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1 DETERMINING THERMAL LETHALITY TO REDUCE PRESENCES OF POTENTIAL PATHOGENIC VIBRIO SPP. IN OYSTERS, CRASSOSTREA VIRGINICA By CHRISTOPHER W. HANNA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PART IAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Christopher W. Hanna
3 To my friends, brothers and sisters who always su pported me through my endeavors
4 ACKNOWLEDGMENTS I would like to thank my major professor Dr. Steve Otwell and his knowledgeable lab co workers Laura Garrido and Victor Garrido. Without their help I would never ha ve been able to accomplish this work Dr. Otwell has stood behind my work, helped me to make the connect ions needed to be able to enter restaurants to conduct studies, and helped fund and support me throughout the entire endeavor. I also acknowledge the patient help provided Dr. Wright and Dr. Schneider who taught me microbiology and helped me through ever y major step of this process. Dr. Wright generously allowed the use of her lab and facilities, while Dr. Schneider provided specific and continuing advice on protocols and thermal assessments. Without their help this never would have been possible. I would like to thank my sisters Laura and Jenna for their support and help through college. I would like to show my appreciation for Zina Williams and Charlene Burke for helping me organize and execute my travels and lab work. I would have gone crazy without you r patience and knowledge, and for that I thank you. Lastly, I would like to thank all my Fraternity Brothers of the Delta Zeta chapter of Delta Tau Delta: Dedicated to Lives of Excellence.
5 TABLE OF CONTENTS page ACKNOWL EDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATI ONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 2 LITERATURE REVIEW ................................ ................................ .......................... 17 3 OBJECTIVES AND HYPOTHESIS ................................ ................................ ......... 25 4 MATERIALS AND METHODS ................................ ................................ ................ 26 Bac terial Growth Curves ................................ ................................ ......................... 26 Thermal Applications in Media ................................ ................................ ................ 28 Determining D and z Values in PBS Media ................................ ........................... 29 Thermal Assessments with Oysters ................................ ................................ ........ 33 Assessing Commercial Cooking Procedures ................................ .......................... 35 Assessing Re gulatory Guidelines ................................ ................................ ........... 37 5 VIBRIO SPP. GROWTH CURVES ................................ ................................ ......... 38 6 D AND Z VALUE ASSESMENTS ................................ ................................ .......... 41 7 THERMAL RECOVERY STUDY ................................ ................................ ............ 50 8 ASSESSING COMMERICAL COOKING PROCEDURES ................................ ...... 53 9 DISCUSSION ................................ ................................ ................................ ......... 58 LIST OF REFERENCES ................................ ................................ ............................... 63 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 66
6 LIST OF TABLES Table page 2 1 Heat resistance of V. cholerae in shrimp homogenate conducted by Hinton and Grodner, 1985. ................................ ................................ ............................ 18 2 2 Heat resistance of V. cholerae in crabmeat homogenate cond ucted by Shultz et al. (1984) ................................ ................................ ................................ ........ 19 6 1 D and z values for V. vulnificus, V. parahaemolyticus, and V. cholerae Letters are used to denote statistical differences with a p 0.05 of the mean. .... 49 7 1 Overnight Temperature Abused Oyster MPN Calculations for V. vulnificus, V. parahaemolyticus and total bacteria in MPN/mL. ................................ ............... 50 7 2 Log unit reduction of Heat Treated Temperature Abused Oysters for V. vulnificus, V. parahaemolyticus and total bacteria in MPN/mL.. ......................... 51 8 1 Initial concentrations and Log unit Reduction of Temperature Abused Oyster V. vulnificus, V. parahaemolyticus and total bacteria after a 200 o F heat treatment in CFU/mL.. ................................ ................................ ........................ 55 9 1 D values for V. cholerae ................................ ................................ ..................... 58 9 2 D values for V. parahaemolyticus. ................................ ................................ ...... 59 9 3 D values for V. vulnificus. ................................ ................................ ................... 59
7 LIST OF FIGURES Figure page 5 1 Growth curve of Vibrio cholerae N16961 in L Broth.. ................................ ......... 38 5 2 Growth curve of Vibrio vulnificus CMCP6 in L Broth. The lin e at 8 hr displays the end of exponential growth and the beginning of the stationary phase. The line at 12 hr displays the beginning of mid stationary phase. ............................. 39 5 3 Growth curve of Vibrio para haemolyticus TX2103 in L Broth. The line at 9 hr displays the end of exponential growth and the beginning of the stationary phase. The line at 12 hr displays the beginning of mid stationary phase. .......... 40 6 1 Vibrio cholerae N16961 D48 time versus average log 10 CFU/mL graph with standard deviation. R 2 =0.9923 ................................ ................................ ........... 41 6 2 Vibrio vulnificus CMCP6 D48 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9916 ................................ ................................ ........... 42 6 3 Vibrio parahaemolyticus TX2103 D48 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9887. ................................ ................................ .......... 43 6 4 Vibrio cholerae N16961 D50 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9659. ................................ ................................ ......................... 44 6 5 Vibrio vulnificus CMCP6 D50 time versus average log 10 CFU/mL with standard devia tion. R 2 =0.9501. ................................ ................................ .......... 44 6 6 Vibrio parahaemolyticus TX2103 D50 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9041. ................................ ................................ .......... 45 6 7 Vibrio cholerae N16961 D55 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9885. ................................ ................................ ......................... 46 6 8 Vibrio vulnificus CMCP6 D55 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9738. ................................ ................................ .......... 46 6 9 Vibrio parahaemolyticus TX2103 D55 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9885. ................................ ................................ .......... 47 8 1 Internal temperature of half shucked oyster on an open chargrill gas grill conducted on site in a commercial environment. ................................ ................ 53 8 2 Internal Oyster Temperatures during on site trials conducted o n the restaurant level.. ................................ ................................ ................................ 54
8 9 1 Illustration of a possible HACCP plan for cooking oysters in a commercial restaurant operation. ................................ ................................ .......................... 62
9 LIST OF ABBREVIATION S APW Alkaline Peptone Water BAM bacteriological Analytical Manual CDC Center for Disease Control and Prevention CFU Colony Forming Unit CL Critical Limit CT Cycle Threshold DI Deionized Water EPIPT End Point Internal Product Temperature mm Millimeter FDA Food and Drug Administration g Grams HACCP Hazardous Analysis and Critical Control Points ISSC Interstate Shellfish Sanitation Conference LA Luria Burtani Broth with NaCl Agar LBN Luria Burtani Broth with NaCl LB Luria Burtani Broth mL Milliliters m CPC Modified Cellobiose Polymyxin B Colistin MPN Most Probable Number NACMCF National Advisory Committee on Microbiological Criteria for Foods NMFS National Marine Fisheries Service PBS Phosphate Buffer Saline PHP Post Harvest Processing qPCR Quantit ative Real Time Polymerase Chain Reaction
10 RPM Rotations Per Minute SCP Safe Cooking Practices TCBS Thiosulfate Citrate Bile Salts Sucrose TSA Tryptic Soy Agar V. Vibrio VC Vibrio cholera VP Vibrio parahaemolyticus VV Vibrio vulnificus vol Volume w t Weight
11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science DETERMINING THERMAL LETHALITY TO REDUCE PRESENCES OF POTENTIAL PAT HOGENIC VIBRIO SPP IN OYSTERS, CRASSOSTREA VIRGINICA By Christopher W. Hanna December 2012 Chair: Steve Otwell Major: Food Science and Human Nutrition C ontrols are necessary to prevent illness associated with Vibrio spp. in oysters, Crassostrea virgi nica The primary bacterial pathogens associated with human disease attributed to oysters are Vibrio parahaemolyticus, V. vulnificus, and V. cholerae serogroup s The most commonly recommended control has been cooking but descriptions of the specific applic ation of this control are limited. The following D and z values were c alculat ed based on thermal consequences in a phosphate buffered saline (PBS) : V. vulnificus CMCP6 were as follows: D 48 =2.24 min, D 50 =2.05 min, D 55 =0.50 min and z value=10.19 o C, V. choler a 01 N16961: D 48 =2.36 min, D 50 =1.96 min, D 55 =0.52 min, and a z value=10.31 o C and V. parahaemolyticus TX2103 was the most heat stable with D 48 =3.02 min, D 50 =1.99 min, D 55 =0.72 min with a z value=11.3 o C. Trials conducted at the same temperatures on whole oys ter s to demonstrate the protective effects of the food matrix and suggest s that at lower temperatures the food matrix provided a protective effect but at 55 o C internal temperatures the protective effect was diminished.
12 Further trials assessing the effect iveness of routine commercial cooking procedures and the (FDA) recommendations in the U S Food C ode (2009 ) was conducted on chargrilled half shell shucked oysters containing Vibrio spp. Field trials confirmed routine chargrill ing and frying exceed internal product temperatures of 200 o F (93.3 o C) and >145 o F for 15 seconds. Results indicate restaurant standard cooking practices proved effective in reducing or eliminating the potential Vibrio spp. pathogens. These results can be us ed to validate cooking controls in Hazardous Analysis and Critical Control Points applications.
13 CHAPTER 1 INTRODUCTION Vibrio vulnificus V. cholerae and V. parahaemolyticus are naturally occurring potentially path ogenic bacteria that are commonly found in C rassostrea virginica (oysters) throughout the Gulf Coast (Blake et al., 1979 ) These halophilic, gram negative bacteria are the most common cause of sea food related bacterial illness in the United States but are still considered rare (CDC, 2009). They can be accompanied by additional Vibrio spp. that are more commonly associated with water contaminants of human origin i.e. Vibrio cholerae and other Vibrio spp. depending on the species and serotype present that c an result in non approved harvest conditions. Together these bacteria are commonly referred to as opportunist ic pathogens because healthy persons do not get V. vulnificus fatal infections and Vibrio spp. infections that can be vectored to consumers through consumption of raw or improperly cooked oysters. Vibrio spp. related disease has increased regulatory action for more appropriate controls to reduce or eliminate the presence of these particular Vibrio spp. M any coastal states are actively developing and implementing new recommendations for Vibrio spp. control plans through the Interstate Shellfish Sanitation Conference ( ISSC ) The s e plan s specify controls for shellfish obtained from approved harvest waters through proper product identification (tagging), reducing product temperature s immediately after harvest and throughout processing and distribution, and a possible intervention with innovative post harvest processing (PHP) methods designed to reduce or eliminate potential pathogens (ISSC, 2012) Although the effectiveness of these controls as single or combined procedures is debated, all regulatory and commercial interest s agree that
14 cooking is the safest and most effective control. The ISSC recommends following the US Food Code (2009) for the control of molluscan shellfish (ISSC, 2012) T he Center s for Disease Control and Prevention (CDC) recognizes an urgent need for additional controls in the face of the trend s for increasing oyster borne Vibrio spp. illness es and the i ncreasing percentage of immunoc o mpromised cons umers. According to the CDC (2012) the reported incidences of Vibrio s pp. illnesses had significant ly increase d in 2010 when compared to 2006 2008; with a rise of 39% within 24 months This situation could be further complicated by environmen tal warming trend s (Cox et al., 2000) that could extend the more proble matic summer seasons that favor the presence of Vibrio spp. and correlates with the period of higher occurrence of recorded oyster related illness (CDC, 2012 ) Serious infections from c onsumption of raw oysters harboring the Vibrio s pp. most often occur within individuals that show some underlying condition that compromises the defenses of the host. Common underlying conditions include diabetes, liver disease, immuno compromised, the eld erly or very young, alcoholic cirrhosis or hemochromatosis (Blake et al., 1979). The se immune compromised conditions appear to be increasing in our aging society (CDC, 2009) and the persistent occurrence of Vibrio spp. illness and environmental trends sugg est s a need for better controls to reduce or eliminate Vibrio spp. encounters by oyster consumers In response, the ISSC and the CDC continues to recommend methods to minimize the risk through the use of controls or hurdles to prevent bacterial presence an d growth Their recommended options include: rapidly refrigerating oysters after harvest, treating the oysters at the processin g plant with heat, freezing or high pressure, and finally by thorough cooking (CDC, 2012).
15 The use of cooking to control bacteri al infections from seafood is not always apparent in terms of actual procedures. The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) was asked by the F ood and Drug Administration (FDA) and the National Marine Fisheries Service (N MFS) to provide advice o n cooking seafood in order to provide consumer message s and directions necessary to ensure the safety of seafood ( NACMCF, 2008 ) Several conclusions were drawn, but overall and most importantly for proper cooking of oysters, the com mittee stated that there is a lack of thermal inactivation data for relevant pathogens in appropriate seafood due, at least in part, to the wide variety of products available and the many methods of cooking that are commonly applied to these products. The committee agrees that the microbial safety of seafood is enhanced greatly when it is properly handled, cooked, served, and stored ; however, the committee still recognizes the fact that some consumers prefer to eat certain seafood products raw or undercooke d. The committee comment s that cooking methods for seafood products differ and often are not necessarily based on scientific data. Although seafood cooking recommendations are widely available, there is no easy, practical measurement or indicator for the c onsumer to objectively determine sufficient cooking in order to ensure the safety of fishery products (NACMCF, 2008) The inactivation of infectious non spore forming pathogens using a heat treatment is a critical control point in the safe preparation of many foods I nsufficient processing, cooking or reheating are often contributi ng factors in food poisoning ou t b reaks (Rob er t s, 1991). Many food processing systems contain a heating step to reduce the number of bacteria in a product to enhance food safet y and increases overall shelf life of the
16 product (Asselt and Zwietering, 2005) Many of these cooking parameters are based on challenge tests, legislation and overall common experience. Heat resistance and i njury of food borne pathogens such as S almonella e Staphylococcus aureus, and C lostridia have been studied extensively; yet, much less attention has been given to the thermal stability characteristics of Vibrio spp. (Beuchat and Worthington, 1976).
17 CHAPTER 2 LITERATURE REVIEW Current thermal inactivat ion data involving Vibrio spp. is limited and shows substantial variability depending on the medium and species used in the trial studies. Vibrio parahaemolyticus is commonly referred to as one the most heat stable Vibrio spp. with V. cholerae and V. vuln ificus being closely related in terms of thermal liability (Joseph and Colwell, 1982). Further studies need to be conducted because no common medium or standard procedure has been used for all three species that would allow a more comprehensive comparison between the species. Hinton and Grodner (1985) purchased fresh shrimp from local seafood stores and created a shrimp homogenate using three parts peeled shrimp and one part distilled water. An addition of the Vibrio cholera e organism provided a final inoc ulated homogenate concentration of approximately 10 6 CFU/gram. Four grams of inoculated homogenate was then added to Pyrex test tubes fitted with a copper constantan to monitor the heating rate and history. Fifty gram samples of the injected shrimp were th en cooked using one of two methods: boiling water or steam (100 o C) for 10 min. Table 2 1 shows the thermal resistance of V. cholera e conducted by Hinton and Grodner (1985) using the Most Probable Number (MPN) technique based on the previously listed method s.
18 Table 2 1 Heat resistance of V. cholerae in shrimp homogenate conducted by Hinton and Grodner, 1985. Temperature D value (min) 48.9 9.17 54.4 0.43 60 0.39 65.5 0.32 71.1 0.31 76.7 0.30 82.2 0.28 The data displays a drastic drop from 48.9 o C w ith a D value of 9.17 min to only 0.43 min at 54.4 o C without the remaining data following thermal death trends. Also, these results do not coincide with a study conducted on Vibrio cholera in blue crab homogenates (Shultz et al., 1984). Raw crabmeat was pl aced in a sterile Warring Blender in 150g quantities and three mL of a 1:3 dilution of the V. cholerae inoculums were added to achieve a final concentration of 10 6 CFU/g; followed directly by homogenization for 2 min (Shultz et al., 1984). Next, 4 g of hom ogenate was aseptically placed in glass tubes and sealed with a surface mix gas oxygen torch. Fifteen total tubes, six of which were equipped with thermocouples, were totally submerged in a water bath for various time increments at 49, 54, 60, 66, and 71 o C Upon removal, the inoculated tubes were cooled rapidly in a water bath for 30 sec and the contents were aseptically transferred to alkaline peptone water (APW). The APW tubes were then incubated at 35 o C for 6 8 hr and recovery techniques taken from the F Bacteriological Analytical Manual (BAM) were used to determine the final bacteria counts. Table 2 2 below displays the results.
19 Table 2 2 Heat resistance of V. cholerae in crabmeat homogenate conducted by Shultz et al. (1984) Temperature D Value 4 9 8.15 54 5.02 60 2.65 66 1.60 71 0.30 V. cholerae experiments with shrimp (1985) at similar temperatures to the aforementioned studies conducted by Shultz et al. (1984), it is noticeable that a margin of differenc e in the D values. At 48.9 o C Hinton and Grodner (1985) found a D value of 9.17 min, whereas at 49 o C Shultz et al. (1984) found the D value to be 8.15 min. This is similar enough to be associated with the medium itself attributing to this difference, but wh en comparing the remaining values, the trend differs exponentially. At 54.4 o C the shrimp homogenate changes from 9.17 to 0.43 min (Hinton and Grodner, 1985); whereas, in the crabmeat homogenate, a D value of 8.15 min at 49 o C only drops to 5.02 min at a sim ilar 5 o C increase to 54 o C. Even with different media, the bacteria themselves should act similarly within one medium. As the temperature increases again the values differ even more drastically. Hinton and Grodner (1985) found that at a temperature of 60 an d 71.1 o C the respective D values were 0.39 and 0.31 min; while Shultz et al. (1984) reports a D value at 60 and 71 o C to be 2.65 and 0.30 min. The values start similarly at lower temperatures, veer off correlation towards the middle temperature range, and t hen end nearly identically. This matter is not expected in normal D value studies. Beuchat and Worthington (1976) conducted another study examining the thermal inactivation of V. parahaemolyticus where exponential phase cells, or bacterial cells in
20 the re production stage of life, were transferred to 190 mL of 0.1 M potassium phosphate containing 0.5%, 3.0% or 7.5% (wt/vol) NaCl at a pH of 7.2 with a concentration of approximately 10 7 CFU/mL for 3% NaCl TSB and 10 7 CFU/mL for 0.5% and 7.5%. These mixtures w ere heated under constant agitation and samples were withdrawn at appropriate times, dispensed in a sterile chilled test tube and then serially diluted in 0.1 M potassium phosphate containing 3.0% NaCl. A tryptic soy agar (TSA) consisting of 1.2% agar and 3.0% NaCl was tempered at 42 o C and colonies were counted after 18 24 hr at 35 o C. Heat survivor curves of log 10 viable number per milliliter versus time at 47 o C were plotted. With a growth temperature of 37 o C, 7.5% NaCl was inoculated and heated at 47 o C and produced a D value of 65.1 min (Beuchat and Worthington, 1976). When compared to a study conducted in 1992 on V. vulnificus in a buffered saline solution at the same 47 o C, a D value of 2.40 min was calculated (Cook and Ruple, 1992). This shows a differenc e of 62.7 min between similar mediums and subspecies that again shows a substantial variation between studies. Thermal inactivation is not solely dependent upon the bacterial organism in question. Other factors for overall inactivation include: exposure t ime and temperature, pH, fatty acid composition, protein insulation and evaporative cooling. In addition bacterial concentration and location inside the specific mediums, water activity, rapid/slow cooling and heat penetration are also extremely important in the overall production of a safe product (Blackburn et al., 1997) For example, the thermal inactivation of Vibrio spp. in PBS may vary slightly or drastically based upon the protective effects of the medium. PBS consists mostly of water, with low conce ntrations of phosphate and saline which should not play a major factor in the overall protection of
21 the bacteria from death, however, inside of an oyster exist large quantities of dispersed water, proteins and fats, as well as thicker portions of meats in which bacteria may be located that may inhibit heat penetration and ultimately shield and protect the Vibrio spp. In the absence of adequate pathogen inactivation data for various seafood products, the current FDA recommendation for safe seafood is heating all parts of the food to 145 o F (63 o C) or above for 15 sec (FDA, 2009). These recommendations are listed in the US Food Code (2009), cooking section 3 401.11 for raw animal foods as compiled by the FDA working in company with the conference of Food Protect ion. The FDA base this decision on lethality data for Salmonella a potential pathogenic bacterial contaminant commonly associated with seafood. The utility of these recommendations is not known relative to cooking oysters to eliminate Vibrio spp. but the expectation is that these recommendations will be sufficient in reducing potentially pathogenic Vibrio spp The second option offered in place of the monitoring of the internal time and temperature is mentioned in the Fish and Fishery Products Hazards and Controls compliance with the seafood HACCP regulations (21CFR Part 123). The HACCP manual provides options for simply monitoring End Point Internal Product Temperatures (EPIPT) in stead of continuous time and temperature monitoring during cooking or pasteurization. This is useful when reaching higher internal temperatures, mostly noted with deep frying, where shorter times are compensated by extremely high temperatures that bacteria cannot survive. Although this currently is offered as an option solely for
22 processors, restaurant settings may benefit from regulations that follow this manner as an alternative option to internal time and temperatures currently in place. The use of HACCP concepts in restaurant operations is encouraged in the US Food Code (2009) (FDA, 2009). The Hazardous Analysis and Critical Control Points (HACCP) for Seafood recommends processes that create a 6D, or 6 log reduction, of potential pathogens for processor s or distributors, but these do not apply to the restaurant level. Restaurants are not held to these standards because the food is cooked for immediate consumption. After processors reduce bacterial concentrations, the product may be transferred or stored for hours or days, allowing the surviving bacteria the time they need to regrow and increase concentrations, which is why the processors have such high D requirements. This is not the case with restaurant foods because they are consumed immediately after b eing cooked. This does not allow the time needed for any surviving bacteria to replicate again to the concentrations normally required for pathogenicity after cooking has occurred. Current oyster cooking methods commonly used in restaurant industry range from chargrilling on an industrial gas grill, to steaming and deep frying; however, many restaurants now solely offer chargrilled and deep frying for several factors including ease and convenience. Also, average consumer demand is higher for these methods over steaming. Household consumers also grill and fry oysters, but most are limited on their ability to steam simply due to their lack of equipment. Overall, mindful of prior restrictions and complications with confounding factors, additional research is necessary to better substantiate the recommendations and
23 dependence on appropriate cooking methods to control potential illness due to Vibrio spp. in raw oysters. Determination of thermal death tim e (D values) and corresponding z values are appropriate m ethods to evaluate the performance of a thermal inactivation process. The se concept s di ctate required temperature and exposure time to effectively reduce or eliminate particular bacteria. These data can then be applied and altered to obtain thermal inactiv ation in different mediums in a laboratory or in food processing and handling situations The same approach can be used to determine required cooking procedures for a retail or restaurant operation that prepares food for immediate consumption. The D value refers to the reduction time required at a certain temperature to kill 90%, or 1 log unit of the organism in question. Upon obtaining several D values, a thermal destruction curve can be created by graphing the corresponding D values to their appropriate times. The z value is a temperature that is required for the thermal destruction curve to move 1 log cycle. Z values can be calculated by taking the reciprocal of the slope resulting from the plot of the logarithm of the D value versus the temperature at w hich the D value was obtained. While the D value gives the time needed to destroy organisms at a specific temperature, the z value relates the resistance of an organism to differing temperatures (McLandsborough, 2004). Mindful of the risk associated with potential pathogenic species of Vibrio bacteria vectored by oyster consumption this study was conducted to provide more reliable measures for effective thermal treatments for cooking controls in restaurant and food service operations preparing oysters fo r immediate consumption The scope of work
24 includes determinations of basic thermal parameters for bacterial lethality and comparisons against recommended FDA guidelines for general cooking of seafood and actual commercial (restaurant) practices.
25 CHAPTER 3 OBJECTIVES AND HYPOTHESIS T he main objective was to determine the thermal inactivation of pathogenic Vibrio spp. in order to obtain reliable and comprehensive pathogenic inactivation data to better direct appropriate cooking of oysters. The hypotheses f or this work included: Vibrio vulnificus CMCP6, Vibrio parahaemolyticus TX2103, and Vibrio cholerae N16961 have similar thermal inactivation requirements Protective food matrix effects may occur in whole oysters, but this will not be a sufficient enough b arrier to prevent bacterial death during common industry cooking standards Cooking to an internal temperature of 145 o F for 15 sec as recorded in the US Food Code (2009) will sufficiently eliminate all three potentially pathogenic Vibrio spp. pathogenic thr eats in oysters, C rassostrea virginica
26 CHAPTER 4 MATERIALS AND METHODS The thermal parameters for bacterial lethality were first determined with media grown sources based on prior efforts to assure proper growth stages and conditions to monitor bacteri al survival. The resulting thermal parameters were then compared with similar measures for thermal resistances during actual commercial operations to determine the resulting thermal parameters and the effectiveness of routine cooking procedures and recomme nded guidelines Bacterial Growth Curves Growth curves are extremely important in determining the phase of bacterial growth. Many bacteria have different growth curves; however, nearly all follow the same pattern of growth: a lag, logarithmic, stationary and death phase. Pra c tically, the mid stationary phase is the most useful in these experiments because of the cells ability to withstand a greater range of stresses with a lower susceptibility to death. Most bacteria in foods tend to be in this phase. I t is important to ensure that the bacteria used in the experiment is in a strong, mid stationary phase for several reasons: it decreases the likelihood of variability during heat treatments, ensures the bacteria is metabolically sound with a majority of its energy going towards survival as opposed to reproduction, and finally to have a known quantity of bacterial concentration as a starting point. Triplicate experiments were conducted and the average log 10 CFU/mL was plotted versus time to give the growth cur ve graph. The selected bacterial strains included species previously associated with raw oysters. They include: V vulnificus CMCP6, V. parahaemolyticus TX2103, and Vibrio cholerae N16961 and were obtained from the University of Florida Food Science and
27 Hu man Nutrition Department Strains were stored as frozen stocks at 80 o C in Luria Burtani Broth with NaCl (LBN) and 50% glycerol with a pH of 7.5 and streaked onto LBN Agar (LA) for isolation and incubated at 37 o C overnight for each individual study An iso lated colony selected from the overnight LA was placed in 50mL of Luria Burtani Broth (LB) and incubated at 37 o C in a shaking incubator set t o 90 rotations per minute (RPM) for 24 hr to ensure viable culture growth Next, 1 mL of the incubated sample was i noculated into 50 mL of LB and placed into the shaking incubator with the same parameters. Every hr including a time 0, serial dilutions of the sample were conducted in PBS test tubes i n a ratio of 1:10 and 0.1 m L was aseptically spread plated onto LA in order to obtain an accurate measurement of the growth of the specific strains. Each sample was conducted with three replications to obtain the most solid statistical model of growth. After each time point the LA plates incubated at 37 o C for 24 hr to all ow accurate growth of colonies. On the 24 hr mark, the plates were taken from the incubator and all individual viable colonies were counted and recorded for each time point at a dilution that met the requirements of being within 25 300 colony forming units ( CFU ) per plate. The recorde d data were then converted into l og 10 CFU and graphed versus time to create a growth curve. These growth curves were used to determine the growth phase ( e. g. lag, exponential, stationary and death phases) of the bacteria throu ghout their replication process. For many human pathogens, the capacity to survive physical challenges during food processing is a critical step in their transmission to the host by the food borne route (Rees et al., 1995). Stationary phase cells are gene rally more resistant to a range of
28 stresses and inimical processes, environmental changes, temperature and pH alterations, and are known to be capable of surviving in conditions where logarithmic phase cells would tend t o be more susceptible to death since a majority of their energy is put into reproduction as opposed to survival (Rees et al. 1995). Therefore, cells from early stationary phase were used to determine bacterial survival in these studies. Thermal Application s in Media Following the substantia tion for stationary growth, preliminary work was necessary to determine the conditions for thermal exposure of the Vibrio spp. in media. A come up time or the time required for the medium to reach the desired temperature, was determined for 5 .0 mL of PBS in an identical test tube that was used during the thermo tests for each temperature (48, 50, 55 o C) The se heating temperatures were chosen based on previous studies that show ed death occurring at these temperatures at a rate large enough to allow progress ive measurements for change in bacterial levels. PBS ( 5 .0 mL ) a t room temperature (21.2 o C 0.3) was dispensed into 16x125MM test tubes and the rate of temperature change in the tube of media. New, calibrated thermocouples were connected to an OCTTEMP 2000 and secured in the center of the test tube without contact to the side. A circulating water bath was filled with approximately 2.5 3.0 L of deionized water (DI water) and allowed to reach the respective temperature ( 48 50, 55 o C) Fifteen test tubes fille d with 5.0 mL of PBS at room temperature were placed in a test tube rack and two thermo couple s w ere placed in two test tube s at random to accurately create a thermo profile and ensure no overloading of the water bath and its temperature would occur. A thi rd thermocouple was placed inside the water bath to ensure the temperature remained constant.
29 Finally, an ice slush was used for rapid the cool ing and a fourth temperature probe was placed inside the ice slush to verify the temperature remained at ~0 o C 0 .4 o C. Once the circulating water bath reached the appropriate temperature, the test tube rack was placed in the center of the water bath and the timing began. T he time was recorded for the PBS tubes to reach the desired temperature 0.4 o C with three rep lications to accurately identify the come up time at individual temperature s; o nce the last of the two test tubes containing the thermo couple had reached the designated t emperature the entire test tube rack was removed from the water bath and instantly pu t into the ice slush. The time required to reduce th e temperature of the PBS from the set point to 25 o C was recorded. Three replications were conducted, with two test tubes monitored per replication, giving a total of 6 temperature trials per study At 55 o C, the come up time was long enough to where the previous experiments (48 and 50 o C) provided data suggesting an elimination of a majority of the bacterial concentration before testing could begin. A n additional study was conducted using an identical method to 48 and 50 o C with time point s 0, 30 and 60 sec The results proved that too large of a quantity of the Vibrio spp. were inactivated for this test to accurately define the lethality over the period of time required Therefore, all studies conducted at 55 o C used pre warmed PBS (4.5 mL) that were inoculated with 0.5 mL of bacteria was pipetted into each tube, individually, from a master mix. This ensured that testing could begin at a time zero, where no ba cteria was lost in the come up and enough would rema in to obtain a reliable death curve. Determining D and z Values in PBS Media The first step in obtaining accurate D values was to assure uniform methods across all experiments Once the freezer stock bacteria was plated onto L Agar and an
30 isolated colony was select ed from the overnight growth, colonies w ere then allowed to culture overnight in L Broth for 11 13 hr to ensure the bacteria had reached stationary phase as previously established in preliminary studies. After t he bacteria reached mid stationary phase at approximately hr 16, it had the desired properties for overall strength and survival needed to conduct an accurate thermal death matrix. Freezer stock samples of each individual bacteria specimen were plated for isolation on LA and allowed to g row overnight at 37 o C in a standard incubator. An isolated colony of each was then inoculated into 50mL of LB for 16 hr which was determined to be early stationary phase of based on prior analysis of the growth curves. This was perfo rmed to ensure all bacteria are in a strong metabolic, non reproductive phase to guarantee the greatest heat resistance during trials and the least amount of variabilit y possible between replications and experiments. Next, 20mLs of the inoculated broth was put into a 50mL conical and spun in a centrifuge for 15 min at 3000 RPMs. The supernatant LB was then discarded, and the remaining sp ecimen was re suspended in 20 mL of PBS solution and vortexed until homogenized. The new solution was then used to cre ate a master solution with a 1:10 dilution in PBS. Portions ( 5mL ) of th is solution was serologically pipetted into 5 sterile test tubes, after which the pipette tip was discarded and the master solution was re vortexed in order to ensure even distribution of t he sample This procedure was repeated until the required amount of test tubes were filled. Upon completion, the concentration of bacteria in the master samp le was determined by serially diluting with a 1: 10 ratio in PBS and spread plated onto LA before h eat was administered. T he same procedure was followed and plated on differential
31 media based on the subspecies: modified cellobiose polymyxin B colistin (mCPC) for V. vulnificus CMCP6 thiosulfate citrate bile salts sucrose (TCBS) for V. cholerae N16961, o r CHROMagar for V. parahaemolyticus TX2103 and the results were compared to the counts on non selective media to verify no contamination. Once the overnight sample was plated, the circulating water bath was brought to the appropriate temperature, the ice slush was prepared and the test tubes were racked in an identical setup to the preliminary come up time procedure. The inoculated test tubes were then placed in the middle of the water bath and the time be gan. A fte r 2 min at 48 o C a time 0 test tube was p ulled to account for the initial death occurring during the initial heat ing After achieving the desired temperature every 4 min utes test tubes were pulled, cooled down in the ice slush for 15 sec and placed inside a new, dry test tube rack. The cool dow n period is used to immediately stop thermal related death and is as minimal as possible to prevent any death occurring from cold shock. After completion of all time intervals, test tubes wer e then serially diluted at a 1:10 ratio in PBS and 0.1mL of the h eat treated samples were plated onto LA and placed in a standard non motion incubator over night at 37 o C. After 24 hr the plates were counted in the standard range and the data was recorded. Finally, the colony numbers were converted into CFU/ml, then Log 10 CFU/ml and graphed versus time. A linear trend of best fit was then applied to the graph to determine the D and z value s. The D values were based on the thermal death that occurred at a specific temperature, whereas the z value was determined based off of the results of all three D value studies.
32 Identical methods were followed for preparation at 50 o C as 48 o C with t he come up time varying slightly, from 2 min to 2 .5 min with the same 15 sec cool down period. The procedure for D and Z values at 50 o C fol lowed the methods at 48 o C with only the time interval altering from 4 min at 48 o C to 1 min intervals at 50 o C. This change allows for a more accurate death calculation with the increased death occurring from higher temperatures and energy in the system Due to the heat sensitivity of Vibrio spp. the normal come up time proce dure was altered at this higher temperature in order to prevent high levels of death before reaching the desired time points. In order to prevent this, 4.5mL of PBS was pipetted into tes t tubes and the appropriate quantity of test tubes were then racked and placed in a circulating water bath set to 55 o C. Two temperature probes were inserted at random into two test tubes and the PBS was allowed to come up to 55 o C. Once the last monitored t est tube reached 55 o C, an additional 5 min was given in order to prevent any tubes from hovering below the required temperature. A portion ( 0.5mL ) of the overnight sample which was prepared and quantified identically to D48 noculated into the 4.5 mL heat treated PBS producing a 1:10 dilution of the overnight sample. The time points were set to 30 sec intervals and upon reaching the time point, the test tube was pulled and instantly iced down in the same ice slush for 15 sec i n order to instantaneously end kill. The same quantification methods as the previous D48 and D50 were then followed to calculate the survival of the bacteria at this temperature. Each sample was conducted in triplicate, and then the average CFU/mL was calc ulated as well as a standard deviation for each trial. The results were then plotted
33 on a time versus average log 10 CFU/mL. Once plotted, a linear regression for the average log was plotted and used to calculate the D value by solving the equation with a 1 log unit reduction, or a reduction of the y axis by 1 log unit Thermal Assessment s with Oysters Food products are commonly known to create a protective effect of bacteria lethality for many reasons including: evaporative cooling, protection from proteins lipids and water in the systems, insulation from thicker or more dense portions of the product and the ability for heat to transfer to evenly throughout the system. Because of this phenomenon, trials were conducted in oysters at 48, 50 and 55 o C in order to evaluate the overall protective effect encountered in oysters at these lower heat temperatures. P reliminary tests were conducted on oyster homogenates (1:1 in PBS) ; however, protein separation, gelling and layering occurred. Further separation between the medium and the oysters as time and heat increased. Due to the non uniformity of the homogenate after the heating process heating who le oysters as opposed to the homogenate s were used in subsequent studies in order to provide greater accura cy of the the rmal inactivation and the homogenate method was abandoned in favor of the use of whole oysters. L ive oyster samples were purchased from a local vendor in Apalachicola and levels of naturally occurring Vibrio spp. in oysters were increased by temperature a bused using incubation at 26 o C for 24 h r T he oysters were subsequently stored at room temperature ( ca. 21 o C). Initial Vibrio spp. concentration in oysters was determined from a standard weight of oysters ( 100g ; ~ 10 12 oysters) that were shucked and placed into a Warring Blender and mixed with an equivalent amount of P BS to produce a 1:2 homogenate The mixture (20 mL) was ad ded to 80mL of PBS to form a 1:10 dilution of
34 oyster to PBS. Serial 10 fold dilutions were performed by taking 11 mL of sample to 99 m L of PBS (1: 10 ) out to the 10 6 dilution. The se dilutions were inoculated into APW with a pH of 8.5 0.2 in triplicate, placed in an incubator set to 37 o C and allowed to grow overnight. A ll test tubes that were vi sually positive for growth in APW were rec orded and processed through the DuPont Vibrio BAX system (Dupont, Wilmington, US) system for real time qPCR of Vibrio spp. All results were obtained using the qPCR were then manually ana lyzed and all positives with a c ycle t hreshold (CT) value above 32 wer e excluded and considered a false positive. A ll positive APW test tubes were compared to confirmed Vibrio spp. positives from the BAX, and any qPCR Vibrio negatives based on the analyzed BAX results were removed in the calculation of MPN in order to obtain an overall death of Vibrio only and t o eliminate any other bacteria that may be producing a positive in later dilutions that were not necessarily Vibrio spp This was repeated in triplicate with four separate temperature abused samples in order to obtain the most likely amount of Vibrio bacteria in the samples. A sample ( 100 g ) of live oysters from the same temperature abused sampling were shucked and put into a 1 gallon size Ziploc (Johnsons & S on, Racine, US) brand bag. The bags were fitted with three t emperature probes attached to an OCTTEMP2000 (Thermoworks, Lindon, US) the temperature recorder. The probes were inserted into three random oysters and the bag was inserted into a circulating water bath set to 48 o C. Once the last probed oyster (considered the worst case scenario) reached an internal temperature of 48 o C, a timer set fo r 9 min and 4 sec began which was determined from a three log reduction based on the PBS D values of V. parahaemolyticus conducted in previous experiments This control point was chosen due to its heat stability over V.
35 vulnificus and V. cholerae. Once time had expired, the samples were immediately placed in an ice slush to instantly stop death. The same MPN procedure as the overnight experiments was then followed, and false Vi brio positives were removed. The same procedure was conducted for the 50 o C heat treatment as the 48 o C with the exception of the water bath and internal temperature being set to 50 o C and the time point altered to 5 min and 58 sec based on the three log redu ction D50 value conducted in PBS. The same MPN procedure was used in all heat treatment studies. The identical procedure was conducted for the 55 o C heat treatment as the previous two heat treatments at 48 and 50 o C with the same alteration for water bath an d internal temperature. The new set point for cooking was also changed based on a 3 log unit reduction in PBS from the D55 study from V. parahaemolyticus equating to a new time of 2 min 10 sec The same MPN procedure as the previous two h eat treatment stud ies was used to calculate the quantity of surviving bacteria. Assessing Commercial Cooking Procedures In order to compare lab based results with actual commercial cooking procedures a series of trials were conducted to monitor thermal history of oysters w hen cooked by chargrilling or frying relative to bacterial reduction The approached involved measuring thermal consequence in actual restaurant settings and using these results to confirm the bacterial consequences in controlled trials. The cooking method of choice was chargrilling. The chargrill method has recently become one of the most popular restaurant forms and appears to be replacing the traditional steamed form. Chargrilling in a restaurant setting involves shucking the oyster on the half shell, th en cooking the half shell shucked oyster directly on a gas chargrill. R estaurants may flavor and butter the product differently ; however, they all have the similar grills and cooking procedures.
36 Recording of the internal temperatures during routine commer cial practices was conducted on site at reputable restaurants (Drago s Seafood Restaurant & Oyster Bar and Acme Oyster House New Orleans) as the actual cooking procedures occurred to determine if common industry cooking practices meet, fall short of, or e xceed lethal expectations and requirements based on the newly calculated D and z values The results from the on site determinations were used to stage a series of similar trials measuring the consequence for bacterial loads in the same oysters, raw and af ter chargrilling. Raw oysters were purchased directly from distributers in Apalachicola, Florida and were temperature abused at 26 o C overnight in an incubator. The next morning, bacterial concentrations were conducted by taking 100 g of shucked oyster, s erially diluting out to 10 6 followed by spread plat ing on both selective media (mCPC and CHROMagar (CHROMagar, Paris, France) ) as well as non selective media ( LA ) CHROMagar was used to calculate the concentration of presumptive V. parahaemolyticus (ident ified by a mauve color), mCPC was used to calculate presumptive V. vulnificus (yellow colonies) and LA was used for total bacterial concentrations. Once spread plating was completed, the inoculated plates were stored overnight in an incubator set to 37 o C a nd the next morning the plates were counted and the CFU/mL was recorded. The assumption that there is no V. cholerae in natural oysters was applied and TCBS was not used to calculate V. cholerae concentrations in order to prevent false positives from p rote us/enterococci on the media and skewing final results A n industrial gas grill was used to simulate the pop u lar chargrilled method conducted onsite in many commercial settings The center of the grill was recorded to
37 have temperatures exceeding 450 o F, whi le the outer skirts ranged from 420 4 45 o F, never dipping below 420 o F on any portion of the grills surface. The temperature abused oysters were then shucked to the half shell and temperature probes were inserted into the oyster. Twelve oysters, three with t emperature probes, were then placed (shell side down) on the grill near the center, identically to how it was observed inside a restaurant setting. The oysters were allowed to come to 200 o F, then were promptly removed from the grill and allowed to cool nat urally to near room temperature. The oysters were then aseptically shucked into a sterile, stomacher bag and the contents were then blended in a Warring Blender PBS dilutions and plating procedures were identical to those of the overnight temperature abus ed samples (mCPC, CHROMagar and LA). Assessing Regulatory Guidelines The final examination was conducted on a process many comm ercial settings follow from the 2009 US Food Code: an internal temperature of 145 o F (62.78 o C) for 15 sec that is commonly based on the inactivation of S almonella ( FDA, 2009 ). Overnight temperature abused oysters were again shucked and left on the half shell. Again, twelve oysters, three with temperature probes, were placed shell side down on the grill near the center. The oysters were allowed to come up to 145 o F, and upon reaching that internal temperature a timer was started. After 15 sec the oysters were removed from the grill and allowed to cool naturally. The identical steps to the 200 o F test above were conducted and the follo wing morning, all selective and non selective plates were counted to determine overall V ibrio spp. lethality as well as overall bacterial lethality. Three replications of each experiment, including the overnight, w ere conducted to increase overall reliabil ity of the data as well as statistical analysis.
38 CHAPTER 5 VIBRIO SPP GROWTH CURVES Initial work determined the parameters for reaching a stationary phase prior to use in thermal trials. Figure 5 1 shows V. cholerae N16961 entering stationary phase aft er approximately 9 hr. The mid stationary phase for this bacteria was determined to be between hr 11 13 with an approximate concentration of 10 10 CFU/mL or very high 10 9 CFU/mL Figures 5 2 and 5 3 show V. cholerae, V. vulnificus CMCP6 and V. parahaemolyti cus TX2103 with a final concentration of 10 9 with the same 11 13 hr mid stationary phase. All bacteria have extremely similar growth patterns, times and final concentrations based on these initial studies Figure 5 1. Growt h curve of Vibrio cholerae N169 6 1 in L Broth. The line at 6 hr displays the end of exponential growth and the beginning of the stationary phase. The line at 10 hr displays the beginning of mid stationary phase.
39 Figure 5 2. Growth curve of Vibrio vulni ficus CMCP6 in L Broth. The line at 8 hr displays the end of exponential growth and the beginning of the stationary phase. The line at 12 hr displays the beginning of mid stationary phase.
40 Figure 5 3. Growth curve of Vibrio parahaemolyticus TX2103 in L Broth. The line at 9 hr displays the end of exponential growth and the beginning of the stationary phase. The line at 12 hr displays the beginning of mid stationary phase.
41 CHAPTER 6 D AND Z VALUE ASSESMENTS The D values were determined using Vibrio spp. cultures in the mid stationary growth phase. In trials at 48 o C using media it was noted that V. parahaemolyticus had the longest survival with a D value of 3.02 min ; V. vulnificus and V. cholerae w ere equally heat liable at 2.36 min Figure 6 1 thro ugh 6 3 display the average concentration of bacteria v ersu s time. Figure 6 1. Vibrio cholerae N16961 D48 time v ersu s average log 10 CFU/mL graph with standard deviation. R 2 =0.9923 Figure 6 1 shows that from a n initial concentration of 1.8x10 8 V cho lerae a 1 log unit reduction every 2.36 min at 48 o C occurred After 20 min at this temperature, all V. cholerae in the sample had been eliminated. This is nearly identical to that for V. vulnificu s, which had a starting concentration of 2.87x10 8 CFU/mL, an d also showed no survival after the 20 min mark and a nor mal 1 log unit reduction at 2.36 min
42 Figure 6 2. Vibrio vulnificus CMCP6 D48 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9916 Figure 6 3 shows the slight heat stable advantag e V. parahaemolyticus has over the other two subspecies. At this temperature, V. parahaemolyticus displayed a greater thermal resistance compared to the other species with a D value of 3.02. This is roughly 3 .02 min versus V. vulnificus and V. cholerae hav ing approxi mately 2 .37 min respectively.
43 Figure 6 3 Vibrio parahaemolyticus TX2103 D48 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9887. All of these experiments were extremely robust as interpreted from the small standard deviat ions of the repetitions. The line of best fit produced by all three graphs was also accurate with the lowest being 0.9887 for V. parahaemolyticus and even higher for V. cholerae and V. vulnificus producing R 2 values greater than 0.99. S imilar results occu rred between the thermal inactivation at 48 o C and 50 o C (Figure 6 4 through Figure 6 6) All three Vibrio spp. had a relatively similar thermal resistance at this temperature, varying only slightly. V ibrio v ulnificus was the most heat stable at this tempera ture displaying a thermal resistance of 2.05 min per log unit reduction with V. parahaemolyticus at 1.99 min and V. cholerae with 1.96 min Figures 6 4, 6 5 and 6 6 display the time versus average log for V. cholerae V. vulnificus, and V. parahaemolyticu s respectively.
44 Figure 6 4 Vibrio cholerae N1696 1 D50 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9659. Figure 6 5 Vibrio vulnificus CMCP6 D50 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9501.
45 Figure 6 6 Vibrio parahaemolyticus TX2103 D50 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9041. T he R values displays minimal variability in these trials; however the standard deviations throughout the experiments display the capability of the se pr ocedure s to be replicated with accuracy The overall low variation of the standard deviation of these experiments coupled with the R values give support to the accuracy of the experiments conducted As the temperature increases, it is commonly expected to have an exponential reduction in bacterial counts over the same time period. Because of this, the normal D value procedure was altered from having a come up time with the lower temperatures, to being inoculated directly into the already heated tubes. Du e to the exponential increase in death over a shorter period of time, t he time poi nts were decreased from 4 min per time at 48 o C point to only 30 sec at 55 o C and afte r only three min nearly all bacteria were destroyed. Figure 6 7, 6 8 and 6 9 display the death versus time graph of V. cholerae, V. vulnificus, and V. parahaemolyticus respectively.
46 Figure 6 7 Vibrio cholerae N16961 D55 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9885. Figure 6 8 Vibrio vulnificus CMCP6 D55 time ve rsus average log 10 CFU/mL with standard deviation. R 2 =0.9738.
47 Figure 6 9 Vibrio parahaemolyticus TX2103 D55 time versus average log 10 CFU/mL with standard deviation. R 2 =0.9885. The low variability of the standard de viations coupled with the high R 2 val ues show that these experiments were reproducible as well as follow a n accurate linear path that is expected in all the thermal death studies that were conduct e d. The pathogens followed similar suit to previous expectations with V. vulnificus and V. choler ae having very similar D values with V. parahaemolyticus being the m ost heat stable with 0.72 min compared to V. vulnificus at 0.50 and cholerae at 0.53. Prior research has shown that at ~48 o C 1 o C, Vibrio spp. can have a D value of 8.15 min in crabmeat (Shultz et al ., 1984) for V. cholerae 0.82 min for V. parahaemolyticus in a clam homogenate (Delmore and Chrisley, 1979), or even as high as 65.1 min in 7.5% NaCl (Beuchat and Worthington, 1976). No prior research conducted had the ability to compare the thermal resistance of the Vibrio spp. simply because no prior research had conducted the same methods or the same media across all three pathogens in question. Using the same methodology and medium between V. vulnificus, V. parahaemolyticus, and V. choler ae allows for a better overall
48 understanding of the pathogens as well as the ability to cross reference and compare the three species. After obtaining a set of three reliable D values per organism, the z value was calculated by taking the reciprocal of the slope resulting from the plot of the logarithm of the D value versus the temperature at which the D value was obtained. This is equivalent to the temperature required for a 1 log unit reduction in the D value. Table 6 1 displays the calculated z values pe r organism based on the obtained D values.
49 Table 6 1 D and z values for V. vulnificus, V. parahaemolyticus, and V. cholerae Letters are used to denote statistical differences with a p 0.05 of the mean. Species D48 (min) D50 (min) D55 (min) z value ( o C) V. vulnificus CMCP6 2.24 B 2.05 CD 0.5 0 E 10.19 V. parahaemolyticus TX2103 3.02 A 1.99 CD 0.72 E 11.3 V. cholerae N16961 2.36 BC 1.96 D 0.52 E 10.31 Once the D values were calculated, a one way ANOVA analysis was carried out used for multiple mean comparisons using statistical analysis system software, version 9.1.3 (SAS Institute Inc., Cary, NC). Differences are noted by different letters and were determined by a statistical difference with a p 0.05 of the mean. Based on these trials, V. parahaemolyticus is significantly different from both V. vulnificus and V. cholerae at 48 o C. As the temperature increases to 50 o C, V. cholerae is significantly different from V. parahaemolyticus as well as V. v ulnificus However, once the temperature reaches 55 o C, all three bacteria show no significant difference. With all three bacteria acting in this manner, V. parahaemolyticus could be considered the target organism due to its heat stability over V. vulnificu s and V. cholerae at lower temperatures and no difference at higher temperatures All Vibrio spp. experimented on displayed minimal thermal survival capacities that will directly result in their ability to be lowered or eliminated during standard cooking p rocedures.
50 CHAPTER 7 THERMAL RECOVERY STU DY Thermal abuse (26 o C for 24 hr) elevated the level of Vibrio spp. for heating trials using whole oysters (Table 7 1) The overnight bacterial concentration was conducted by MPN and verified by the BAX Vibrio spp system. Each individual Vibrio spp. concentration was calculated, however, no V. cholerae cultures were found in any samples at any dilution. T he MPN calculations for V. vulnificus V. parahaemolyticus and overall MPN for total aerobic bacterial counts (bacteria that are capable of growth in APW at 37 o C) are probable in raw oysters harvested from approved waters for commerce (ISSC, 2012). Table 7 1 Overnight Temperature Abused Oyster MPN Calculations for V. vulnificus, V. parahaemolyticus and total bac teria in MPN /mL Sample V v ulnificus V. p arahaemolyticus Total Aerobic Plate Count 1 7.3x10 1 2.4x10 4 >2.4x10 5 2 2.3x10 1 2.4x10 4 >2.4x10 5 3 4.3x10 1 9.3x10 3 >2.4x10 5 4 2.3x10 1 2.4x10 4 >2.4x10 5 Mean 4.0x10 1 2.0x10 1 2.0x10 4 6.4x10 3 >2.4x10 5 0.0 *Total aerobic plate counts were calculated by bacteria that are capable of growth in APW at 37 o C incubation overnight. After determining the initial concentration, the overall reduction after the heating process was calculated to determine if there was any protective effect from the oyster on the survival of the Vibrio spp A lower final concentration of V. vulnificus was expected due in part to decreased heat tolerance a s well as its lower starting concentration versus V. parah aemolyticus The oysters that were heat treated at 48 o C showed a complete reduction of V. vulnificus ; however, V. parahaemolyticus had survivors This could be due to a low er starting concentration of V. vulnificus versus V. parahaemolyticus as well as the more heat stable V. parahaemolyticu s This
51 experiment was arranged to ensure at least a 3 log unit reduction in the bacteria. Although this shows a nearly complete inactivation of V. vulnificus, it is apparent that there is little increase in the inactiva tion of V. parahaemolyticus or total bacteria counts. This suggests that there are significant protective effects from the oyster at this temperature, which was fully expected. Table 7 2 Log unit reduction of Heat Treated Temperature Abused Oysters for V vulnificus, V. parahaemolyticus and total bacteria in MPN /mL Sample V. v ulnificus V. p arahaemolyticus Total Aerobic Plate Count Overnight Mean 4.0x10 1 2.0x10 1 2.0x10 4 6.4x10 3 >2.4x10 5 0.0 48 o C Mean Reduction 1 .0 0 .0 0 .0 0 .0 0.3 0. 6 50 o C Mean Reduction 1.0 0.0 1.3 0.6 2.0 1.0 55 o C Mean Reduction 1.0 0.0 2.7 0.6 2.7 0.6 *Total aerobic plate counts were calculated by bacteria that are capable of growth in APW at 37 o C incubation overnight. Table 7 2 show the log unit re ductions of V. parahaemolyticus, V. vulnificus and total bacteria after the 50 and 55 o C treatment respectively. At the 50 o C treatment, again, all the V. vulnificus was completely eliminated, however, an average of a 1.3 log unit reduction was observed for V. parahaemolyticus. Again, with the expectation of a minimum of a 3 log unit reduction, we can conclude that the protective eff ect at this temperature results in a reduction in the overall inactivation of V. parahaemolyticus and thus not safe for human co nsumption. It is important to n ote that the protective effect at 50 o C was less than at the previous 48 o C temperature, which could suggest that as the temperature increases, the ability for the oyster to provide a protective effect may be minimized. At 55 o C, however, the protective effect becomes even less apparent than in the previous two cases. Using the mean of the overnight sample s V. parahaemolyticus concentrations (2.0x10 4 ), a 3 log unit reduction in tria ls 1 and 2 and a 2 log unit in trial 3
52 is foun d The mean shows a 2. 7 0.6 log unit reduction, displaying that the protective effect at this temperature has be en reduced. Again, V. vulnificus was completely eliminated, most probably related to the low initial starting concentration. This temperature not only significantly reduced the Vibrio spp. but also reduced the total bacterial count by 2. 7 0.6 log units A pattern can be noticed that as the temperature increases, the protective effect appears to be minimized, thus suggesting the higher the temp erature the less likely protective effects influence the survival of these potential pathogens. Overall, the study conclude s that cooking to a 3 log unit reduction at lower temperatures is not sufficient in eliminating the test organisms due to the protec tive effect of the oyster. As the temperature increas ed it was observed that protection from the food system was reduced and a safer product is the end result. Because of this issue, safe cooking parameters were not recommended on these temperatures, and other studies were conducted to allow safe cooking practices based on higher temperatures.
53 CHAPTER 8 ASSESSING COMMERICAL COOKING PROCEDURES With the results from the lower temperatures providing evidence of a protective effect the next step was determ ining if the standard cooking methods inside of restaurants were sufficient in the overall thermal inactivation of potential pathogens. All the temperatures for commercial chargrilling reached an end point internal temperature of at least 200 o F (93.3 o C) w hile during frying all internal temperatures reached a minimum of 340 o F (171.1 o C). Figures 8 1 and 8 2 display the internal temperatures of chargrilling and deep frying, respectively. Figure 8 1 Internal temperature of half shucked oyster on an open ch argrill gas grill conducted on site in a commercial environment. Each trial was conducted with a minimum of 12 oysters with three temperature probes per run. Each run is the average of the three temperature probes per run with the final average calculated and added to the graph.
54 Figure 8 2. Internal Oyster Temperatures during on site trials conducted on the restaurant level. Twelve oysters were dropped per frying batch with each run having three temperature probes per 12 oysters. Each run is the averag e of those 3 trials and then the average of those trials was averaged. These results should differ from the lower temperature if follow ing the assumption that as the temperature increases the overall protective effects of the oyster will decrease as noted in previous experiments. Using the D and z values determined in a PBS solution it was hypothesized that if mimicking the lowest internal temperature recorded in commercial restaurant operations (200 o F), enough energy should enter the system to sufficientl y eliminat e potential V ibrio spp. pathogens The staged trials used whole shucked oysters with elevated levels of V. vulnificus and V. parahaemolyticus prepared using identical temperature abuse procedures as described for the thermal
55 assessment at interna l temperatures of 48, 50 and 55 o C (Table 8 1 ) The resulting levels exceeded 10 3 CFU/mL V. vulnificus and V. parahaemolyticus. The staged chargrill system provided direct heating between 420 450 o F. The oysters all reached an internal temperature of 20 0 o F b efore removal from the grill, and they were allowed to cool (for handling) as would customarily occur in a restaurant setting. Total bacteria were calculated by bacteria that are capable of growth in APW at 37 o C incubation overnight. It is important to not e that the V. vulnificus concentrations in these trials were much higher than in in i tial thermal recovery study (10 3 CFU/mL versus 10 1 CFU/mL ). This helped to also determine the overall reduction of V. vulnificus as well as V. parahaemolyticus where the pr ior study could only hypothesize that if V. parahaemolyticus was reduced, V. vulnificus should be reduced at least to an equivalent rate Table 8 1 Initial concen trations a nd Log unit Reduction of Temperature Abused Oyster V. vulnificus, V. parahaemolyti cus and total bacteria after a 200 o F heat treatment in CFU/mL. Sample V v ulnificus V. p arahaemolyticus Total Aerobic Plate Count* Initial concentration 6.0x10 3 4.0x10 3 7.3x10 5 Mean log unit reduction at 200 o F 3 0.0 3 0.0 4. 7 0. 6 Mean log unit re duction at 145 o F 3 0.0 3 0.0 1.7 0.6 *Total aerobic plate counts were calculated by bacteria that are capable of growth in APW at 37 o C incubation overnight. The results suggests that when reaching an internal temperature of 200 o F in a standard cooki ng practice as followed by many reputable restaurants, not only are the potential Vibrio spp. pathogens eliminated, so are nearly all other bacterial counts (Table 8 1 )
56 Mindful of the FDA US Food Code (2009) recommendations to cook seafood to 145 o F inter nal for 15 sec the final trial intended to demonstrate the bacterial influence of this procedure. The approached used the same batch of overnight temperature abused oysters that were shucked on the half shell and placed on the grill H owever, the product was only allowed to reach and internal temperature of 145 o F After 15 sec the product was removed from the grill (temperatures reached greater than 145 o F internally from the extra 15 sec on the grill) and t he product was then allowed to cool to room temp erature identically as used in the 200 o F trials. Table 8 1 displays the t emperature a bused o yster mean a erobic p late c ounts for V. vulnificus, V. parahaemolyticus and total bacteria after a 145 o F for 15 sec heat treatment in CFU/mL as well as the total l og unit reduction after heat treatment *Total bacteria were calculated by bacteria that are capable of growth in APW at 37 o C incubation overnight. Heating to an internal temperature of 145 o F for 15 sec with a starting concentration of ~10 3 CFU/mL will eli minate potential Vibrio spp. pathogens (Table 8 1 ) Interestingly, although there were no Vibrio spp. survivors that grew on the selective media, some bacteria did survive and grew on standard non selective media APC at 37 o C. With this data, it can be conc lusively stated that with an internal temperature of 200 o F or 145 o F for 15 sec a passive cool down, and a common 3 log unit initial bacterial count for Vibrio spp. will produce a saf e product for human consumption. Another important note about these trial s is the amount of energy entered into the 200 o F system is drastically greater in terms of reducing Vibrio spp. than in the 145 o F.
57 Since both reduced the Vibrio spp. concentrations by at least 3 log units it is reasonable to assume that cooking to 200 o F w ill eliminate even greater than 3 log unit s simply because of the time and energy in the system after the 145 o F temperature has been reached
58 CHAPTER 9 DISCUSSION Vibrio vulnificus, V. parahaemolyticus and s erogroup 01 V. cholerae are halophilic, potenti al pathogenic mesophiles commonly found inside of Crassostrea virginica Although consumption of this product includes raw to steamed fried and chargrilled products, little h as been documented on how to create safe cooking parameters to eliminate potentia l pathogenic threats. This study provides detailed information about D and z values in a common PBS medium and relates that with the actual food commonly associated with human infections. T he findings determined in this study are similar to some previous studies ( Tables 9 1, 9 2 and 9 3 ). Table 9 1 D values for V. cholerae Temp erature D Value Medium Reference ( o C) ( o F) (min) 48 118.4 2.36 PBS Solution Hanna and Otwell, unpublished 48.9 120 9.17 Shrimp Homogenate Hinton and Grodner, 1985. 49 120 .2 8.15 Crabmeat Shultz et al ., 1984 50 122 1.96 PBS Solution Hanna and Otwell, unpublished 54 129.2 5.02 Crabmeat Shultz et al ., 1984 54.4 129.9 0.43 Shrimp Homogenate Hinton and Grodner, 1985 55 131 0.52 PBS Solution Hanna and Otwell, unpublished 60 140 2.65 Crabmeat Shultz et al ., 1984 60 140 0.39 Shrimp Homogenate Hinton and Grodner, 1985 65.5 149.9 0.32 Shrimp Homogenate Hinton and Grodner, 1985 66 150.8 1.60 Crabmeat Shultz et al ., 1984 66 150.8 1.22 Crayfish Homogenate Grodner and Hinton, 19 85 71 159.8 0.30 Crabmeat Shultz et al ., 1984 71 159.8 0.30 Crayfish Homogenate Grodner and Hinton, 1985 71.1 160 0.31 Shrimp Homogenate Hinton and Grodner, 1985 76.7 170.1 0.30 Shrimp Homogenate Hinton and Grodner, 1985 77 170.6 0.27 Crayfish Homogen ate Grodner and Hinton, 1985 82 179.6 0.27 Crayfish Homogenate Grodner and Hinton, 1985 82.2 180 0.28 Shrimp Homogenate Hinton and Grodner, 1985
59 Table 9 2 D values for V. parahaemolyticus. Temperature D Value Medium Reference ( o C) ( o F) (min) 47 116.6 65.1 7.5% NaCl Beuchat and Worthington, 1976 48 118.4 3.02 PBS Solution Hanna and Otwell, unpublished 49 120.0 0.82 Clam Homogenate Delmore and Chrisley, 1979 50 122 1.99 PBS Solution Hanna and Otwell, unpublished 51 123.8 0.66 Clam Homogen ate Delmore and Chrisley, 1979 53 127.4 0.40 Clam Homogenate Delmore and Chrisley, 1979 55 131 0.29 Clam Homogenate Delmore and Chrisley, 1979 55 131 0.72 PBS Solution Hanna and Otwell, unpublished Table 9 3 D values for V. vulnificus Temperature D Value Medium Reference ( o C) ( o F) (min) 47 116.6 2.4 Buffered Saline Cook and Ruple, 1992 48 118.4 2.24 PBS Solution Hanna and Otwell, unpublished 50 122 1.15 Buffered Saline Cook and Ruple, 1992 50 122 2.05 PBS Solution Hanna and Otwell, unpubli shed 55 131 0.50 PBS Solution Hanna and Otwell, unpublished The lack of literature on D values for based on media as opposed to a food matrix makes such comparisons difficult. A n apparent food protective effect is noticed in the shrimp hom ogenate at 48.9 o C when compared directly to this study s value at 48 o C (9.17 min in the shrimp homogenate versus 2.36 min in PBS). This is expected as protective barriers found in foods can vary based on food product type, density, shape and related compos ition. Similar discrepancies were noticed with V. parahaemolyticus (Table 9 2) A difference was noted when heating Vibrio spp. at 48 o C in PBS (Table 9 2) versus (1976) trials in 7.5% NaCl at 47 o C. Beuchat and Worthington (1976) r ecorded a 65.1 min D value at this temperature, where as this study resulted i n a 3.02 min D value in PBS, a similar media.
60 At certain temperatures, t he values in this study correspond closely to values in previous literature for V. vulnificus (Table 9 3) Cook and Ruple (1992) noted a 2.4 min D value in a buffered saline at 47 o C, where as this study noted a 2.24 min D value at 48 o C. As the temperatures increased to 50 o C, Cook and Ruple (1992) noted a 1.15 min (in a buffered saline) where as this study record ed a 2.05 min D value (in PBS). Because of the significant role of the food matrix in the protection of the bacteria during cooking processes, the thermal recovery studies were essential to determining how the PBS D values would compare to trials conducte d in oysters The t hermal recovery studies show that at lower temperatures (48 and 50 o C), protective effects from the oyster are greater than protective effects at higher temperatures (55 o C+) This justifies cooking to higher temperatures in order to produ ce a product safe for human consumption. These studies also provide directions to develop cooking control programs in actual restaurant operations that can support HACCP based concepts Every trial conducted on site at multiple locations had an internal te mperature reaching a minimum of 200 o F. Using this as a standard, this study proves that not only were all potential Vibrio spp. pathogenic threats reduced from concentrations commonly found in oyster products (~10 3 CFU/mL ) but other bacterial counts were reduced as well Another extremely beneficial aspect of this study is the validation of the US Food (2009) internal temperature of 145 o F for 15 sec as a safe guard for reducing pathogenic threats. Results from this study conclude that following the (2009) recommendation eliminates all potential Vibrio spp. pathogenic threats to a th reshold of a minimum of 3 log unit s which is a normal concentration of Vibrio spp. commonly associated with oysters.
61 This research demonstrated that foll owing the parameters of cooking to an internal temperature of either 145 o F for 15 sec or 200 o F will sufficiently eliminate potential pathogenic threats of Vibrio spp. Because of these data it can be stated that using oyster products with natural Vibrio spp loads (~10 3 CFU/mL) can be cooked to a point of safe for human consumption with little risk of infection. With this knowledge, a HACCP plan for the restaurant level can be recommended and standard cooking procedures can be applied and validated. Restaura nts following these validated methods can assume that their product is safe for human consumption as long as it follows the criteria in the validated methods. Restaurants can rely on these science based results as a validation of proper cooking procedures For example, one well established famous oyster restaurant in New Orleans has traditionally followed in house protocols or recipes to cook their most popular oyster dish, chargrilled oysters. They train their staff on very specific cooking instructions w ith visual aids to help ensure their methods are followed. They recommend placing the required amount of oysters onto a preheated grill and allowing the oysters to heat to the point that they begin to expel some of their water, using the visual aid of the separation and expansion of the edges of the oyster to help determine when this point is reached. Next, they tip and remove the liquor (commonly called the liquid) of the oyster, and continue to cook for approximately 6 min After applying proprietary seas onings and butter sauces, they allow to cook for an a dditional 1 min and 30 sec and then remove from the heat. They state that at this point the oyster should be the color of a brown paper bag and are cooked to an internal temperature of >200 o F at this poi nt.
62 The results from this thesis can be integrated with traditional recipes to build an applicable HACCP program (Figure 9 1). The program would have a validated critical limit (CL) of an internal temperature of >145 o F for 15 sec This CL is consistent wit h existing 2009 US Food Code regulations (FDA, 2009). This thesis provides the validation for compliance monitoring and it would generate necessary records to provide evidence of compliance. This approach is innovative and new for the oyster industry. C CP 1 Critical Control Point (CCP) (1) Cooking Significant Hazard (2) V. vulnificus, V. parahaemolyticus, V. cholerae Critical Limits (3) 145 o F for 15 sec + cool down Monitoring What (4) Internal Product Temperature How (5) Internal Temperature Probes When (6) 3 trials daily Who (7) Cook manager Corrective Actions (8) If internal product temperature is not reached, continue cooking until reaching 145 o F for 15 sec Records (9) Daily cooking log Supervisor training records Verification (10) Valid ation study on file Figure 9 1. Illustration of a possible HACCP plan for cooking oysters in a commercial restaurant operation. Additional recommended research based on this study should include D and z values conducted on Vibrio spp. inside of a whole o yster product.
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66 BIOGRAPHICAL SKETCH Christopher W. Hanna attended the University of Florida from 2005 2009 f or his Bachelor of Science in f ood s cience and h uman n utrition. He completed courses ranging from c hemistry 1 o rganic 2, b iology 1 and 2, m icrobiology, b iochemistry, p hysics 1 2, n utrition courses, and general electives. He then took a year off and applied to the f ood s cience graduate program at the University of Florida. During his graduate degree he received all A marks in his core classes: a dvanced f ood c hemistry, a dvanced f ood m icrobiology, a dvanced f ood p rocessing, p roduct d evelopment, and s ensory a nalysis. Chris completed this course work under Dr. Wade Yang initially, and transferred to the a quatics s eafood d epartment and began his thesis with Dr. Steve Otwell as his chair. His appointed committee members include Dr. Keith Schneider Dr. Anita Wright, and Dr. Chuck Adams. Chris eventually desires to pursu e a career as a technical sales associate, however, he hope to one day end up managing and becoming the vice president of sales at a large company.