024012 9.134017 pH 6.424003 6.234005 6.024003 5.464020 4.834006 4.264014 T(*Native contaminating bacterial count in milk: 1.424080) 110 Table 28 Time - (h) l, MTPA 1 . MTPNA ‘ Injured . Starter l 18” (*Native Table 29 Time (h) MTPA Table 28: Fate of Uninjured L. monocytogenes in Raw Milk at a 1.0% Starter Inoculum Time —-> 0 2 4 6 8 24 (h) MTPA 5.894037 7.544021 8.024046 8.674043 8.864039 9.024031 MTPNA 5.714040 6.344035 6.584030 6.634012 6.674014 6.544007 Injured 5.404031 7.504023 8.004047 8.674043 8.864039 9.024031 Starter 6.554005 7.794021 8.604019 8.834007 9.014004 9.094003 pH 6.424002 6.204002 5.884003 5.1 140.03 4.414008 4.544121 (*Native contaminating bacterial count in milk: 2.024058) Table 29: Fate of LHI L. monocytogenes in Raw Milk at a 1.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 (h) MTPA 6.024050 7.354024 8.184056 8.834065 8.984049 9.104035 MTPNA 2.964022 4.454029 5.284026 5.504007 5.494006 5.514004 Injured 6.024050 7.354024 8.184056 8.834065 8.984049 9.104035 Starter 6.684028 7.554051 8.384022 8.734025 8.934013 9.044009 pH 6.404002 6.184004 5.934005 5.134008 4.514014 3.984010 (*Native contaminating bacterial count in milk: 1.8041 . 12) Table 30: Fate of HHI L. monocytogenes in Raw Milk at a 1.0% Starter Inoculum Time —-> 0 2 4 6 8 24 (h) MTPA 5.614018 7.024052 7.864020 8.554025 8.744026 8.894025 MTPNA 2.824007 4.004009 5.054017 5.364010 5.364007 5.294005 Injured 5.614018 7.024052 7.864020 8.554025 8.744026 8.894025 Starter 6.624032 7.474033 8.154010 8.694017 8.954002 9.014001 pH 6.414003 6.184002 5.934006 5.224004 4.694008 4.054006 (*Native contaminating bacterial count in milk: 1.784042) 11] Table 31: Fate of Uninjured L. monocytogenes in Raw Milk at a 2.0% Starter Inoculum Time 44, 0 2 4 6 8 24 (h) MTPA 5.924042 7.634015 8.334040 8.894037 8.904036 8.894026 MTPNA 5.844044 6.894015 7.104058 7.374019 7.294014 7.334015 Injured 5.104027 7.544015 8.304039 8.874039 8.894037 8.884027 Starter 6.654004 7.634057 8.564036 8.794017 9.014003 9.094002 pH 6.424005 6.224006 5.884004 4.954036 4.404008 3.904010 (*Native contaminating bacterial count in milk: 2.614001) Table 32: Fate of LHI L. monocytogenes in Raw Milk at a 2.0% Starter Inoculum Time —4p 0 2 4 6 8 24 (h) MTPA 6.014026 7.324022 8.214042 8.704042 8.844036 8.864029 MTPNA 2.854013 4.214030 5.074030 5.264017 5.324018 5.234013 Injured 6.014026 7.324022 8.214042 8.704042 8.844036 8.864029 Starter 6.614008 7.764031 8.324038 8.734019 8.884014 9.014005 pH 6.424001 6.184004 5.924007 5.224004 4.594006 4.024010 (*Native contaminating bacterial count in milk: 1.754044) Table 33: Fate of HHI L. monocytogenes in Raw Milk at a 2.0% Starter Inoculum Time —» 0 2 4 6 8 24 (h) MTPA 6.054022 7.144023 8.054028 8.814034 8.934023 8.954020 MTPNA 2.674035 3.834006 4.904011 5.244010 5.234013 5.214004 Injured 6.054022 7.144023 8.054028 8.814034 8.934023 8.954020 Starter 6.594063 7.474041 8.424009 8.734026 9.004009 9.014004 pH 6.444003 6.204001 5.934004 5.204009 4.714005 4.084003 (*Native contaminating bacterial count in milk: 1.824050) 112 I Table 3f r—\ '1 Time \th MTPA \ , MTPN; l Injured ix ; 0/0 anur x (THO n; Table 3 Table 34: Fate of Uninjured L. monocytogenes in LHT Milk without Starter Culture Time .— (h) + 0 2 4 6 8 24 MTPA 6.014032 7.294021 8.524020 8.864012 9.074002 9.184006 MTPNA 5.934031 7.234021 8.454021 8.804013 8.984003 9.124008 Injured 5.274035 6.424023 7.694018 7.944028 8.344001 8.274016 18.09416 % Injury 13.57418 15.32456 13.6447.1 18.6941.4 13.40463 (*No native bacteria detected in milk after heat treatment) Table 35: Fate of LHI L. monocytogenes in LHT Milk without Starter Culture Time — (h) 0 2 4 6 8 24 MTPA 5.864016 6.944009 7.754007 8.714007 8.924006 9.094016 MTPNA 3.064010 5.214001 6.944006 8.424005 8.824007 9.014018 Injured 5.864016 6.934009 7.684008 8.394008 8.254004 8.274011 % Injury 99.84401 98.09404 84.47432 48.65416 21.27414 15.56446 (*No native bacteria detected in milk after heat treatment) Table 36: Fate of HHI L. monocytogenes in LHT Milk without Starter Culture Time — (h) + 0 2 4 6 8 24 MTPA 5.644023 6.784024 7.764007 8.604011 8.884012 9.064006 MTPNA 2.874011 4.324024 6.324028 8.134011 8.724011 8.974007 Injured 5.644023 6.784024 7.744006 8.424011 8.384014 8.294006 % Injury 99.82401 99.64401 96.1141.7 66.10413 31.53415 17.23428 (*No native bacteria detected in milk after heat treatment) 113 Table 37: Fate of Uninjured L. monocytogenes in LHT Milk at a 0.5% Starter Inoculum Time —-> 0 2 4 6 8 24 (h) MTPA 5.824019 7.034033 7.864016 8.524020 8.774028 8.874016 MTPNA 5.674018 6.824027 7.504014 8.02401 1 7.724007 7.544006 Injured 5.284022 6.584042 7.594024 8.324031 8.724030 8.854017 Starter 6.904054 7.544066 8.284028 8.784018 8.974010 9.024002 pH 6.454003 6.264003 5.954006 5.424016 4.794019 4.134005 (*No native bacteria detected in milk after heat treatment) Table 38: Fate of LHI L. monocytogenes in LHT Milk at a 0.5% Starter Inoculum Time —_> 0 2 4 6 8 24 (h) MTPA 5.854006 7.144011 8.124018 8.624010 8.874008 8.974002 MTPNA 2.844006 4.264033 5.254024 5.384013 5.354005 5.314010 Injured 5.854006 7.144011 8.124018 8.624010 8.874008 8.974002 Starter 6.644007 7.364056 8.234051 8.644043 8.92401 1 8.954012 pH 6.424004 6.264008 6.014005 5.334026 4.884014 4.394013 (*No native bacteria detected in milk after heat treatment) Table 39: Fate of HHI L. monocytogenes in LHT Milk at a 0.5% Starter Inoculum Time —+ 0 2 4 6 8 24 (h) MTPA 5.674023 7.214026 8.094029 8.744032 8.814025 8.904022 MTPNA 2.534021 3.864012 4.914014 5.304011 5.264004 5.194001 Injured 5.674023 7.214026 8.084029 8.744032 8.814025 8.904022 Starter 6.914030 7.474039 8.434006 8.804011 8.924010 9.034005 EpH 6.464004 6.264007 6.004008 5.324018 4.794014 4.184005 (*Native bacterial count in milk after heat-treatment: 0524000) 114 Table 40: Fate of Uninjured L. monocytogenes in LHT Milk at a 1.0% Starter Inoculum Time .4) 0 2 4 6 8 24 (h) MTPA 5.984020 7.244036 7.834031 8.474033 8.574028 8.724023 MTPNA 5.854020 7.094033 7.514021 7.504012 7.434016 7.414009 Injured 5.394023 6.724043 7.534041 8.424035 8.544029 8.704023 Starter 6.714016 7.614047 8.414040 8.754024 8.944010 9.064006 pH 6.434001 6.184003 5.844004 5.084006 4.364006 3.864004 (*No native bacteria detected in milk after heat treatment) Table 41: Fate of LHI L. monocytogenes in LHT Milk at a 1.0% Starter Inoculum Time — 0 2 4 6 8 24 (h) MTPA 5.964011 7.274030 8.034023 8.734010 8.894008 8.964001 MTPNA 2.844024 4.094040 4.984031 5.154017 5.204019 5.184015 Injured 5.964011 7.274030 8.034023 8.734010 8.894008 8.964001 Starter 6.764008 7.824014 8.294024 8.724006 8.914008 9.004004 pH 6.454003 6.204003 5.934008 5.234016 4.554019 4.014012 (*No native bacteria detected in milk after heat treatment) Table 42: Fate of HHI L. monocytogenes in LHT Milk at a 1.0% Starter Inoculum Time ——p 0 2 4 6 8 24 . (h) MTPA 5.894014 7.274024 8.224025 8.834017 8.884015 8.954008 MTPNA 2.504017 4.364014 4.934003 5.174003 5.214001 5.544057 Injured 5.894014 7.274024 8.224025 8.834017 8.884015 8.954008 Starter 6.454017 7.544043 8.254040 8.774024 8.934010 9.034003 pH 6.434003 6.214004 5.974005 5.284005 4.724007 4.044005 (*No native bacteria detected in milk after heat treatment) 115 Table 43: Fate of Uninjured L. monocytogenes in LHT Milk at a 2.0% Starter Inoculum Time -> 0 2 4 6 8 24 (10 MTPA 5.644017 7.534019 8.114030 8.724028 8.774030 8.954026 MTPNA 5.474016 7.294023 7.624016 7.534005 7.454005 7.414003 Injured 5.134020 7.154015 7.924038 8.684031 8.754033 8.944027 Starter 6.884035 7.904001 8.554013 8.924005 9.024003 9.094002 pH 6.414001 6.164002 5.904003 5.114008 4.384003 3.894006 (*No native bacteria detected in milk after heat treatment) Table 44: Fate of LHI L. monocytogenes in LHT Milk at a 2.0% Starter Inoculum Time -—-> 0 2 4 6 8 24 (h) MTPA 6.004011 7.384011 8.024018 8.654013 8.764010 8.984008 MTPNA 3.014024 4.174037 5.174021 5.514017 5.244008 5.194006 Injured 6.00401 1 7.384011 8.024026 8.654023 8.764014 8.984006 Starter 6.684020 7.754013 8.344026 8.754023 8.944014 9.034006 pH 6.564012 6.194002 5.924008 5.164008 4.624009 4.064005 (*No native bacteria detected in milk after heat treatment) Table 45: Fate of HHI L. monocytogenes in LHT Milk at a 2.0% Starter Inoculum Time —--p 0 2 4 6 8 24 (h) MTPA 5.994003 7.344042 8.084031 8.684004 8.874005 9.024002 MTPNA 2.724022 3.864012 4.574033 5.184001 5.234006 5.154005 Injured 5.994003 7.344042 8.084031 8.684004 8.874005 9.024002 Starter 6.404044 7.684044 8.634028 8.794023 8.984010 9.044006 pH 6.434002 6.214003 5.954003 5.224008 4.664022 4.054009 (*No native bacteria detected in milk after heat treatment) 116 Table 46: Fate of Uninjured L. monocytogenes in HHT Milk without Starter Culture Time ——p 0 2 4 6 8 24 (h) MTPA 5.934026 7.174018 8.314015 8.784011 8.974005 9.104003 MTPNA 5.864028 7.124016 8.214015 8.724011 8.914004 9.044003 Injured 5.074012 6.114039 7.604013 7.934010 8.084014 8.234006 % Injury 14.28448 10.1547.0 19.59426 14.19413 13.00425 13.30413 (*No native bacteria detected in milk after heat treatment) Table 47: Fate of LHI L. monocytogenes in HHT Milk without Starter Culture Time .4; 0 2 4 6 8 24 (h) MTPA 5.784018 6.934014 7.704010 8.484023 8.914012 9.104012 MTPNA 3.004004 4.674015 7.004024 8.194022 8.824012 9.034013 Injured 5.784018 6.934014 7.594008 8.164023 8.174011 8.304007 % Injury 99.83401 99.44401 79.01465 48.56410 18.51423 16.20440 Table 48: Fate of HHI L. monocytogenes in HHT Milk without Starter Culture Time —-> 0 2 4 6 8 24 (h) ' MTPA 5.584017 6.674019 7.584018 8.564010 8.914011 9.054012 MTPNA 2.744013 4.314012 6.424019 8.094011 8.734010 8.964010 Injured 5.584017 6.664019 7.554018 8.384009 8.454013 8.294018 % Injury 99.85401 99.53402 92.97403 66.09416 34.84427 17.64428 (*No native bacteria detected in milk after heat treatment) 117 Table 49: Fate of Uninjured L. monocytogenes in HHT Milk at a 0.5% Starter Inoculum Time .4. 0 2 4 6 8 24 (h) MTPA 5.794025 68540.36 7.704025 83940.23 85940.37 8.794021 MTPNA 5.674026 6.654038 73440.22 78840.06 7.704005 76140.05 Injured 5.144024 64140.36 74440.30 82040.33 8.514041 8.764023 Starter 6.644013 7.544066 8.384046 8.744013 9.044009 9.054003 pH 6.414003 6.284005 59440.15 54340.07 46440.07 4.1 140.07 (*No native bacteria detected in milk after heat treatment) Table 50: Fate of LHI L. monocytogenes in HHT Milk at a 0.5% Starter Inoculum Time —-> 0 2 4 6 8 24 (h) MTPA 5.76401 1 7.254010 8.124022 8.494019 8.774017 8.864015 MTPNA 2.804004 4.274039 5.254023 5.434005 5.414005 5.294013 Injured 5.764011 7.254010 8.124022 8.494019 8.774017 8.864015 Starter 6.844030 7.614055 8.344046 8.794026 8.994013 9.034010 pH 6.464002 6.264001 6.004003 5.294021 4.724024 4.204017 (*No native bacteria detected in milk after heat treatment) Table 51: Fate of HHI L. monocytogenes in HHT Milk at a 0.5% Starter Inoculum Time —--> 0 2 4 6 8 24 (h) MTPA 5.624018 7.124019 8.044029 8.674030 8.814025 8.874020 MTPNA 2.584027 3.824024 4.804014 5.114006 5.134011 5.104019 Injured 5.624018 7.124019 8.044029 8.674030 8.814025 8.874020 Starter 6.894033 7.634052 8.364040 8.754026 8.964014 9.064010 pH 6.444003 6.264008 6.014002 5.38401 1 4.834012 4.264009 (*No native bacteria detected in milk after heat treatment) 118 Table 52: F ate of Uninjured L. monocytogenes in HHT Milk at a 1.0% Starter Inoculum Time —hp 0 2 4 6 8 24 (h) MTPA 5.884019 7.334014 7.924022 8.424021 8.704029 8.924019 MTPNA 5.714020 7.034019 7.554028 7.604016 7.564010 7.474015 Injured 5.384018 7.034011 7.684018 8.354022 8.664030 8.904019 Starter 6.644014 7.694038 8.554018 8.824004 8.944008 9.044005 pH 6.454006 6.194003 5.884004 5.124013 4.454013 3.944005 (*No native bacteria detected in milk after heat treatment) Table 53: Fate of LHI L. monocytogenes in HHT Milk at a 1.0% Starter Inoculum Time —~L> 0 2 4 6 8 24 (h) MTPA 5.914008 7.364011 8.014018 8.634015 8.804007 8.954008 MTPNA 2.764015 4.174037 5.174021 5.354003 5.244008 5.194006 Injured 5.914008 7.364011 8.014018 8.634015 8.804007 8.954008 Starter 6.824020 7.754013 8.344026 8.784016 8.934016 9.034007 pH 6.454003 6.224004 5.96003 5.264006 4.584006 4.084004 (*No native bacteria detected in milk after heat treatment) Table 54: Fate of HHI L. monocytogenes in HHT Milk at a 1.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 (h) MTPA 5.974009 7.244042 8.024031 8.854012 8.904011 8.984010 MTPNA 2.654030 3.994027 4.814024 5.254005 5.204004 5.164002 Injured 5.974009 7.244042 8.024031 8.844012 8.90401 1 8.984010 Starter 6.624012 7.874009 8.444010 8.764003 8.904011 9.004007 pH 6.424005 6.184003 5.874005 5.074005 4.434003 3.994003 (*No native bacteria detected in milk after heat treatment) 119 Table 55: Fate of Uninjured L. monocytogenes in HHT Milk at a 2.0% Starter Inoculum Time -—-> 0 2 4 6 8 24 (h) MTPA 5.774033 7.284014 7.964017 8.644032 8.814029 8.944023 MTPNA 5.654036 7.044017 7.334017 7.364007 7.534005 7.464001 Injured 5.134021 6.904016 7.854017 8.614033 8.784031 8.934024 Starter 6.774015 7.954008 8.364010 8.774026 8.974012 9.054008 pH 6.424003 6.184003 5.904009 5.224020 4.584022 4.014009 (*No native bacteria detected in milk after heat treatment) Table 56: Fate of LHI L. monocytogenes in HHT Milk at a 2.0% Starter Inoculum Time 14} 0 2 4 6 8 24 (h) MTPA 6.124009 7.294023 8.044016 8.774011 8.864010 8.974010 MTPNA 2.984009 4.124035 4.974021 5.214008 5.244009 5.224008 Injured 6.124009 7.294023 8.044016 8.774011 8.864010 8.974010 Starter 6.484009 7.504036 8.314026 8.774026 8.934004 9.014001 pH 6.444003 6.194001 5.924004 5.194009 4.594004 4.054005 (*No native bacteria detected in milk after heat treatment) Table 57: Fate of HHI L. monocytogenes in HHT Milk at a 2.0% Starter Inoculum Time —_p 0 2 4 6 8 24 (h) MTPA 5.914010 7.224021 8.044017 8.564017 8.874015 8.974010 MTPNA 2.644019 4.374014 4.934003 5.084008 5.244015 5.154007 Injured 5.914010 7.224021 8.044017 8.564017 8.874015 8.974010 Starter 6.654034 7.544043 8.224035 8.734019 8.974012 9.024003 pH 6.424002 6.204003 5.924005 5.174001 4.574013 4.004008 (*No native bacteria detected in milk after heat treatment) 120 Table 58: Fate of Uninjured L. monocytogenes in Pasteurized Milk without Starter Culture Time ——> 0 2 4 6 8 24 (h) MTPA 5.914026 7.294025 8.514022 8.864014 9.074005 9.184008 MTPNA 5.844029 7.214023 8.444021 8.804015 8.994008 9.1 140.09 Injured 5.034012 6.494035 7.684029 7.934009 8.244010 8.394003 % Injury 14.05453 16.32443 15.51459 12.04425 15.61450 16.05419 (*No native bacteria detected in milk after heat treatment) Table 59: Fate of LHI L. monocytogenes in Pasteurized Milk without Starter Culture Time ——Lp 0 2 4 6 8 24 (h) MTPA 5.834014 6.944008 7.814005 8.664005 8.924008 9.074022 MTPNA 3.064006 5.39401 1 7.144003 8.404004 8.834008 9.014021 Injured 5.834014 6.934009 7.714007 8.324007 8.22401 1 8.164027 % Injury 99.83401 97.15408 78.51430 45.68415 19.91430 12.2541.7 (*No native bacteria detected in milk after heat treatment) Table 60: Fate of HHI L. monocytogenes in Pasteurized Milk without Starter Culture Time -~-> 0 2 4 6 8 24 (h) MTPA 5.624023 6.784021 7.824014 8.574012 8.934010 9.104007 MTPNA 2.834016 4.384023 00.14 8.134012 8.734011 9.004007 Injured 5.624023 6.784021 7.804014 8.384012 8.494009 8.394009 % Injury 99.83401 99.57402 95.4141.0 64.09410 36.80426 19.654l.l (*No native bacteria detected in milk after heat treatment) 121 Table 61: Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 0.5% Starter Inoculum Time —_> 0 2 4 6 8 24 (h) MTPA 5.894029 6.914042 7.744017 8.414026 8.824015 8.984010 MTPNA 5.754031 6.644037 7.284007 7.904004 7.744004 7.644006 Injured 5.364026 6.584047 7.554022 8.224038 8.784016 8.964010 Starter 6.924058 7.544069 8.294020 8.794026 9.014016 8.794023 pH 6.414003 6.274003 6.014014 5.394021 4.764022 4.074009 (*No native bacteria detected in milk after heat treatment) Table 62: Fate of LHI L. monocytogenes in Pasteurized Milk at a 0.5% Starter Inoculum Time _1, 0 2 4 6 8 24 (h) MTPA 5.984033 7.424028 8.394056 8.814044 9.054049 9.154028 MTPNA 2.834012 4.224048 5.274025 5.484009 5.554019 5.364018 Injured 5.984033 7.424028 8.394056 8.814044 9.054049 9.154028 Starter 6.604013 7.584062 8.134072 8.734031 8.944013 9.034008 pH 6.434002 6.254007 6.004007 5.264027 4.704024 4.154005 (*No native bacteria detected in milk after heat treatment) Table 63: Fate of HHI L. monocytogenes in Pasteurized Milk at a 0.5% Starter Inoculum Time —L> 0 2 4 6 8 24 (h) MTPA 5.684021 7.374039 8.204041 8.744028 8.994025 9.144018 MTPNA 2.634035 3.814020 4.574033 5.184003 5.234006 5.154005 Injured 5.684021 7.374039 8.204041 8.744028 8.994025 9.144018 Starter 6.744003 7.704040 8.654025 8.854007 8.974012 9.054005 pH 6.444003 6.214004 6.014002 5.404005 4.874005 4.104004 (*No native bacteria detected in milk after heat treatment) 122 141 Table 64: Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 1.0% Starter Inoculum Time —+ 0 2 4 6 8 24 (h) MTPA 5.984029 7.334011 7.964018 8.514028 8.814025 8.964030 MTPNA 5.804033 7.044017 7.334018 7.564015 7.574009 7.464001 Injured 5.494021 7.014005 7.844018 8.454030 8.794026 8.944031 Starter 7.144022 7.954007 8.404009 8.954008 9.044004 9.094006 pH 6.434002 6.214002 5.924003 5.124005 4.444004 3.904006 (*No native bacteria detected in milk after heat treatment) Table 65: Fate of LHI L. monocytogenes in Pasteurized Milk at a 1.0% Starter Inoculum Time 4 0 2 4 6 8 24 (h) MTPA 5.944015 7.294023 8.144032 8.674021 8.774014 8.894010 MTPNA 2.944013 4.124035 4.994023 5.234016 5.25401 1 5.224008 Injured 5.944015 7.294023 8.144032 8.674021 8.774014 8.894010 Starter 6.654008 7.504036 8.314026 8.754009 8.934004 9.014001 pH 6.454003 6.194002 5.904005 5.144005 4.464008 3.944003 (*No native bacteria detected in milk after heat treatment) Table 66: Fate of HHI L. monocytogenes in Pasteurized Milk at a 1.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 (h) MTPA 5.974013 7.304049 8.144027 8.804014 8.904015 8.994013 MTPNA 2.544058 3.834015 4.474016 5.224008 5.224008 5.164007 Injured 5.974013 7.304049 8.144027 8.804014 8.904015 8.994013 Starter 6.694009 7.504034 8.194010 8.634012 8.894005 9.034001 pH 6.424001 6.204001 5.954004 5.204006 4.694009 4.094012 (*No native bacteria detected in milk after heat treatment) 123 Table 6. A 5‘ ‘1 .— Table 67: Fate of Uninjured L. monocytogenes in Pasteurized Milk at a 2.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 (h) MTPA 5.824031 7.284015 7.914022 8.674021 8.834020 8.884014 MTPNA 5.694036 7.084014 7.644013 7.644013 7.574009 7.494003 Injured 5.204020 6.834023 7.544034 8.624025 8.814022 8.864015 Starter 6.614005 7.574025 8.464018 8.734001 8.964004 9.034003 pH 6.394003 6.164004 5.924006 5.224007 4.554007 4.054004 (*No native bacteria detected in milk after heat treatment) Table 68: Fate of LHI L. monocytogenes in Pasteurized Milk at a 2.0% Starter Inoculum Time —-+ 0 2 4 6 8 24 (h) _ MTPA 6.154009 7.314026 8.034014 8.794009 8.924008 8.974004 MTPNA 2.944019 4.104023 5.034016 5.264012 5.394007 5.384006 Injured 6.154009 7.314026 8.034014 8.794009 8.924008 8.974004 Starter 6.694025 7.754021 8.36401 1 8.774012 8.974003 9.054004 pH 6.454003 6.174003 5.914009 5.134012 4.504013 3.994007 (*No native bacteria detected in milk after heat treatment) Table 69: F ate of HHI L. monocytogenes in Pasteurized Milk at a 2.0% Starter Inoculum Time -—+ 0 2 4 6 8 24 (h) MTPA 6.044023 7.274056 8.224039 8.654026 8.894015 8.974008 MTPNA 2.624015 3.954023 5.034015 5.214005 5.264007 5.234004 Injured 6.044024 7.274056 8.224039 8.654026 8.894015 8.974008 Starter 6.754008 7.604041 8.274035 8.734010 8.934010 9.024003 pH 6.454004 6.204003 5.874009 5.26401 1 4.554006 4.014006 (*No native bacteria detected in milk after heat treatment) 124 Table 7". 1 Time (h) 1 TPA L___v.— 1 1184 l_—_ ' Injured l 0 Injur; ("N0 11‘- Table 70: Fate of Uninjured L. monocytogenes in UHT Milk without Starter Culture Time 4+ 0 2 4 6 8 24 (11) TPA 5.174051 6.034057 7.194045 7.864009 8.204032 8.454046 TPNA 5.124050 5.994057 7.154045 7.824009 8.154029 8.414045 Injured 4.274063 4.904061 6.154042 6.774007 7.204064 7.344051 % Injury 12.76435 76642.1 93042.3 8.41418 11.51465 79641.5 (*No native bacteria detected in milk after heat treatment) Table 71: Fate of LHI L. monocytogenes in UHT Milk without Starter Culture Time —+ 0 2 4 6 8 24 (h) TPA 4.294059 4.404058 4.624054 5.174036 6.054008 7.534012 TPNA 2.054065 2.834075 4.034058 4.924037 5.964009 7.474014 Injured 4.294059 4.394057 4.494052 4.824034 5.334006 6.60400 % Injury 99.42401 97.1741.l 74.27430 44.06415 19.28415 12.13433 (*No native bacteria detected in milk after heat treatment) Table 72: Fate of HHI L. monocytogenes in UHT Milk without Starter Culture Time —l+ 0 2 4 6 8 24 0!) TPA 3.844059 4.644062 5.094042 5.814091 6.244067 7.464016 TPNA 1.414068 2.954013 3.864034 5.424092 6.084068 7.414017 Injured 3.844059 4.634063 5.064042 5.594090 5.724064 6.524007 % Injury 99.59402 97.04427 93.97413 59.60412 30.5641.7 11.86440 (*No native bacteria detected in milk after heat treatment) 125 “um Vivi-F r I Table 73: Fate of Uninjured L. monocytogenes in UHT Milk at a 0.5% Starter Inoculum Time —-+ 0 2 4 6 8 24 (11) TPA 6.564028 7.454039 8.274043 8.724030 8.924016 8.934012 TPNA 6.464030 7.164036 7.794075 7.894019 7.674042 7.724037 Injured 5.864023 6.934067 7.914026 8.644037 8.894016 8.894015 Starter 7.114041 7.844016 8.514031 8.874001 9.034004 9.014003 pH 6.444002 6.344003 6.13401 1 5.644019 4.944010 4.144019 (*No native bacteria detected in milk after heat treatment) Table 74: Fate of LHI L. monocytogenes in UHT Milk at a 0.5% Starter Inoculum Time —+ 0 2 4 6 8 24 (11) TPA 5.944017 7.474023 7.964013 8.764021 8.874016 8.944006 TPNA 2.984059 4.864013 6.254082 6.614050 6.524054 6.454053 Injured 5.944017 7.474023 7.94401 1 8.754020 8.874015 8.934006 Starter 7.004043 7.544073 8.374037 8.824026 8.974012 8.984023 pH 6.444003 6.314002 6.134003 5.734017 5.034007 4.024011 (*No native bacteria detected in milk after heat treatment) Table 75: Fate of HHI L. monocytogenes in UHT Milk at a 0.5% Starter Inoculum Time 4+ 0 2 4 6 8 24 (11) TPA 5.744023 7.574030 8.354038 8.804032 8.884032 8.824012 TPNA 3.074043 5.094008 5.814023 5.994008 6.094007 6.044009 Injured 5.744023 7.574030 8.344038 8.804032 8.884032 8.824012 Starter 6.904029 7.364043 8.194040 8.544049 8.824023 8.994006 pH 6.454004 6.244003 6.034010 5.274008 4.924008 3.964005 (*No native bacteria detected in milk after heat treatment) 126 Table 76: Fate of Uninjured L. monocytogenes in UHT Milk at a 1.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 01) TPA 6.044016 7.184011 7.724014 8.384030 8.714038 8.864020 TPNA 5.814015 6.424028 6.614022 6.924010 7.194044 6.644022 Injured 5.654021 7.054025 7.684014 8.364031 8.704037 8.864021 Starter 6.924040 7.574070 8.424047 8.624013 8.894005 9.034010 pH 6.424002 6.254003 5.994005 5.154008 4.564008 3.894002 (*No native bacteria detected in milk after heat treatment) Table 77: Fate of LHI L. monocytogenes in UHT Milk at a 1.0% Starter Inoculum Time —+ 0 2 4 6 8 24 (h) TPA 6.324025 7.784068 8.394053 9.174039 9.154030 9.024015 TPNA 3.564049 4.864053 6.004024 6.35401 1 6.204006 6.094016 Injured 6.324025 7.784068 8.394053 9.174039 9.154030 9.024015 Starter 7.384053 7.99401 1 8.464039 8.67401 1 8.894015 8.974006 pH 6.424002 6.244004 5.954008 5.01401 1 4.524010 3.934010 (*No native bacteria detected in milk after heat treatment) Table 78: Fate of HHI L. monocytogenes in UHT Milk at a 1.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 (11) TPA 5.964027 6.844019 7.884045 8.684036 8.774037 8.894034 TPNA 2.974066 4.204067 4.634057 5.634021 5.494020 5.444019 Injured 5.954027 6.844019 7.884045 8.684036 8.774037 8.894034 Starter 6.954040 7.354056 8.264020 8.764024 8.964012 8.924018 pH 6.414003 6.244004 6.004009 5.214024 4.644021 3.874017 (*No native bacteria detected in milk after heat treatment) 127 Table 79: Fate of Uninjured L. monocytogenes in UHT Milk at a 2.0% Starter Inoculum Time 4L» 0 2 4 6 8 24 (11) TPA 6.474037 8.144043 8.454043 8.704020 8.824007 9.154009 TPNA 6.434039 7.874077 7.574029 7.694023 7.614035 7.784042 _ Injured 5.334014 7.534026 8.384046 8.664020 8.784006 9.134008 Starter 7.124038 8.014018 8.604038 8.854011 9.044002 9.024002 pH 6.444003 6.194004 5.864004 5.054004 4.654012 3.954004 (*No native bacteria detected in milk after heat treatment) Table 80: Fate of LHI L. monocytogenes in UHT Milk at a 2.0% Starter Inoculum Time —+ 0 2 4 6 8 24 (h) TPA 5.894028 7.724005 8.014010 8.414033 8.614031 8.824026 TPNA 3.264023 6.064022 6.224050 6.404058 6.314054 6.184052 Injured 5.894029 7.714004 7.994010 8.404034 8.604032 8.824026 Starter 7.084030 8.014024 8.644041 8.914012 9.064004 9.104003 pH 6.444004 6.194004 5.924002 5.134001 4.714007 3.964004 (*No native bacteria detected in milk after heat treatment) Table 81: Fate of HHI L. monocytogenes in UHT Milk at a 2.0% Starter Inoculum Time 4+ 0 2 4 6 8 24 01) TPA 6.094013 7.264035 8.014054 8.694015 8.714014 8.874011 TPNA 3.444046 5.524035 5.944022 6.244032 6.284054 5.764020 Injured 6.094013 7.264035 8.014054 8.694015 8.714014 8.874011 Starter 7.084013 7.854049 8.494050 8.654054 8.894028 9.054004 pH 6.474004 6.194006 5 .934006 5.154007 4.794004 3 .964004 (*No native bacteria detected in milk after heat treatment) 128 Table 82: Growth of Starter Culture at 0.5% Inoculum Level without L. monocytogenes Time 4+ 0 2 4 6 8 24 0!) Starter 7.014021 7.954014 8.594036 8.934008 9.014002 9.064005 pH 6.434001 6.314002 6.164006 5.784012 5.024007 4.204017 Table 83: Growth of Starter Culture at 1.0% Inoculum Level without L. monocytogenes Time —+ 0 2 4 6 8 24 0!) Starter 6.914022 7.904007 8.394035 8.924004 9.004002 9.034001 pH 6.424002 6.244003 5.984003 5.194006 4.624008 4.034004 Table 84: Growth of Starter Culture at 2.0% Inoculum Level without L. monocytogenes Time —+ 0 2 4 6 8 24 (h) Starter 6.844020 8.074007 8.814009 8.954002 9.034004 9.064003 pH 6.434002 6.224003 5.904009 5.064009 4.574006 3.974005 Table 85: Acid-Injury of L. monocytogenes in Tryptose Phosphate Broth (pH 3.5) Time (min) TPA TPNA % Injury 0 7.674024 7.614026 15.134665 15 7.604025 7.504031 249641106 30 7.504027 7.384030 26.114589 45 74240.27 72440.28 35.554456 60 72240.11 6.914010 503241.71 Mean4standard deviation (n=4) 129 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII lllllllllllllllllllllllllllllllllll