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Seasonal Occurrence and the Use of Non Thermal Technologies to Control Growth and Toxin Formation of Bacillus cereus in Tuna

Permanent Link: http://ufdc.ufl.edu/UFE0021452/00001

Material Information

Title: Seasonal Occurrence and the Use of Non Thermal Technologies to Control Growth and Toxin Formation of Bacillus cereus in Tuna
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Cevallos, Juan M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bacillus, cereus, nonthermal, tuna
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: There is an increasing concern about the hazardous presence of B. cereus in seafood. Some outbreaks caused by a diarrheal toxin produced by B. cereus in fish have been reported. For this reason all seafood processors must follow FDA guidelines which specify that to avoid outgrowth of B. cereus, the period of time for holding seafood after cooking must not exceed 3 hours. In this investigation tuna samples of the species Skip Jack, Yellow Fin, and Big Eye were taken from the coasts of Ecuador and analyzed for presence of B. cereus vegetative cells and spores every other month during one year. A significant (p < 0.05) seasonal variation of the amount of B. cereus vegetative cells was found in the months June- August as oppose to the rest of the months in each of the species. No significant (p > 0.05) difference was found in the amount of B. cereus vegetative cells among tuna species. No significant occurrence (p > 0.05) was found on the levels of B. cereus spores in any of the sampled months. In order to find an efficient non-thermal technology capable to reduce B. cereus vegetative cells in tuna, Yellow Fin tuna samples were inoculated with known amounts of B. cereus vegetative cells and treated with ultraviolet (UV) light, Ozone, and lactoperoxidase system. The first set of samples was treated with 8.5 mJ/cm2 and 17 mJ/cm2 doses of UV light. A significant (p < 0.05) reduction in the number of B. cereus vegetative cells was achieved with these doses. No significant (p < 0.05) difference was found between the two doses. The second set of samples was treated with 0.65 mg/L of Ozone. A significant (p < 0.05) reduction was achieved with this treatment. The third set of sample was treated in a one liter solution containing 100 ?l of a 1mg/ml solution of the enzyme lactoperoxidase, 20 ?l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. No significant (p > 0.05) reduction in the number of B. cereus vegetative cells was achieved by this treatment. The effects of these treatments on the color of the tuna were also evaluated. All treatments showed a significant (p < 0.05) variation in the a* value when compared with untreated tuna.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Juan M Cevallos.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Rodrick, Gary E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021452:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021452/00001

Material Information

Title: Seasonal Occurrence and the Use of Non Thermal Technologies to Control Growth and Toxin Formation of Bacillus cereus in Tuna
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Cevallos, Juan M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bacillus, cereus, nonthermal, tuna
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: There is an increasing concern about the hazardous presence of B. cereus in seafood. Some outbreaks caused by a diarrheal toxin produced by B. cereus in fish have been reported. For this reason all seafood processors must follow FDA guidelines which specify that to avoid outgrowth of B. cereus, the period of time for holding seafood after cooking must not exceed 3 hours. In this investigation tuna samples of the species Skip Jack, Yellow Fin, and Big Eye were taken from the coasts of Ecuador and analyzed for presence of B. cereus vegetative cells and spores every other month during one year. A significant (p < 0.05) seasonal variation of the amount of B. cereus vegetative cells was found in the months June- August as oppose to the rest of the months in each of the species. No significant (p > 0.05) difference was found in the amount of B. cereus vegetative cells among tuna species. No significant occurrence (p > 0.05) was found on the levels of B. cereus spores in any of the sampled months. In order to find an efficient non-thermal technology capable to reduce B. cereus vegetative cells in tuna, Yellow Fin tuna samples were inoculated with known amounts of B. cereus vegetative cells and treated with ultraviolet (UV) light, Ozone, and lactoperoxidase system. The first set of samples was treated with 8.5 mJ/cm2 and 17 mJ/cm2 doses of UV light. A significant (p < 0.05) reduction in the number of B. cereus vegetative cells was achieved with these doses. No significant (p < 0.05) difference was found between the two doses. The second set of samples was treated with 0.65 mg/L of Ozone. A significant (p < 0.05) reduction was achieved with this treatment. The third set of sample was treated in a one liter solution containing 100 ?l of a 1mg/ml solution of the enzyme lactoperoxidase, 20 ?l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. No significant (p > 0.05) reduction in the number of B. cereus vegetative cells was achieved by this treatment. The effects of these treatments on the color of the tuna were also evaluated. All treatments showed a significant (p < 0.05) variation in the a* value when compared with untreated tuna.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Juan M Cevallos.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Rodrick, Gary E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021452:00001


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SEASONAL OCCURRENCE AND THE USE OF NON THERMAL TECHNOLOGIES TO CONTROL GROWTH AND TOXIN FORMATION OF Bacillus cereus IN TUNA By JUAN MANUEL CEVALLOS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007 1

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2007 Juan Manuel Cevallos 2

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To my parents and family in Ecuador 3

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ACKNOWLEDGMENTS I would like to thank my committee chairman and major advisor Dr. Gary Rodrick. Without his guidance and support this work would not have been possible. I would also like to thank my supervisory committee members, Dr. Ronald Schmidt and Dr. Sally Williams for their guidance and help. Extensive thanks are due to the people from the Microbiology Department of Sociedad Ecuatoriana de Alimentos y Frigorificos Manta C.A. (Stalin Mendoza and Roberto Benitez) in Manta, Ecuador who collaborated in the seasonal assessment of Bacillus cereus in tuna in my study. Without them, the seasonal assessment would not have been completed. Special thanks are also given to my coworkers Amanda Thompson and Jillian Fleisher for their invaluable help in the execution of this project. In conclusion, I would like to thank my parents Esperanza Cevallos and Eddie Cevallos, and my siblings Maria Susana, Eddie Andres, Maria Del Pilar, Maria Esperanza, Maria Isabel, and Maria Ines for all their unconditional love and support that they have always given me. Finally I want to thank God. It is right to say that with no doubt nothing in my life could have been achieved without Gods help. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................................10 CHAPTER 1 REVIEW OF LITERATURE.................................................................................................12 Bacillus Cereus.......................................................................................................................12 General Characteristics....................................................................................................12 Reservoirs.................................................................................................................12 Diseases....................................................................................................................12 Diarrheal Toxin...............................................................................................................13 Emetic Toxin...................................................................................................................14 Spore................................................................................................................................15 Foodborne Outbreaks......................................................................................................15 Potential Problems in the Seafood Industry....................................................................16 Methods for B. cereus Inhibition in Food...............................................................................17 Sorbates...........................................................................................................................17 Temperature.....................................................................................................................17 Ultraviolet Light..............................................................................................................18 Ozone...............................................................................................................................19 Lactoperoxidase System..................................................................................................19 Tuna Processing......................................................................................................................20 Overview.........................................................................................................................20 Canned Tuna....................................................................................................................20 Precooked Tuna Loins.....................................................................................................23 Objectives...............................................................................................................................24 2 MATERIALS AND METHODS...........................................................................................25 Microbiological Materials and Methods.................................................................................25 Media...............................................................................................................................25 Microorganisms...............................................................................................................25 Enumeration....................................................................................................................25 Seasonality Analysis...............................................................................................................26 Spore Assessment...................................................................................................................26 Growth Assessment................................................................................................................26 Bacillus cereus Inactivation by Processing Methods.............................................................27 Ultraviolet Light Processing............................................................................................27 5

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Ozone Processing............................................................................................................27 Lactoperoxidase System Processing................................................................................28 Color Analyses........................................................................................................................28 Statistical Analysis..................................................................................................................28 3 RESULTS...............................................................................................................................29 Seasonality and Spore Assessment.........................................................................................29 Bacillus Cereus Growth Assessment in the Yellow Fin Tuna Specie....................................33 Bacillus Cereus Inactivation...................................................................................................34 Inactivation by Ultraviolet Light.....................................................................................34 Bacillus Cereus Inactivation by Ozone...........................................................................35 Bacillus Cereus Inactivation by Lactoperoxidase System..............................................36 Color Analysis of Tuna after Each Treatment........................................................................36 Color Analysis after UV Treatment................................................................................37 Color Analysis after Ozone Treatment............................................................................38 Color Analysis after Treatment with Lactoperoxidase System.......................................40 Plan HACCP Flow Chart and Critical Control Points for Processing of Tuna......................41 4 DISCUSSION.........................................................................................................................44 Seasonality and Spore Assessment.........................................................................................44 Growth Assessment................................................................................................................45 Bacillus Cereus Inactivation and Color Analysis...................................................................46 Ultraviolet Light..............................................................................................................46 Ozone...............................................................................................................................47 Lactoperoxidase System..................................................................................................47 5 SUMMARY AND CONCLUSIONS.....................................................................................49 APPENDIX ...52 LIST OF REFERENCES...............................................................................................................55 BIOGRAPHICAL SKETCH.........................................................................................................57 6

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LIST OF TABLES Table page 3-1 January 2006 occurrence of B. cereus in the Skip Jack, Yellow Fin, and Big Eye tuna species ..............................................................................................................................30 3-2 March 2006 occurrence of B. cereus in the Skip Jack, Yellow Fin, and Big Eye tuna species ..............................................................................................................................31 3-3 May 2006 occurrence of B. cereus in the Skip Jack, Yellow Fin, and Big Eye tuna species ..............................................................................................................................31 3-5 October 2006 occurrence of B. cereus in the Skip Jack, Yellow Fin, and Big Eye tuna species........................................................................................................................32 3-6 December 2006 occurrence of B. cereus in the Skip Jack, Yellow Fin, and Big Eye tuna species........................................................................................................................33 3-7 Bacillus cereus inactivation by Ultraviolet light in Yellow Fin tuna at 28 C ...................34 3-8 Reduction of B. cereus in Yellow Fin tuna at several ozone concentrations.....................35 3-9 Reduction of B. cereus at several contact times by lactoperoxidase ............................36 3-10 Average results of the L*, a*, and b* values of the UV treated and non treated Yellow Fin tuna samples....................................................................................................38 3-11 Average results of the L*, a*, and b* values of the ozone treated and non treated Yellow Fin tuna samples....................................................................................................39 3-12 Average results of the L*, a*, and b* values of the lactoperoxidase system treated and non treated Yellow Fin tuna samples..........................................................................41 3-13 Precook tuna loins HACCP summary................................................................................43 7

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LIST OF FIGURES Figure page 1-1 Survival of spores exposed to 95 C for 0, 5, 10 and 15 minutes .....................................17 1-2 D values for different strains of spores of B. cereus .........................................................18 3-1 The seasonal occurrence of B. Cereus in the species skip jack (SJ), yellow fin (YF), and Big eye (BE) tuna........................................................................................................29 3-2 Exponential growth of B. cereus in tuna at 28 C. The regression equation and coefficient are also shown..................................................................................................34 3-3 Inactivation of B. cereus in Yellow Fin tuna by several doses of ultraviolet light............35 3-4 Photographs of typical tuna samples before and after UV treatment................................37 3-5 Photographs of typical tuna samples before and after ozone treatment.............................39 3-6 Photographs of typical tuna fillet samples before and after lactoperoxidase system treatment............................................................................................................................40 3-7 Hazard Analysis and Critical Control Points flow chart....................................................42 8

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LIST OF ABBREVIATIONS BAM Bacteriological analytical manual BE Big eye CFU Colony forming units FDA Food and Drug Administration MYP Mannitol egg yolk polimixin agar SJ Skip jack STD Standard deviation TFTC Too few to count UV Ultraviolet YF: Yellow fin 9

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SEASONAL OCCURRENCE AND THE USE OF NON THERMAL TECHNOLOGIES TO CONTROL GROWTH AND TOXIN FORMATION OF Bacillus cereus IN TUNA By Juan Manuel Cevallos August 2007 Chair: Gary E. Rodrick Major: Food Science and Human Nutrition There is an increasing concern about the hazardous presence of B. cereus in seafood. Some outbreaks caused by a diarrheal toxin produced by B. cereus in fish have been reported. For this reason all seafood processors must follow FDA guidelines which specify that to avoid outgrowth of B. cereus, the period of time for holding seafood after cooking must not exceed 3 hours. In this investigation tuna samples of the species Skip Jack, Yellow Fin, and Big Eye were taken from the coasts of Ecuador and analyzed for presence of B. cereus vegetative cells and spores every other month during 1 year. A significant (P < 0.05) seasonal variation of the amount of B. cereus vegetative cells was found in the months June to August as opposed to the rest of the months in each of the species. No significant (P > 0.05) difference was found in the amount of B. cereus vegetative cells among tuna species. No significant occurrence (P > 0.05) was found on the levels of B. cereus spores in any of the sampled months. To find an efficient non-thermal technology capable to reduce B. cereus vegetative cells in tuna, Yellow Fin tuna samples were inoculated with known amounts of B. cereus vegetative cells and treated with ultraviolet (UV) light, Ozone, and lactoperoxidase system. The first set of samples was treated with 8.5 mJ/cm 2 and 17 mJ/cm2 doses of UV light. A significant (P < 0.05) reduction in the number of B. cereus vegetative cells was achieved with 10

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these doses. No significant (P > 0.05) difference was found between the two doses. The second set of samples was treated with 0.65 mg/L of Ozone. A significant (P < 0.05) reduction was achieved with this treatment. The third set of samples was treated in a one liter solution containing 100 L of a 1 mg/mL solution of the enzyme lactoperoxidase, 20 L of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. No significant (P > 0.05) reduction in the number of B. cereus vegetative cells was achieved by this treatment. Effects of these treatments on the color of the tuna were also evaluated. All treatments showed a significant (P < 0.05) variation in the a* value when compared with untreated tuna. 11

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CHAPTER 1 REVIEW OF LITERATURE Bacillus Cereus General Characteristics Bacillus cereus is a gram-positive, spore-forming, aerobic bacterium that grows well anaerobically. B. cereus produces two kinds of foodborne illnesses: diarrheal and emetic. The diarrheal toxin is heat labile and has to be produced in the small intestine to cause the disease whereas the emetic toxin is heat stable and can be produced in some foods to cause intoxication (Granum 2001). Besides food borne illnesses B. cereus may also cause diseases like endocarditis and endophthalmitis (Drobniewski 1993). Reservoirs Bacillus cereus is ubiquitous, but is most commonly found in soils and growing plants from which it can be spread to foods. It can also be found in fish and other marine species (Granum 2001). Diseases Bacillus cereus is the etiologic agent of two types of food-borne disease, diarrhea and vomiting illnesses. Both types are caused by toxins: the diarrheal type by protein toxins which must be formed in the intestinal tract by growing organisms, and the emetic type by a peptide toxin that is preformed in the food (Agata and others 1995). The diarrheal type of disease is characterized by diarrhea within 6 to 15 hours after consumption of the suspected food (FDA 2003). Other symptoms are abdominal cramp and pain, and nausea. The emetic type of disease is characterized by vomiting and nausea within half to 6 hours after the consumption of the suspected food. For both types of disease the total duration of clinical signs is about 24 hours (FDA 2002; Kramer and Gilbert 1989). 12

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Diarrheal toxin The diarrheal type of disease is caused by B. cereus species that are able to produce enterotoxins. There are four toxins that are produced by B. cereus and cause diarrhea: Haemolytic BL toxin (HBL), Non Haemolytic Enterotoxin (NHE), enterotoxin T, and cytotoxin K. Three of these toxins are related to food borne outbreaks; the fourth, enterotoxin T, is not (Granum and Lund 1997;Lund and others 2000;Agata and others 1995). Haemolytic BL toxin is a hemolytic toxin consisting of three protein subunits: B, L1 and L2. The protein subunit B is the one that binds the toxin to the cells whereas the proteins L1 and L2 cause the cellular lysis (LM Wijnands and others 2002). The toxin shows dermonecrotic activity as well as activity towards vascular permeability, and causes fluid accumulation in ligated rabbit ileal loops (Granum and Lund 1997). All three components are necessary for maximal enterotoxic activity (Beecher and others 1995). Non Haemolytic Enterotoxin consists also of three protein subunits: nheA, nheB and nheC. In this case the nhC protein is the binding factor whereas the other two are cause lysis (LM Wijnands and others 2002). Although binary combinations of the subunits show some biological effect, maximal activity is achieved when all three components are present (Lund and Granum, 1997). Enterotoxin-T has been named such based on cloning and immunoblot experiments. This toxin has not been related to outbreaks of food borne disease so far. It is a single component protein enterotoxin with activity towards vascular permeability. Enterotoxin T causes fluid accumulation in the ligated rabbit ileal loop test, and is lethal to mice after intravenous injection (Agata and others 1995a). Cytotoxin-K is the most recently described enterotoxin from B. cereus. It was detected after a food poisoning outbreak in an elderly home in France. In total 44 people were ill, 6 of 13

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these patients had bloody diarrhea, and three of these six died (Lund and others 2000 reviewed by LM Wijnands and others 2002). Cytotoxin-K is a single component protein enterotoxin showing necrotic and hemolytic activity, and is highly toxic to epithelial cells (Hardy and others 2001 reviewed by LM Wijnands and others 2002). Emetic Toxin Emetic poisoning is characterized by vomiting and occurs within 1 to 5 hours after ingestion of contaminated foods (Kramer and Gilbert 1989). Emetic syndrome is usually mild but rare fatal cases have been reported (Malher and others 1997). The emetic toxin cereulide is produced in the food and poisoning occurs after ingestion of the toxin. The emetic toxin cereulide is a small cyclic peptide (Agata and others 1994). Because cereulide is very stable, it may persist in heat treated foods after death of the B. cereus cells. All B. cereus strains involved in emetic foodborne infections produce cereulide. Emetic B. cereus are unable to hydrolyze starch, so incidence of starch negative B. cereus could provide an estimate of emetic B. cereus. Starch negative B. cereus represented at most 2% to 11% of strains isolated from dairy products and from dairy farms (Te Giffel and others 1995). Since emetic intoxication occurs through ingestion of emetic toxin (cereulide) preformed in the food, determining conditions in the foods that would lead to production of cereulide by emetic B. cereus is important for risk assessment of emetic intoxication. Cereulide is not easily destroyed by heat treatments. For instance, it can resist 90 min at 126C (Turnbull and others 1979; ICMSF 1996). It is also resistant to acid conditions. Cereulide will therefore not be eliminated from foods in which it had been produced. Conditions permitting emetic toxin production in foods by emetic strains of B. cereus are still not elucidated. 14

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The few conclusions that can be drawn from published work are the following: Cereulide became detectable at the end of the growth of B. cereus (Hggblom and others 2002). The range of conditions permitting cereulide production is narrower than conditions permitting growth of B. cereus. Anaerobic conditions and temperatures above 37C did not permit cereulide production (Finlay and others 2000, Finlay and others 2002, Jskelinen and others 2004). Not all foods permit cereulide production even if growth of B. cereus is possible (Agata and others 2002). Milk, cooked rice and pasta supported important cereulide production at 30C (Finlay and others 2002). Production of cereulide below 10C does not seem possible. This clearly shows that presence and even growth of emetic B. cereus does not always mean cereulide accumulation in foods. Spore B. cereus is a sporeformer microorganism. Sporulation is stimulated under low nutrient conditions (Granum 2001). Phase-contrast microscopic studies have indicated that the germination response of a single B. cereus spore consists of a lag-phase (microlag) and a biphasic event in which the actual germination reactions take place (Hashimoto and others 1969 reviewed by Vries 2006) Heat activation stimulates the germination of spores primarily by reducing the microlag times, and the kinetics of germination of spore suspensions are most critically influenced by the microlag time of each member of that population. More recent analysis confirmed that the microlag was affected by heat activation treatment and indicated that the germination phase varied considerably with germination temperature (Vries 2006). Foodborne Outbreaks Most outbreaks produced by this microorganism have been related to rice and other starchy foods, but recently it has been taken into consideration B. cereus presence in other kind of food like fish (National Restaurant Association 2004). 15

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It is well known that B. cereus diarrheal infection can be associated with the consumption of high amounts of B. cereus in fish (Granum 2001); however the required levels of B. cereus to cause infection have not been established yet. Some outbreaks have been related to B. cereus in fish. For example in 1996 some cases of fish borne intoxication with B. cereus were reported in Japan (FAO 1998). Other outbreaks have been reported in small numbers in USA, The Netherlands, Canada and England (Notermans and others 1998). Potential Problems in the Seafood Industry The ubiquitous nature of B. cereus suggests that it is present in a vegetative form the fish before catching, then during freezing it sporulates, so it can resist the low temperatures. Further research is needed to understand how B. cereus reaches the fish, in which part of the fish it is mainly located; and to confirm the hypothesis that B. cereus is present in a vegetative form in the fish after catching and before freezing. The main problem in fish processing is that many fish processes just require a mild cooking i.e. 130 F during 50 minuteswhich only can kill other competitive bacteria. On the other hand this kind of heat treatments causes B. cereus spore germination (Granum 2001). As a preventing measure, producers must not exceed a 3 hours holding time at room temperature after mild cooking (FDA 2001). This short amount of time is known to cause some loss in texture quality and efficiency, and hence an increase in production costs. No data exist on the quantification of the presence of B cereus in tuna. It is also necessary to know if B cereus is present in tuna under vegetative or spore conditions. For this reason it is necessary to find out other ways to control B. cereus growth. Additional heating, pH and Aw reduction will alter in some ways the sensory quality of the fish, 16

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so it would be necessary to try out other kind of treatments such as ozone, UV light, freezing, and lactoperoxidase treatments. Methods for B. cereus Inhibition in Food Sorbates Smoot and Pierson (1981) were the first to report that 3900 micrograms of sorbate per milliliter of sodium-potassium buffer at a pH of 5.7 will inhibit B. cereus growth in culture media. This amount of sorbate is too high compared to the levels sorbates are commonly used in the food industry. Temperature Vries (2006) determined the D values of several B. cereus spores. Those results are shown in Figure 1-1. Figure 1-1. Survival of spores exposed to 95 C for 0, 5, 10 and 15 minutes (Vries 2006) Based on the results obtained in Figure 1-1, Vries (2006) determined the D values for each analyzed strain. Those results are shown in Figure 1-2. Data in Figure 1-2 show a high variability of the resistance of B. cereus to heat treatment, being the highest D value of 80 minutes a very 17

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large amount of time to treat a food. For this reason it is important to find alternatives to reduce B. cereus in food. Figure 1-2. D values for different strains of spores of B. cereus (Vries 2006). Ultraviolet Light Ultraviolet processing involves the use of radiation from the ultraviolet region of the electromagnetic spectrum in order to reduce the number of microorganisms in the surface of a food. Typically, the wavelength for UV processing ranges from 100 to 400 nm. This range may be further subdivided (Bolton 1999 cited by FDA 2000) into UVA (from 315 to 400 nm), UVB (280 to 315 nm); UVC (from 200 to 280 nm) called the germicidal range since it effectively inactivates bacteria and viruses, and the vacuum UV rangefrom 100 to 200 nmthat can be absorbed by almost all substances and thus can be transmitted only in a vacuum (FDA 2000). 18

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The germicidal properties of UV irradiation are mainly due to DNA mutations induced through absorption of UV light by DNA molecules. Benoit and others (1990) found that B. cereus spores can be reduced in a 90% by applying UV doses of the order of 738 J/m2 in distilled water and several culture broths; however no experiments in tuna have been done to inactivate B. cereus. Ozone Another important alternative is the use of ozone. The strong antibacterial characteristics of ozone are due to a combination of its high oxidizing potential and its ability to diffuse through biological membranes (Hunt and Marinas 1996). Oxidation reactions of ozone in water follow two major pathways: direct oxidation by molecular ozone and indirect oxidation by free radical species formed from the auto decomposition of ozone. Also reactions between ozone and some inorganic and organic compounds can produce important antibacterial compounds (Hoigne and Bader 1976 cited by Hunt and Marinas 1997). Broadwater and others (1973) found that ozone concentrations of 0.19 mg/L or higher can inactivate vegetative cells of B. cereus in water; however no complete data exist regarding inactivation of this microorganism in tuna. Lactoperoxidase System Finally, an important tool against gram positive pathogens is the use of the enzyme lactoperoxidase. Lactoperoxidase (LPO) is an enzyme that catalyses some oxidation reactions at the expense of hydrogen peroxide. This enzyme is widely distributed in the nature and can be found in plants and animals including man. LPO is a part of some human secretions such as saliva and tears (Kussendager and van Hooijdonk 2000). This enzyme has been evaluated as an antimicrobial agent, and its use has been suggested as a preservative in food (Bosch and others 2000). 19

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LPO catalyses the following reaction (Kussendager and van Hooijdonk 2000): 2SCN + H 2 O 2 + 2H + LPO (SCN) 2 + 2H 2 O (SCN) 2 + H 2 O HOSCN + H + + SCN HOSCN OSCN + H + Hypothiocyanite (OSCN ) and hypothiocyanus acid (HOSCN) are oxidizing products of this reactions and are responsible of the inhibition of some microorganisms by the oxidation of sulphydryl groups of important bacterial proteins (Kussendager and van Hooijdonk 2000). Tenovuo and others (1985) proved the efficacy of the lactoperoxidase system against B. cereus in phosphate buffer, but no research in tuna has been done. Tuna Processing Overview The term tuna correspond to several species of fish from the family Scombridae and the genus Thunnus. One of tunas most known characteristic is that its flesh is redas oppose many other fish that have a white fleshbecause of its higher amount of myoglobin in tunas tissues (Wikipedia 2007). Tuna can be processed in several ways, the most common tuna products are canned tuna, precooked tuna loins, and raw tuna. According to Foodmarket Exchange (2003) countries with the highest level of caught tuna are Japan and Taiwan. Ecuador and Mexico have the highest level of caught tuna in the eastern Pacific. Data from Foodmarket Exchange (2003) shows that US is the world's largest market for canned tuna, with consumption estimated at about 46 million cases in 2001, or 28 percent of the worlds consumption. Canned Tuna Canned tuna is one of the most popular ways of processing tuna since it has a longer shelf life and can be shipped to longer distances. Canned tuna is produced from fresh or frozen tuna in 20

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accordance with requirements in the Canned Tuna Standard of Identity, 21 CFR 161.190 (FDA 2001). Several tuna species are packed in water or oil and may be seasoned with salt, vegetable broth, hydrolyzed protein, or other optional ingredients. Bones, scales, skin, and other undesirable fish portions are removed from tuna. The filled cans are hermetically sealed and processed in retorts in accordance with processes scientifically designed to render them commercially sterile. The finished products are shelf stable low acid canned foods with pH above 6.0. Tuna species used in tuna canning operations in Ecuador include Albacore, Yellow fin, Skipjack, and Big Eye. Other species provided for in the Canned Tuna Standard of Identity may be used (FDA 2001). Tuna are caught via a variety of methods, including long line, jig boat (hook and line), pole and line, and purse seine nets. Fishing boats are equipped with blast or brine freezing systems to quickly lower backbone temperatures of fish to the freezing point and below. Some fish are delivered to canneries directly from the boats utilized to catch them. Some are transferred from catch vessels to refrigerated carriers or vans and then delivered to canneries. Tuna are unloaded from fishing boats, refrigerated carriers, or vans into steel fish boxes. During unloading, the fish are segregated into lots that identify the supplier, well, or van from which the fish were unloaded, and species. To be considered acceptable in the FDA standard, tuna lots must have histamine levels below FDAs established guidelines for canned tuna which is 50 ppm (2001). Tuna boxes are transferred to cannery cold storage and maintained at temperatures near 0F (-18C) until needed for processing (FDA 2001). When tuna lots are scheduled for processing, tuna boxes are brought out of cold storage and thawed to backbone temperatures sufficient to facilitate evisceration and organoleptic 21

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evaluation. After thawing, viscera are removed and trained staff for physical characteristics associated with decomposition or contamination evaluates fish. Any fish exhibiting unacceptable characteristics is rejected. After evisceration and organoleptic evaluation, fish are placed on racks and transferred to large ovens called precookers where they are processed until backbone temperatures are sufficient to facilitate cleaning of the fish. Precooked fish are cooled under controlled conditions and then transferred to the packing room for cleaning. The cleaning operation consists of manual removal of the head, tail, skin, bones, and dark flesh known as red meat. Cleaned loins are fed into filling machines where prescribed amounts of fish are placed into cans. Using a separate system, empty cans are conveyed to filling machines after being inverted and flushed with air jets and/or water sprays. When cans leave the filling machine, they are conveyed to locations where packing media and other ingredients are added. Several types of packing media are used in canned tuna processing. These include spring water, water or vegetable oil used alone or in combination with hydrolyzed protein and/or vegetable broth. Sodium acid pyrophosphate and/or salt may also be added. Filled cans are conveyed to seaming machines where ends are put in place and the cans hermetically sealed. Each can or end is affixed with a production code that identifies manufacturing plant, product, date packed, batch, and other information necessary for product tracing purposes. Seamed cans are then retorted under controlled conditions designed by process authorities to render the can contents commercially sterile. Both the seaming and retorting 22

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operations are carried out in strict compliance with Low Acid Canned Food regulations in 21 CFR 113 (FDA 2001). After retorting, cans are partially cooled, in accordance with good manufacturing practice GMP establish in 21 CFR 113.5 (FDA 2001), and removed from retorts. Cans are then further cooled and delivered to labeling lines and are labeled, cased, and palletized. Cases and pallets are appropriately marked to facilitate product tracing and pallets are either shipped or staged in warehouses for later shipment. Precooked Tuna Loins Tuna Loins are produced from fresh or frozen tuna in accordance with requirements in the Canned Tuna Standard of Identity, 21 CFR 161.190 (FDA 2001). Tuna species, color designations (e.g., white or light), and forms of pack (e.g., solid or chunk) are as provided for in the standard. Bones, scales, skin, and other undesirable fish portions are removed from tuna, in keeping with good manufacturing practices, and the cleaned fish is packed into plastic bags from which most of the air is removed. Tuna are unloaded from fishing boats, refrigerated carriers, or vans into steel fish boxes. During unloading, the fish are segregated into lots that identify the supplier, well, or van from which the fish were unloaded, and species. Lots considered potentially acceptable must have histamine levels below FDAs established guidelines for canned tuna. Tuna boxes are transferred to cannery cold storage and maintained at temperatures near 0F (-18C) until needed for processing. When fish lots are scheduled for processing, fish boxes are brought out of cold storage and thawed to backbone temperatures sufficient to facilitate evisceration and organoleptic evaluation. After thawing, viscera are removed and fish are evaluated by trained staff for physical 23

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characteristics associated with decomposition or contamination. Any fish exhibiting unacceptable characteristics is rejected. After evisceration and organoleptic evaluation, fish are placed on racks and transferred to large ovens called precookers where they are processed until backbone temperatures are sufficient to facilitate cleaning of the fish. Precooked fish are cooled under controlled conditions and then transferred to the packing room for cleaning. The cleaning operation consists of manual removal of the head, tail, skin, bones, and dark flesh known as red meat. The precooked cleaned loins may be packed into plastic bags from which most of the air is removed, frozen and transferred by frozen containers to another plant for thawing and packing as canned tuna. Objectives The overall objective of the research was to determine the seasonal occurrence of B. cereus spores and vegetative cells in Skip Jack, Yellow Fin, and Big Eye tuna; as well as achieve B. cereus inactivation using non thermal technologies, and develop a HACCP plan. Objective 1: to develop the B. cereus growth curve in tuna at 28C Objective 2: to determine the doubling time of B. cereus in tuna at 21, 28, and 37 C Hypothesis 1: tuna can be held for more than 3 hours at ambient temperatures under adequate conditions without any risk of emetic toxin formation. Hypothesis 2: B. cereus is present only as vegetative cellsno as sporesin tuna. Several samples of tuna are to be analyzed for presence of spores in a monthly basis. Hypothesis 3: B. cereus presence in tuna is affected by the season in which the tuna is caught. Tuna samples are to be taken in different seasons and analyzed for presence of B. cereus. Hypothesis 4: B. cereus can be inactivated in tuna by non thermal treatments. Ozone, UV light, freezing and lactoperoxidase system are going to be tested to determine which of these alternatives is the best for reducing the number of B. cereus cells 24

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CHAPTER 2 MATERIALS AND METHODS Microbiological Materials and Methods Media Mannitol Egg Yolk Polimixin agar (MYP agar) was obtained from Oxoid Ltd., Lenexa, Kansas; polimixim B sulfate from Fisher Scientific Inc., Miami, Oklahoma; phosphate buffer from Fisher Scientific Inc., Miami, OK; and the egg yolk emulsion was prepared by soaking raw eggs in 70% ethanol, cracking the eggs, separating the yolk, and mixing it with equal volume of a 0.85% sodium chloride solution as described in the US Food and Drug Administration Bacteriological Analytical Manual (2001). Microorganisms Bacillus cereus strains were isolated from tuna samples from Seafman C.A.a tuna processing company located in Manta, Ecuadoras follows: Tuna samples of the species Katsuwonus pelamis (skipjack tuna), Tunnus albacares (yellow fin tuna), and Tunnus obesus (big eye tuna) were obtained in the Pacific coasts of Ecuador in South America. Bacillus cereus was isolated from these samples by inoculation in MYP agar enriched with egg yolk emulsion and polimixim B sulfate. Confirmation test were executed by following FDA guidance on B. cereus given in the Bacteriological Analytical Manual (2001). Enumeration One hundred grams of each sample was dissolved in 900 ml of phosphate buffer to achieve 10 -1 dilution. Following dilutions were made by adding 1 ml of the previous dilution to 9 ml of phosphate buffer. Each sample was diluted and run in triplicate by adding 0.1 ml of the dilution to the solidified MYP agar and spreading it with a glass spreader. Growth was observed after 24, 48, and 72 hours and reported as colony forming units per gram of tuna (cfu/g). Plates yielding 25

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colony numbers from 25 to 200 were counted. Dilution was noted and used to calculate colony forming units per gram of tuna. Seasonality Analysis For the seasonal analysis, 10 samples of each of the species Katsuwonus pelamis (skipjack tuna), Tunnus albacares (yellow fin tuna), and Tunnus obesus (big eye tuna) were taken per month in two month intervals during one year. The samples were taken in the Pacific coasts of Ecuador. Levels of B. cereus were determined by surface plating on MYP agar as described above. Spore Assessment Ten samples of each of the three tuna species mentioned above were taken in two month intervals during one year. The samples were taken in the pacific coasts of Ecuador. One hundred grams of each sample was dissolved in 900 ml of phosphate buffer to achieve 10 -1 dilution. This dilution was divided in two halves: the first half was further diluted by adding 1 ml of the previous dilution to 9 ml of phosphate buffer and inoculating by triplicate in MYP agar as described above. The second half was heat treated at 80C for 60 seconds and then diluted and inoculated as done with the first half. The difference in growth between the first and second half is the number of spores present in the sample) Growth Assessment Tuna samples of the specie Tunnus Albacares (Yellow fin tuna) were obtained from Norwest Seafood in Gainesville, Fl. The samples were inoculated with a known concentration of B. cereus cells and incubated at 28C. Fifty grams of the sample were taken every 30 minutes during 6 hours and analyzed for B. cereus quantification by using Mannitol Egg Yolk Polimixin Agar as described in the FDA Bacteriological Analytical Manual (2001). 26

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The growth curve and the doubling time of B. cereus in tuna were determined by using the traditional equations: tNoNlnln (equation 1) 693.0td (equation 2) Where, N = Final amount of B. cereus colonies per gram of tuna after the period of time t No = Initial amount of B. cereus colonies per gram of tuna after the time t t= Time in hours in which the exponential growth part of the curve is clear. Td = Doubling time. Amount of time in hours that B. cereus needs in order to duplicate its number. = slope of the exponential logarithmical exponential growth curve. Bacillus Cereus Inactivation by Processing Methods Ultraviolet Light Processing Samples of Yellow Fin tuna were inoculated with known amounts of B. cereus. The inoculated samples received several doses of ultraviolet light irradiation (250 nm) and the amount of surviving B. cereus was measured by spread plating in MYP Agar as described above. The 250 nm of wavelength ultraviolet light source was a 20 watts lamp from a Type 3 safety cabinet from Fischer Scientific Inc., Miami, OK. The amount of ultraviolet radiation was measured using an Ultraviolet Light Meter (Professional Equipment Inc., Janesville, WI). Ozone Processing Yellow Fin tuna samples were inoculated with known levels of B. cereus vegetative cells and treated with different levels of ozone. The ozone generator was from Tersano Inc., Buffalo, New York. There were two groups of Yellow Fin tuna samples. The first group was inoculated 27

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with a known number of B. cereus vegetative cells and treated with several concentrations of ozone. The second group was inoculated with known amounts of B. cereus vegetative cells, frozen, and then treated with the same ozone concentrations used for the sample that was not frozen. In both cases, the number of surviving B. cereus was measured by spread plating on MYP agar as described above and reported in colony forming units per gram of tuna. Lactoperoxidase System Processing Yellow Fin tuna samples were inoculated with known amounts of B. cereus vegetative cells and treated in a one liter solution containing 100 l of a 1mg/ml solution of the enzyme lactoperoxidase, 20 l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. The samples were treated and analyzed after zero, fifteen, and thirty minutes of contact time. The number of B. cereus surviving were measured by spread plating on MYP agar and were reported in colony forming units per gram of tuna. Color Analyses Yellow Fin tuna samples were analyzed before and after each of the three treatments by using a Color Machine Vision system from the Department of Food Science and Human Nutrition at University of Florida, Gainesville, Florida. The L*(light-dark scale), a* (red-green scale), and b* (yellow-blue scale) values were recorded and analyzed looking for statistical differences. Statistical Analysis All experiments were run in triplicates. The means of microbial counts of all repetitions were calculated for the seasonality, spore assessment, and for all the treatments. All statistical analysis were run by using the Statistical Analysis System software SAS 9.0 from SAS Institute Inc., Cary, North Carolina, and significance was reported at levels of P value lower than 0.05. 28

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CHAPTER 3 RESULTS Seasonality and Spore Assessment In order to find the effect of the sea temperature in the occurrence of B. cereus in the species skip jack, yellow fin, and big eye ten samples of each species were taken every two months and analyzed for presence of B. cereus vegetative cells and spores. The sampling and analysis procedure are detailed in the materials and methods section. All the samples that tested positive, all the B. cereus vegetative cells were found in the surface of the tuna only. Figure 3-1 shows the seasonal occurrence of B. cereus in the most commonly used tuna species: skip jack (SJ), yellow fin (YF), and big eye (BE) 700 600 500 CFU/g SJ 400 YF 300 BE 200 100 0 March Decembe r January May A ugust October Figure 3-1. The seasonal occurrence of B. Cereus in the species skip jack (SJ), yellow fin (YF), and Big eye (BE) tuna Data in Figure 3-1 reveals that the highest levels of B. cereus are generally present in the yellow fin specie, except during the month of March, in which the big eye specie had the highest level of B. cereus. The data show a clear seasonality trend in B. cereus levels in the Skip Jack specie. 29

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The highest B. cereus levels for the Skip Jack specie occurred in the months of January and December, in which the water temperature is the highest of the year (about 32C). The coldest month of the yearfrom May to Augustshow the lowest amount of B. cereus in the Skip Jack specie. The Big Eye specie also shows a seasonality pattern similar to that of the Skip Jack specie. The highest values of B. cereus in the Big Eye specie were found in the month of Januarythe Big Eye specie was not sampled and analyzed in the month of Decemberand the lowest levels were also found between the months of May and August. The Yellow Fin specie did not show the same seasonality trend as the Skip Jack and Big Eye species did, however the highest levels of B. cereus were found in the months of January and December, and the lowest in the month of August. The individual bimonthly data are detailed in Tables 3-1 to 3-6 Table 3-1. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of January 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 9 310 136 a 0 Yellow Fin 6 633 175 a 0 Big Eye 9 544 305 a 0 Values with the same subscript a, b, or c are not significant different (P > 0.05) Table 3-1 shows the results for the month of January. All the samples were collected in the same location in the Ecuadorian sea. The average weight of the Skip Jack, Yellow fin, and Big Eye samples was 10 lb, 50 lb, and 30

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20 lb respectively. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species. Table 3-2. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of March 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown. Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 10 323 188 a 0 Yellow Fin 9 369 319 a 0 Big Eye 9 512 297 a 0 Values with the same subscript a, b, or c are not significant different (P > 0.05) Table 3-2 shows the results for the month of March. The average weight of the Skip Jack, Yellow fin, and Big Eye samples was 10 lb, 50 lb, and 20 lb respectively. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species Table 3-3. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of May 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 6 112 112 a 0 Yellow Fin 3 475 150 a 0 Big Eye 5 133 189 a 0 Values with the same subscript a, b, or c are not significant different (P > 0.05) Table 3-3 shows the results for the month of May. All the samples were collected in the same location in the Ecuadorian sea. The average weight of the Skip Jack, Yellow fin, and Big Eye samples was 10 lb, 50 lb, and 20 lb respectively. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species. 31

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Table 3-4. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of August 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown. Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 5 112 98 a 0 Yellow Fin 4 301 145 a 0 Big Eye 5 154 202 a 0 Values with the same subscript a, b, or c are not significant different (P > 0.05) Table 3-4 shows the results for the month of August. All the samples were collected in the same location in the Ecuadorian sea. The average weight of the Skip Jack, Yellow fin, and Big Eye samples was 10 lb, 50 lb, and 20 lb respectively. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species. Table 3-5. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of October 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 5 112 12 a 0 Yellow Fin 9 401 202 a 0 Big Eye 5 305 98 a 0 Values with the same subscript a, b, or c are not significant different (P > 0.05) Table 3-5 shows the results for the month of October. All the samples were collected in the same location in the Ecuadorian sea. The average weight of the Skip Jack, Yellow fin, and Big Eye samples was 10 lb, 50 lb, and 20 lb respectively. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species 32

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Table 3-6. Bacillus cereus occurrence in the Skip Jack, Yellow Fin, and Big Eye tuna species in the month of December 2006. All values are the means of the positive samples out of 10 samples expressed as colony forming units per gram of tuna. Standard deviations are also shown Specie Positive samples Average vegetative cells (cfu/g) Average spores Skip Jack 10 445 198 a 0 Yellow Fin 10 605 312 a 0 Big Eye NA* NA* NA* *NA = Not analyzed. Values with the same subscript a, b, or c are not significant Different (P > 0.05) Table 3-6 shows the results for the month of December. All the samples were collected in the same location in the Ecuadorian sea. The Skip Jack samples weighed 10 pounds on average, the Yellow Fin ones 50 pounds and the Big Eye ones 20 pounds. No significant difference (P > 0.05) was found among the levels of B. cereus in any of the species. Bacillus Cereus Growth Assessment in the Yellow Fin Tuna Specie. In order to determine the doubling time of B. cereus in Yellow Fin tuna under processing temperature (28C), samples of Yellow Fin tuna were inoculated with B. cereus and its growth at 28C was monitored every 30 minutes. Figure 3-2 shows the results at 28C. From the data point in Figure 3-2 the slope valueor can be obtained from the regression equation: = 0.34 hours -1 ; then the doubling time was calculated using the value: hourstd234.0693.0 This information can be used to calculate the time that B. cereus needs to increase from 10 2 to 10 4 CFU/gram by working equation 1 (see materials and methods section) for t: ht54.1334.010ln10ln24 33

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This result shows that if we have an initial population of B. cereus of 10 2 CFU/g it would take it 13.54 hours to reach the critical value of 10 4 CFU/g 8.0 7.0 6.0 Log 5.0 CFU Figure 3-2. Exponential growth of B. cereus in tuna at 28C. The regression equation and coefficient are also shown. Bacillus Cereus Inactivation Inactivation by Ultraviolet Light To evaluate the effects of ultraviolet light against B. cereus in Yellow Fin tuna, several Yellow Fin tuna samples were inoculated with known levels of B. cereus. These samples were then treated with several ultraviolet radiation doses and the surviving amount of B. cereus was measured in colony forming units per gram of Yellow Fin tuna. Table 3-7 shows the different ultraviolet radiation doses that were used in this study. Table 3-7. Bacillus cereus inactivation by Ultraviolet light in Yellow Fin tuna at 28C. Before Treatment After 8.5 mJ/cm 2 After 17 mJ/cm 2 7.04 a 5.85 b 5.48 b 6.36 a 4.92 b 4.60 b 7.98 a 4.70 b 3.0 b Values with the same subscript a, b, or c are not significant different (P > 0.05). All the values in the Table are in Log 10 of colony forming units of B. cereus per gram of Yellow Fin tuna. y = 0.3402x + 4.8973 3.0 4.0 = 0.9798 2 R 2.0 1.0 0.0 0.0 1.0 6.0 2.0 3.0 4.0 5.0 7.0 Time (hours) 34

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Data in Table 3-7 show that a significant (P < 0.05) reduction can be achieved by ultraviolet light. Table 3-7 also shows that more than 2 log reduction can be obtained by the ultraviolet light doses shown in the Table. There is no significant difference (P > 0.05) between treatments at 8.5 mJ/cm 2 and 17 mJ/cm 2 Figure 3-3 shows the progressive reduction of B. cereus vegetative cells in Yellow Fin tuna at 28C. 8 7 6 5 Log 4 CFU/g 3 2 1 0 0 Figure 3-3. Inactivation of B. cereus in Yellow Fin tuna by several doses of ultraviolet light. As we can see in Figure 3-3 as we plot the logarithm of the colony forming units per gram of tuna versus the UV doses, the plot follows a straight line pattern. Bacillus Cereus Inactivation by Ozone Yellow Fin tuna samples were inoculated with known amounts of B. cereus vegetative cells and treated with different concentrations of ozone. Table 3.8 shows the results of several concentrations treatments. Table 3-8. Reduction of B. cereus in Yellow Fin tuna at several ozone concentrations Ozone concentration (mg/L) Treatment time (min) Log reduction 0.3 30 0.138 a 0.4 30 0.45 b 0.5 30 0.98 c Values with the same subscript a, b, or c are not significant different (P > 0.05) 2 4 16 6 8 18 10 12 14 mJ/cm2 35

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Data in Table 3-8 shows that the log reduction increased as the ozone concentration increased. There is a significant difference (P < 0.05) between each of the treatments at the ozone concentrations specified in the Table Bacillus Cereus Inactivation by Lactoperoxidase System Yellow Fin tuna samples were inoculated with known amount of B. cereus vegetative cells and treated in a one liter solution containing 100 l of a one mg/ml solution of the enzyme lactoperoxidase, 20 l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. The samples were treated and analyzed after zero, fifteen, and thirty minutes of contact time. Table 3-9 shows the results of the treatment. Table 3-9. Reduction of B. cereus at several contact times by a solution containing 100 l of a one mg/ml solution of the enzyme lactoperoxidase, 20 l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN Contact time Initial count of B. cereus (Log 10 CFU/g) Final count of B. cereus (Log 10 CFU/g) Log reduction 15 min 5.08 a 4.7 a 0.4 b 30 min 5.08 a 4.2 a 0.87 b Values with the same subscript a, b, or c are not significant different (P > 0.05) Data in Table 3-9 shows the amount of log reduction that can be achieved by different contact times by using the lactoperoxidase system described above, however this reduction is not significant (P > 0.05). Also no significant difference (P > 0.05) was found among the two contact times. Color Analysis of Tuna after Each Treatment In order to evaluate the effects of each of the analyzed treatment on the color and visual appearance, each treated sample was analyzed by using a Machine Vision system, and the results were compared to non treated tuna. 36

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Color Analysis after UV Treatment Figure 3-4 shows photographs of typical tuna loins before and after being treated with the UV light doses described in Table 3-7 Figure 3-4. Photographs of typical tuna samples before (A) and after UV treatment (B) A B 37

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Color variations were analyzed by sampling the tuna before and after treatment with UV light. Table 3-10 shows the average results of the L*, a*, and b* values of the treated and non treated samples. Data in Table 3-10 shows that the a* values after the treatment are significantly higher (P < 0.05) than those of the untreated sample, suggesting a color closer to the brownish region. Table 3-10. Average results of the L*, a*, and b* values of the UV treated and non treated Yellow Fin tuna samples Before treatment UV treated L* 50.77 a 46.88 a A* 23.25 b 28.36 c B* 12.62 d 14.42 e Values with the same subscript a, b, c, d or e are not significant different (P > 0.05) The b* value also presented a significant difference (P < 0.05) among the treated and non treated samples whereas the L* value does not show significant difference (P > 0.05) among the treated and non treated samples. Color analysis after Ozone treatment Color variations were analyzed by sampling the tuna fillets before and after treatment with ozone. Tuna samples were treated with the ozone concentrations shown in Table 3-8 and the a*, L*, and b* values were measured. Table 3-11 shows the average results of the L*, a*, and b* values of the treated and non treated samples. Data in Table 3-11 shows that the a* values after the treatment are significantly higher (P < 0.05) than those of the untreated sample, suggesting a color closer to the brownish region. The L* and b* values do not show significant difference (P > 0.05) among the treated and non treated samples. 38

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Table 3-11. Average results of the L*, a*, and b* values of the ozone treated and non treated Yellow Fin tuna samples Before treatment Ozone treated L* 50.77 a 47.25 a a* 23.25 b 26.42 c b* 12.62 d 12.53 d Values with the same subscript a, b, c or d are not significant different Figure 3-5 shows photographs of typical tuna fillets before and after being treated with the ozone doses described in Table 3-8 Figure 3-5. Photographs of typical tuna samples before (A) and after ozone treatment (B) A B 39

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Color Analysis after Treatment with Lactoperoxidase System Figure 3-6 shows photographs of typical tuna fillets before and after being treated with the ozone doses described in Table 3-9 A B Figure 3-6. Photographs of typical tuna fillet samples before (A) and after lactoperoxidase system treatment (B) 40

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Color variations were analyzed by sampling the tuna before and after treatment with lactoperoxidase system and then tested by using a Machine Vision device to contrast those colors. Table 3-12 shows the average results of the L*, a*, and b* values of the treated and non treated samples. Table 3-12. Average results of the L*, a*, and b* values of the lactoperoxidase system treated and non treated Yellow Fin tuna samples No treatment Lactoperoxidase system treatment L* 50.77 a 47.32 a a* 23.25 b 30.07 c b* 12.62 d 15.53 e Values with the same subscript a, b, c, d or e are not significant different Data in Table 3-12 shows that the a* values after the treatment are significantly higher (P < 0.05) than those of the untreated sample, suggesting a color closer to the brownish region. The b* values of the tuna treated with lactoperoxidase system are also significantly different (P < 0.05) than those of the non treated tuna samples. Only the L* value does not show significant difference (P > 0.05) among the treated and non treated tuna samples. Plan HACCP Flow Chart and Critical Control Points for Processing of Tuna Figure 3-7 shows the flow chart for processing precooked tuna loins. As we can see only one critical control point can be detected. Table 3-13 shows the critical limits, monitoring actions, and corrective actions for the processing of tuna loins. Notice that tuna can also be processed on several other ways, being canned tuna the most processed tuna product in the world; however because of the high temperatures involved, B. cereus may not be a threat in canned tuna. 41

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42 Since only one critical limit was detected (histamine at reception), all the corrective action determined in the HACCP plan are related to the prevention of the occurrence of this hazard. Freezing Cold Stora g e Shipping Fish Receivin g Cold storage Thawin g Rackin g Butcherin g Precoockin g Coolin g Bag Filling/air re duc t i on Skinni g / Cleanin g Figure 3-7. Hazard Analysis and Critical Control Points flow chart Tuna Catching Freezing Shipping 10 hours, -10C 2 hours, 28C Shipping Freezing Catch/harvest 15 days, -10C 2 hours, -10C CCP 1 2 weeks, -15C 1 week, -15C 4 hours, -15C 10 minutes, 28C 1 hour, 28C 10 hours, 28C 2 hour, 60C 30 minutes, 2C 1 hour, 2C 10 hours, 2C 1 month, -10C

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43 Table 3-13. Precooked tuna loins HACCP summary Critical control point Hazard Critical limit(s) Monitoring Corrective actions Records Verification Fish receiving Chemical Histamine levels shall be less than FDA action level of 5 mg% (50ppm) (FDA 2001) Histamine levels of incoming analyze for loin lots. Collect samples and histamine content. Every loin lot. Quality control Reject loins with histamine levels in excess of 4.9 mg% (49 ppm) or divide into sub-lots of 25 TM max. ant test 60 fish for histamine. Reject sub-lot if any histamine greater than 4.9 mg% (49 ppm) (FDA 2001) Histamine analytical results Histamine sub-lots results Verify accuracy of histamine analytical results; Review records within one week

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CHAPTER 4 DISCUSSION Seasonality and Spore Assessment In Figure 3-1 there is a significant (P < 0.05) variation in the number of B. cereus vegetative cells in the months of May to August compared with the rest of the months in all the three species Skip Jack, Yellow Fin, and Big Eye tuna. The months from May, June, July and some days of August are colder in the region of Ecuador compared with the rest of the year. During this time of the year the temperature in the coast of Ecuador is around 20C which is more than 10C lower than the rest of the year. This lower temperature might have caused the number of vegetative cells of B. cereus to be lower in tuna, since they cannot multiply at the same rate they do at above 30C. Another hypothesis is that since during those months, the catching of tuna is not as abundant as the rest of the monthsprobably because many of them migrate to warmer waters, and some times the International Commission of Tuna bans the catching of tuna during those months to assure future sustainabilitytherefore the fishing vessels have lower amount of tuna being cough, so they can handle the tuna in a more quicker and efficient manner thus preventing the exposure of the catch to high temperatures during high periods of time before the tuna is placed in frozen storage. We can also notice from Table 3-1 that the highest amount of B. cereus vegetative cells are present in Yellow Fin tuna, followed by Big Eye tuna, and the last one is Skip Jack tuna. These three species of tuna do not differ much in their composition, and feeding and migrating habits; however one major difference between the three species is the average weight of the caught. The weigh of the Yellow Fin and Big Eye tuna species tend to be larger than the Skip Jack specie. Specifically, the average size of the sampled Yellow Fin tuna was 50 pounds, whereas the Big 44

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Eye tuna specie was 20 pounds, and for the Skip Jack one was 10 pounds. This size differences may have had an influence in the number of B. cereus vegetative cells present in the sample, giving a positive correlation between the amount of B. cereus vegetative cells present in a whole tuna and the original tuna size, since the bigger the tuna the higher the number of B. cereus vegetative cells. No spores were found in any of the samples analyzed. The absence of B. cereus spores may be because vegetative cells of B. cereus have sufficient nutrients and therefore do not need to sporulate in tuna. Another important fact to notice is that the highest level of B. cereus found was in the order of 10 2 colony forming units per gram of tuna, which is not enough to produce the toxin. The lowest level of B. cereus vegetative cells needed to produce the toxin is 10 4 (10,000) colony forming units per gram of food (Granun 2001). Even though those levels are not likely to be found in fresh tuna according to this research, time and temperature abuse may occur during processing, and levels of 10,000 or greater in might be reached in tuna. Growth Assessment From Figure 3-2 we can see that at 28 o C B. cereus needs approximately 2 hours to increase its population in 1 log in Yellow Fin tuna. In other words, since the seasonality data shows that the regular levels of B. cereus in tuna are in the 10 2 (100) levels of colony forming units per gram of Yellow Fin tuna, a time and temperature abuse of two hours at 28C may cause the B. cereus population increases to 10 4 in tuna and cause the illness. This is an important finding because some processors do not control their processing time and therefore the amount of B. cereus may increase in the production lines if they do not hold the tuna at the proper time-temperature while processing it. 45

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Figure 3-2 shows the growth of B. cereus in Yellow fin tuna at 28C. We can see that it is much slower in growth with a doubling time of 2 hours. This might be due to the complexity of the protein composition in Yellow Fin tuna, which forces B. cereus to use its proteolytic enzymes in order to obtain free amino acids that can be used by the bacteria as food. Yellow Fin tuna samples have a moisture contend of about 75% and a slightly acid pH of 6.5 as an average. These conditions are conducive for B. cereus enzymes to break down nutrients; however those conditions are not optimal Bacillus Cereus Inactivation and Color Analysis Ultraviolet Light As mentioned earlier, all the samples that were found to be contaminated with B. cereus were only contaminated in the surface, therefore any antibacterial port harvest treatment that affects the surface of the tuna may be successful in lowering the number of bacteria. In addition, the seasonality study revealed that no spores are found under regular catching and processing circumstances, so the post harvest antibacterial treatments were focused to reduce B. cereus vegetative cells only. Figure 3-3 shows the kinetics of inactivation of B. cereus vegetative cells in the surface of Yellow fin tuna. As we can see in Figure 3-3, the higher the dose of UV radiation, the higher the inactivation of B. cereus vegetative cells. There is a significant (P < 0.05) reduction in the number of B. cereus vegetative cells after applying UV treatment. The reduction may be because of the following reasons: Bacillus cereus is mostly present in the surface of the tuna in the early stages of the process Bacillus cereus is found in tuna on its vegetative stage only Tuna surface does not provide a significant protection against UV light, which is usually provided by fatty compounds. 46

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For these reasons the UV radiation can easily penetrate trough B. cereus membrane and cause DNA mutations induced through absorption of UV light by DNA molecule. Another important point is that UV light has a low penetration power, so it would not cause any important change in the tuna composition. However there may be an effect on the myoglobin composition of the tuna since a significant difference (P < 0.05) was found in the a* value of the UV treated tuna when compared to the non treated ones. Some authors have suggested that this phenomenon is due to the fact that UV radiation stimulates the formation of metmyoglobine which has a brownish color (Djenane and others 2001). Ozone Bacillus cereus vegetative cells were targeted in the surface of some Yellow Fin tuna samples. Data in table 3-8 shows that the higher the ozone concentration, the higher the level of inactivation of B. cereus in tuna. The reduction in the levels of this bacterium may be due to direct oxidation of B. cereus membrane and citoplasmatic compounds by molecular ozone and indirect oxidation by free radical species formed from the auto decomposition of ozone and its reaction with some tuna compounds as described in the review of the literature (Hunt and Marinas 1997). One of the main problems found when treating tuna with ozone was the oxidation of some pigmented compounds in the tuna. This caused a significant (P < 0.05) variation in the a* value probably due to oxidation reaction on the myoglobine molecules in the tuna leading to the production of metmyoglobin. Lactoperoxidase System The effect of lactoperoxidase system was found to be not significant (P > 0.05) when compared with untreated controls. The lack of effect of this treatment may be due to the fact that 47

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some bacteria use compounds in the food surface to reduce the contact area and provide protection against lactoperoxidase antimicrobial products as suggested by Min and others (2005). Color changes in the tuna samples were also noticed, due to the oxidation reactions of the hydrogen peroxide which is one of the components of the lactoperoxidase system. A significant difference (P < 0.05) was found in the a* value of the sample treated with lactoperoxidase system compared to tuna samples that were not treated. 48

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CHAPTER 5 SUMMARY AND CONCLUSIONS Concerns about the presence of B. cereus in tuna can be supported by this research since the seasonal results show that the average levels of B. cereus in tuna can be as high as 63 x 10 1 CFU/g of tuna especially in the months of January and December. The Yellow Fin and Big Eye species were the ones that presented the highest levels of B. cereus probably because they are usually bigger in size and weight than the Skip Jack specie. Even though the likelihood of finding B. cereus in tuna is very high, the levels found do not represent any threat to human health, since they were below 10 4 CFU/g which is the lowest amount of B. cereus needed to produce intoxication. For this reason FDA suggests a holding time at room temperature of 3 hours or less, so the B. cereus levels are kept below this value. No spores of B. cereus in tuna were found in a one year period, suggesting that B. cereus has all the nutrients they need in order to stay in a vegetative stage. For this reason all efforts to reduce B. cereus in tuna should be directed to the vegetative stage only. In the second part of this research the doubling time was determined for B. cereus in tuna at room temperature (28C). The doubling time obtained was 2 hours and the value 0.34 h -1 This allows us to calculate the actual amount of time that processors can hold the tuna before it reaches the critical B. cereus level of 10 4 CFU/gram of tuna. The calculated time (assuming a starting level of 10 2 CFU/g) is 13.5 hours. This shows that the 3 hours limit may actually be underestimated. The third part of the research was aimed to find a method to reduce the levels of B. cereus in tuna with the least change in the color of the tuna. The processing methods used in this study were non thermal, since the final tuna product is intended for raw or precook usages. 49

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The first processing method we tested was ultraviolet light. Ultraviolet light is known to be effective when the microorganism is mainly present in the surface of the product (which is the case of B. cereus in tuna). Several ultraviolet doses (doses between 8.5 mJ/cm 2 and 17 mJ/cm 2 were used) were tested producing significant (P < 0.05) reduction in the levels of B. cereus in tuna. The doses needed to produce this effect were achieved by a regular ultraviolet light lamp from a type 2 laminar flow cabinet. This suggests that the dosages needed are too low, so it would be something of low cost to apply in a bigger industrial scale. Even though UV light achieved a significant log reduction in the levels of B. cereus in tuna, there is an effect on the myoglobin composition of the tuna since a significant difference (P < 0.05) was found in the a* value of the UV treated tuna when compared to the non treated ones. The second processing method we tested was ozone. Relatively low ozone concentrations (between 0.1 and 0.5 ppm of ozone) were tested yielding to significant (P < 0.05) log reduction of B. cereus in tuna. Ozone is known to cause oxidation in several cytoplasmatic compounds of B. cereus. One of the main problems found when treating tuna with ozone was the oxidation of some pigmented compounds in the tuna. This caused a significant (P < 0.05) variation in the a* value probably due to oxidation reaction on the myoglobine molecules in the tuna leading to the production of metmyoglobin. The third processing method used was the lactoperoxidase system. Lactoperoxidase is an enzyme that takes hydrogen peroxide and some tyocianates to produce tyocianic acid, which is further broken into bactericide compounds. Yellow Fin tuna samples were inoculated with known amount of B. cereus vegetative cells and treated in a one liter solution containing 100 l of a one mg/ml solution of the enzyme lactoperoxidase, 20 l of a 30% hydrogen peroxide solution, and 1.8 grams of KSCN. No significant (P > 0.05) log reduction was observed in the 50

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levels of B. cereus in tuna. Color changes in the tuna samples were also noticed, due to the oxidation reactions of the hydrogen peroxide which is one of the components of the lactoperoxidase system. A significant difference (P < 0.05) was found in the a* value of the sample treated with lactoperoxidase system compared to tuna samples that were not treated. In conclusion, treating tuna with moderate doses of UV light (doses between 8.5 mJ/cm 2 and 17 mJ/cm 2 are recommended) is effective in reducing the levels of B. cereus in tuna, however it causes a small change in the a* value. Treating tuna with concentrations above 0.5 ppm of ozone is also effective in reducing the levels of B. cereus in tuna but a small change in the a* value is also observed. Further research need to be done to find if this significant (P < 0.05) changes in the color of the tuna are actually perceivable by the human eye. 51

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APPENDIX SEASONALITY AND SPORE ASSESSMENT RAW DATA Table A-1. Raw data for the seasonal analysis for the month of January 2006. Sample Skip Jack Yellow Fin Big Eye 1 269 429 406 2 342 649 599 3 427 905 824 4 66 455 133 5 360 704 647 6 344 656 605 7 445 TFTC 872 8 119 TFTC 8 9 418 TFTC 801 10 TFTC TFTC TFTC Average 310 633 544 STD 135 175 305 TFTC: Too few to count. All values are in CFU/g Table A-2. Raw data for the seasonal analysis for the month of March 2006. Sample Skip Jack Yellow Fin Big Eye 1 297 930 289 2 392 108 663 3 504 207 167 4 82 809 537 5 416 566 728 6 395 266 671 7 528 49 177 8 100 221 319 9 493 164 1057 10 23 TFTC TFTC Average 323 369 512 STD 189 320 298 TFTC: Too few to count. All values are in CFU/g 52

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Table A-3. Raw data for the seasonal analysis for the month of May 2006. Sample Skip Jack Yellow Fin Big Eye 1 75 338 25 2 199 451 129 3 298 636 461 4 25 TFTC 25 5 49 TFTC 25 6 25 TFTC TFTC 7 TFTC TFTC TFTC 8 TFTC TFTC TFTC 9 TFTC TFTC TFTC 10 TFTC TFTC TFTC Average 112 475 133 STD 112 150 189 TFTC: Too few to count. All values are in CFU/g Table A-4. Raw data for the seasonal analysis for the month of August 2006. Sample Skip Jack Yellow Fin Big Eye 1 25 287 25 2 109 359 54 3 262 450 500 4 25 108 25 5 139 TFTC 166 6 TFTC TFTC TFTC 7 TFTC TFTC TFTC 8 TFTC TFTC TFTC 9 TFTC TFTC TFTC 10 TFTC TFTC TFTC Average 112 301 154 STD 98 145 202 TFTC: Too few to count. All values are in CFU/g 53

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Table A-5. Raw data for the seasonal analysis for the month of October 2006. Sample Skip Jack Yellow Fin Big Eye 1 129 323 301 2 114 443 333 3 95 582 401 4 112 84 142 5 110 473 348 6 TFTC 447 TFTC 7 TFTC 612 TFTC 8 TFTC 77 TFTC 9 TFTC 568 TFTC 10 TFTC TFTC TFTC Average 112 401 305 STD 12 202 98 TFTC: Too few to count. All values are in CFU/g Table A-6. Raw data for the seasonal analysis for the month of December 2006. All values are in CFU/g Sample Skip Jack Yellow Fin 1 350 463 2 482 668 3 636 908 4 427 593 5 515 720 6 486 674 7 669 960 8 240 156 9 620 884 10 25 25 Average 445 605 STD 198 312 All values are in CFU/g 54

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LIST OF REFERENCES Agata, N.; Ohta, M.; Arakawa, Y., and Mori, M. 1995. The bceT gene of Bacillus cereus encodes an enterotoxic protein. Microbiology 141(4):983-988. Agata, N.; Ohta, M.; Mori, M., and Isobe, M. 1995. A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiology Letters 129:17-20. Benoit T, Wilson G, Bull D, Aronson A. 1990. Plasmid-Associated Sensitivity of Bacillus thuringiensis to UV Light. App Env Mic 56: 2282 2286 Broadwater W, Hoehn R, Kimng P 1973. Sensitivity of Three Selected Bacterial Species to Ozone. J of App Mic 26: 391-393 Food and Agriculture Organization. 1998. Seafood Safety-Economics of Hazard Analysis and Critical Control Point (HACCP) programmes. 69p Food and Drug Administration, 2000. Kinetics of Microbial Inactivation for Alternative Food Processing Technologies : Ultraviolet Light. http://www.cfsan.fda.gov Food and Drug Administration 2001. Bacteriological Analytical Manual Online. http://www.cfsan.fda.gov Food and Drug Administration. 2001. Fish and Fisheries Products Hazard and Control Guidance. 326p Foodmarket Exchange 2003. Tuna Consumption. http://www.foodmarketexchange.com Djenane D., Sanchez A., Beltran J, Proncales P. 2001. Extension of the Retail Display Life of Fresh Beef Packaged in Modified Atmosphere by Varying Lighting Conditions. J of Food Sci 66: 181 186 Drobniewski, F. A. 1993. Bacillus cereus and related species. Clinical Mic Rev 6(4):324-38. Granum E. 2001. Bacillus Cereus. In: Doyle M. Food Microbiology: Fundamentals and Frontiers. Washington, D.C.: ASM Press. p 373-381 Holbrook, R. and Anderson, J. M. 1980. An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Can J of Mic 26:753-759. Hunt N, Marinas B 1997. Kinetics of E coli Inactivation with Ozone. Wat Res 31: 1355-1362 Kramer, J. M. and Gilbert, R. J. 1989. Bacillus cereus and other Bacillus species. Pages 21-70. Doyle, M. P., ed. Foodborne bacterial pathogens. Marcel Dekker Inc., New York. Kussendrager K, van Hooijdonk A. 2000. Lactoperoxidase: physico-chemical properties, occurrence,mechanism of action and applications. Br J Nutr. 84: S19-S25 55

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LM Wijnands, JB Dufrenne, FM van Leusden 2002. Characterization of Bacillus Cereus. RIVM report 250912002/2002. Marquez V, Mittal, G, Grifiths M. 1997. Destruction and inhibition of bacterial spores by high voltage electric field. Journal of Food Science, 62, 399_401, 409. Min S, Harris L, Krochta J. 2005. Listeria monocytogenes Inhibition by Whey Protein Films and Coatings Incorporating the Lactoperoxidase System. J of Food Sci. 70: M317-M324 Mossel, D. A.; Koopman, M. J., and Jongerius, E. 1967. Enumeration of Bacillus cereus in foods. App Mic 15:650-653. National Restaurant Association. 2004. Serv Safe Essentials. Chicago, IL: Serv Safe Notermans S, Batt C. 1998. A Risk Assessment Approach for Food-Borne Bacillus Cereus and its Toxins. J of App Mic 84: 51s-61s Sale A. and Hamilton W.1967. Effect of high electric fields on icroorganisms. I. Killing of bacteria and yeast. Biochimica et Biophysica Acta, 48, 781_788. Smoot L, Pierson M. 1981. Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination. App Envir Microb 42(3): 477-483 Tenouvo J, Makinen K, Sievers G. 1985. Antibacterial Effect of Lactoperoxidase and Myeloperoxidase Against Bacillus cereus. Antimic Agents and Chem 27: 96-101 Vries Y 2006. Bacillus cereus spore formation, structure, and germination. Thesis Wageningen University, Wageningen, the Netherlands Wikipedia Online 2007. Tuna. http://en.wikipedia.org/wiki/Tuna. 56

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BIOGRAPHICAL SKETCH Juan Manuel Cevallos was born in Manta, Ecuador on August 24 th 1981. He is the sixth of seven children born to Eddie Cevallos and Esperanza Cevallos. He attended Julio Pierregrosse elementary and high school and graduated with honors being the best student graduating in 1999. He was admitted to do an undergrad program in food engineering and received a full tuition waiver at the Escuela Superior Politecnica del Litoral in Guayaquil, Ecuador. He graduated with honors being the best student graduating with a food engineering degree in 2004. In early 2004 he started working in the quality assurance department for one of the biggest tuna processing companies in Ecuador: Sociedad Ecuatoriana de Alimentos y Frigorificos Manta C.A. (SEAFMAN C.A.) In 2005 he was awarded a Fulbright fellowship to attend University of Florida to pursue a Masters degree in food science. At the same time he pursued a masters degree in agribusiness. He was awarded both degrees in 2007 and plans to begin a Ph.D. program in food science at the University of Florida. 57