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Evaluation of oxygen transmission rate of packaging films on growth of clostridium sporogenes and media oxidation reduct...

University of Florida Institutional Repository

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EVALUATION OF OXYGEN TRANSMISSI ON RATE OF PACKAGING FILMS ON GROWTH OF CLOSTRIDIUM SPOROGENES AND MEDIA OXIDATION REDUCTION POTENTIAL IN PACKAGED SEAFOOD SIMULATING MEDIA By JAYASHREE GNANARAJ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ENGINEERING UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Jayashree Gnanaraj

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This thesis is dedicated to my parents a nd my brother who have always supported and encouraged me from near and afar.

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iv ACKNOWLEDGMENTS I am grateful to Dr. Bruce A. Welt, my advisor, supervisor and mentor who taught me more than I hoped to learn here at gr aduate school, without whose support this research work would not have been possible. His work has been my inspiration. This work has been a product of his patience and endu rance. He has inspired me to be a better researcher and also a better person. He understood my problems and helped me to succeed inspite of them. My success is and will be a reflection of his outstanding abilities as a teacher. Nothing short of this will be adequate to express my gratitude to him. I would like to thank Dr. Art A. Teixei ra and Dr. Hordur G. Kristinsson for agreeing to serve on my committee, guiding me and always ready to help. I would like to thank Dr. Steven Otwell for his suggestions I would like to tha nk National Fisheries Institute and Florida Sea Grant for financial assistance without whic h this project would not have been completed. This paper is also result of enduring support and love and cooperation of my parents, Mrs. and Mr. Gnanaraj. I would like to thank my brother Sriram for being there for me. My family members have given me st rength for what I started. I am indebted to them for being there as unsh akeable pillars of support. This thesis is incomplete without acknowledging my friends in Gainesville. Special thanks go to Bob, Billy, Dhuruva, Ralph, Teresa and Vivek. Most of all I would like to thank the faculty and staff in Department of Agriculture and Biological Engineering.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 FDA Alert.....................................................................................................................1 Food-Borne Botulism...................................................................................................2 Significance of Clostridium botulinum..........................................................2 Conducive conditions for growth of C. botulinum........................................3 Reduced Oxygen Packaging.........................................................................................3 Packaging of Horticultural Products..............................................................4 Packaging of Flesh Foods..............................................................................5 Dynamic nature of atmosphere in ROP packaged flesh foods.....................................5 Research Hypothesis.....................................................................................................6 2 EFFECT OF TEMPERATURE AND RELATIVE HUMIDITY ON FILM PERMEABILITY.........................................................................................................8 Materials and Method...................................................................................................9 Results and Discussion...............................................................................................13 3 EFFECT OF FILM OTR, PACKAGE AREA AND TEMPERATURE ON CLOSTRIDIUM SPOROGENES SPORE OUTGROWTH........................................18 Materials and Method.................................................................................................20 Results and Discussion...............................................................................................25 4 CONCLUSION AND FUTURE WORK...................................................................34 Conclusion..................................................................................................................34 Future Work................................................................................................................35

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vi APPENDIX A OXYGEN TRANSMISSION RATE OF PACKAGING FILMS AT DIFFERENT TEMPERATURES AND RELATIVE HUMIDITY..................................................36 B DIGITAL PICTURES OF SPORE OUTGROWTH IN DIFFERENT FILMS AND BAG SIZES.......................................................................................................39 C OXIDATION REDUCTION POTENTIA L WITHOUT PH COMPENSATION...150 LIST OF REFERENCES.................................................................................................152 BIOGRAPHICAL SKETCH...........................................................................................155

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vii LIST OF TABLES Table page 2-1 Film description........................................................................................................10 2-2 Oxygen transmission rates of different f ilms measured at different temperature and relative humidity................................................................................................13 2-3 Comparison of measured oxygen transm ission rates with value reported by manufacturer.............................................................................................................13 2-4 Ea and k0 values for Arrhenius relationship between OTR and temperature for the packaging films at 0% RH.......................................................................................16 2-5 Ea and k0 values for Arrhenius relationship between OTR and temperature for the packaging films at 50% RH.....................................................................................16 3-1 OTR of film used in this study.................................................................................21 3-2 Spore outgrowth over time in regular me dia for various film types at various temperatures.............................................................................................................26 3-3 Spore outgrowth over time in anaerobic media for various film types at various temperatures.............................................................................................................28 3-4 Oxidation reduction poten tial of highly reduced anaerobic media in bags of various film types at different temperatures.............................................................30 A-1 OTR of packaging films at 0% RH..........................................................................37 A-2 OTR of packaging films at 50% RH........................................................................38 B-1 Growth table for C60 8X8 at 15 C Anaerobic Media..............................................40 B-2 Growth table for AET 8x8 at 15 C Anaerobic Media..............................................44 B-3 Growth table for BDF 8x8 at 15 C Anaerobic Media..............................................47 B-4 Growth table for C60 18X14 at 15 C Anaerobic Media..........................................51 B-5 Growth table for AET 18X14 at 15 C Anaerobic Media.........................................55

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viii B-6 Growth table for BDF 18X14 at 15 C Anaerobic Media.........................................59 B-7 Growth table for C60 8X8 at 23 C Anaerobic Media..............................................62 B-8 Growth table for AET 8x8 at 23 C Anaerobic Media..............................................63 B-10 Growth table for C60 18x14 at 23 C Anaerobic Media...........................................65 B-11 Growth table for AET 18x14 at 23 C Anaerobic Media..........................................66 B-12 Growth table for BDF 18x14 at 23 C Anaerobic Media..........................................67 B-13 Growth table for C60 8x8 at 30 C Anaerobic Media...............................................68 B-14 Growth table for AET 8x8 at 30 C Anaerobic Media..............................................69 B-15 Growth table for BDF 8x8 at 30 C Anaerobic Media..............................................70 B-16 Growth table for C60 18x14 at 30 C Anaerobic Media...........................................71 B-17 Growth table for AET 18x14 at 35 C Anaerobic Media..........................................72 B-18 Growth table for BDF 18x14 at 35 C Anaerobic Media..........................................73 B-19 Growth table for C60 8x8 at 35 C Anaerobic Media...............................................74 B-20 Growth table for AET 8x8 at 35 C Anaerobic Media..............................................75 B-21 Growth table for BDF 8x8 at 35 C Anaerobic Media..............................................75 B-22 Growth table for C60 18x14 at 35 C Anaerobic Media...........................................76 B-23 Growth table for AET 18x14 at 35 C Anaerobic Media..........................................76 B-24 Growth table for BDF 18x14 at 35 C Anaerobic Media..........................................77 B-25 Growth table for C60 8x8 at 15 C Regular Media...................................................78 B-26 Growth table for AET 8x8 at 15 C Regular Media..................................................81 B-28 Growth table for C60 18x14 at 15 C Regular Media...............................................87 B-29 Growth table for AET 18x14 at 15 C Regular Media..............................................90 B-30 Growth table for BDF 18x14 at 15 C Regular Media..............................................93 B-31 Growth table for C60 8x8 at 20 C Regular Media...................................................96 B-32 Growth table for AET 8x8 at 20 C Regular Media..................................................99

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ix B-33 Growth table for BDF 8x8 at 20 C Regular Media................................................102 B-34 Growth table for C60 18x14 at 20 C Regular Media.............................................105 B-35 Growth table for AET 18x14 at 20 C Regular Media............................................108 B-36 Growth table for BDF 18x14 at 20 C Regular Media............................................111 B-37 Growth table for C60 8x8 at 30 C Regular Media.................................................114 B-38 Growth table for AET 8x8 at 30 C Regular Media................................................117 B-39 Growth table for BDF 8x8 at 30 C Regular Media................................................120 B-40 Growth table for C60 18x14 at 30 C Regular Media.............................................123 B-41 Growth table for AET 18x14 at 30 C Regular Media............................................126 B-42 Growth table for BDF 18x14 at 30 C Regular Media............................................129 B-43 Growth table for C60 8x8 at 35 C Regular Media.................................................132 B-44 Growth table for AET 8x8 at 35 C Regular Media................................................135 B-45 Growth table for BDF 8x8 at 35 C Regular Media................................................138 B-46 Growth table for C60 18x14 at 35 C Regular Media.............................................141 B-47 Growth table for AET 18x14 at 35 C Regular Media............................................144 B-48 Growth table for BDF 18x14 at 35 C Regular Media............................................147 C-1 Redox potential values wit hout compensating for pH 7...........................................151

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x LIST OF FIGURES Figure page 2-1 Mocon Oxtran 2/20..................................................................................................10 2-2 Film cutting template...............................................................................................11 2-3 A diagram representing gas flow th rough films inside MOCON instrument..........12 2-4 Comparison of 0% and 50% RH of C60..................................................................14 2-5 Arrhenius relationship between OTR and temperature at 0% RH...........................15 2-6 Arrhenius relationship between OTR and temperature at 50% RH.........................15 2-7 Comparison of PE, C60 and C75 FTIR spectra.......................................................17 3-1 Sample of bag sizes used for the experiment...........................................................22 3-2 Rack arrangement inside the chamber.....................................................................22 3-3 Back lighted stand used for taking digital pictures..................................................23 3-4 Fiber optic oxygen sensor system............................................................................23 3-5 Oxygen sampling inside the bag..............................................................................24 3-6 Equipment used to measure ORP and pH................................................................25 3.7 Control plate at 30 C inside anaerobic box..............................................................28 3-8 Headspace oxygen content over time in film types C60 and BDF at 23 C.............31 3-9 Dissolved oxygen content over tim e in highly reduced media at 23 C for film types C60 and BDF..................................................................................................32 3-10 Dissolved oxygen content over tim e in highly reduced media at 35 C for film types C60 and BDF..................................................................................................32

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Engineering EVALUATION OF OXYGEN TRANSMISSI ON RATE OF PACKAGING FILMS ON GROWTH OF CLOSTRIDIUM SPOROGENES AND MEDIA OXIDATION REDUCTION POTENTIAL IN PACKAGED SEAFOOD SIMULATING MEDIA By Jayashree Gnanaraj August 2003 Chair: Dr. Bruce A. Welt Major Department: Agricultur al and Biological Engineering Studies with packaged fish have shown that obvious spoilage can be delayed by removing oxygen. However, anaerobic pathogenic Clostridium botulinum may thrive in reduced oxygen packaging, causing packaged fish to become toxic prior to obvious spoilage. In an attempt to mitigate devel opment of reduced oxygen atmospheres within fresh seafood packaging, FDA has specified a minimum oxygen transmission rate (OTR) for seafood packaging films of 10,000 cc/m2/day at 24 C. However, this specification does not take the actual package design into consideration. It is suspected that a specification that combines film OTR with desc riptive parameters of the package, such as film area, may offer a better structure for sp ecification. Additionally, while it is generally accepted that C. botulinum is an obligate anaerobe, it re mains unclear if a particular concentration of oxygen is capable of preventing toxigenesis. Like C. botulinum C. sporogenes is an obligate anaerobe but nonpathogenic so it was used as a surrogate for C. botulinum in this study.

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xii The objective of this work was to devel op a scientific rationa le for a new seafood package OTR specification, and to study the re lationships among film OTR, package area and storage temperature on C.sporogenes spore outgrowth in regular and anaerobic media. Commercially available packaging films w ith a wide range of OTR were used in the study. OTR as a function of temperatur e was determined in the range of 10-35 C at 0% and 50% relative humidity (RH). Films were converted into packages with areas of 8x8 and 18x14 inches. Inoculated petri dishes were sealed in these packages using multiple vacuum/ nitrogen gas flush cycles. Inoculated packages were incubated at 10, 15, 20, 30 and 35 C. Dynamic oxygen concentrations we re measured in packaged media and package headspace. Oxidation reduction po tentials (ORP) of me dia were measured before and after incubation. As expected, oxygen levels in high OTR film s increased quickly to an approximate level of 12% O2. Oxidation reduction potentials tended to become more positive with rising oxygen levels, suggesting that sample ORP plays an im portant role in predicting potential outgrowth of spores. Results suggest that a critical parameter for inhibiting outgrowth is the time required to raise oxygen concentration sufficien tly to increase ORP above some critical value. It was found that package area, within a practical range of package dimensions, is not sufficiently important to provide an avenue for modifying FDAs OTR guideline. Since film OTR plays a key role in this proce ss, this parameter may continue to offer the most convenient approach toward ensuring safety of fresh seafood.

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1 CHAPTER 1 INTRODUCTION Limited availability and increased trans portation of raw fish and seafood make it important to minimize losses. Improved mana gement and food preservation technology are needed because trends show increased interest in minimally preserved products (Gould, 1996). Annual landed seafood in Florida was estimated to be over $200 million (Welt et al., 2003). Fresh pre-prepared seaf ood items like sushi, raw oysters and clams and use of fish as a substitute for meat ha ve been instrumental in making fish/seafood an everyday alternative. Seafood menu mentions for entrees were up 10.2% over previous year in 2000, growing more than any ot her center-of-the-plate category, including chicken and beef (Sloan, 2000). Determination and prediction of shelf life of fresh fish and lightly preserved seafood has become partic ularly important to prevent losses due to spoilage. FDA Alert Specifics of Alert Section 402 (a) (4) of Food, Drug and Cosme tic Act considers refrigerated fresh fish stored under reduced oxygen conditions such as modified atmosphere packaging (MAP) and vacuum packaging (VP) as adulterated when no controls for Clostridium botulinum toxin liberation are em ployed. FDA issued an import alert which states “Detention without physical exam ination of refrigerated products (not frozen) vacuum packaged or modified atmosphere packaged raw fish and fishery products due to the potential for C. botulinum toxin production” (FDA, 2002).

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2 This alert affects 4100 U.S seafood proce ssors, most of which are small scale businesses responsible for processing over 350 species of fish. The alert also affects foreign seafood processors and U.S seafood im porters. Overall fina ncial impacts caused by these regulations are estimated to be mo re than $1 million per year (Otwell, 2002). FDA identifies the following two ways to package unfrozen fish products safely: Use of packaging film with a minimum OTR of 10,000 cc/m2/day. An indicator can be used in or on the p ackaging to show that the product has not been exposed to time and temperature combination that could result in an unsafe product between the time of packaging and the time of use by the consumer. Food-Borne Botulism Food borne botulism is a seve re type of food poisoning due to ingestion of foods containing potent neurotoxin produced by Clostridium botulinum. Intoxication occurs when toxin enters the body and di rectly affects bodily functions Symptoms of this progressive paralytic disease begin with numbness in the extremities and double vision Death is often slow and typically results from suffocation as control of respiration fails. Though incidence of food borne botulism is lo w, it remains a considerable food safety concern because of high mortality rates. Significance of Clostridium botulinum C. botulinum is a food pathogen that is commo n in the natural environment, particularly in soil and marine and freshwater sediments. This organism is so ubiquitous that it is not possible to exclude it from foods. C. botulinum is a rod shaped gram positive, anaerobic bacteria capable of forming heat resistant spores that withstand long periods of dryness and fairly severe thermal tr eatments. Seven (A, B, C, D, E, F and G) strains are recognized based on their antigenic specificity of toxin. Strains causing human botulism include types (A, B, E and F), while botulism from types C and D occurs in

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3 animals. Given favorable conditions, this organi sm produces a heat labi le neurotoxin that can be destroyed by boiling for 10 minutes or longer (Sumner et al., 1995). An extremely small amount of toxin (few nanograms) has b een shown to be capable of causing illness. In 1987, eight cases of type E botulism that o ccurred due to the consumption of dry salted whole uneviscerated fish (FDA/CFSAN, 1992). Conducive Conditions for Growth of C. botulinum Botulism has been associated with Inadequately processed home canned foods. Foods with water phase salt concentra tions less than 5% (water activity, aW, of 0.97). Almost any type of food that is not very acidic (pH above 4.6) Sausages, meat products, canned vegetabl es and seafood products have been the most frequent vehicles fo r botulism (FDA/CFSAN, 1992). Reduced Oxygen Packaging Altering atmospheres within food packages to extend shelf life is a method of food preservation. Reduced oxygen packaging (R OP) contains little or no oxygen. FDA defines ROP as any package that when sealed, has the potential to result in an internal atmosphere that contains lower concen tration of oxygen than standard ambient conditions. Cook-chill, controlled atmosphere packaging (CAP), modified atmosphere packaging (MAP), sous vide and vacuum packaging (VP) fall under ROP category. Advantages of Reduced Oxygen Packaging Advantages of ROP include Prevents growth of aerobic spoilage micro organisms such as pseudomonas, aerobic yeast and molds which are often responsible for organoleptic spoilage. Shelf-life extension.

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4 Inhibition of oxidative processe s that degrade food quality. Prevents color deterioration in raw m eats during storage and retail display. Reduces product shrinkage by preventing water loss (FDA, 1997). Trends and Rationale for Vacuum Packagi ng and Modified Atmosphere Packaging The principle involved in VP is removal of gases from a package. MAP involves methods to maintain a specific gaseous atmosp here within the packag e that is different from standard atmospheric conditions. MAP in conjunction with refr igeration has been shown to increase shelf life of many types of foods. MAP offers several potential advantages to the seafood industry, including Possibility of centralized production. Reduced economic loss by preventing quality degradation. Increased distribution efficiency due to standardized packaging. Potential shelf-life increases of 50 to 400% (Farber, 1991). Relationship between packaging film perm eability to food safety and quality Ability to establish and maintain a specifi c atmosphere in MAP packaging depends on gas permeation characteristics of the pack aging films particularly with respect to oxygen and carbon dioxide. Packaging of Horticultural Products When applying MAP to horticultural products like fruits and vegetables, it is often desired to maintain low oxygen levels a nd relatively high carbon dioxide levels (Robertson, 1992). Such conditions tend to slow product respiration resulting in extended shelf life. To achieve specific modified at mospheres, a delicate balance between film permeation and product respiration must be es tablished. When this balance is violated, either due to improper packaging films or abusive temperatures, anoxic conditions can

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5 develop which results in rapid product quality loss. As a resu lt of these considerations, highly permeable films are typically used in such applications. Packaging of Muscle Foods Important properties to be considered during packaging of muscle foods are product color and microbial population. Although oxygen may be harmful to red meat product, it is essential for development of th e bright red color that consumer’s desire. Since packaged flesh foods do not respire, MA P of such foods typically involves flushing packages with a specific atmosphere prior to sealing. Use of hi gh barrier films (low permeability) are intended to “trap” injected gases in the package. The primary gases involved are oxygen, carbon dioxide and nitrogen. These pack aging techniques typically utilize high barrier films in an attempt to trap modified atmospheres within package. Dynamic Nature of Atmosphere in ROP Packaged Muscle Foods Flesh foods spoil through the combined e ffects of chemical reaction, biochemical reactions (enzyme activity) and microbial gr owth. These reactions typically consume oxygen, which can lead to anaerobic conditions inside the package. This often leads to progression of microbial activ ity from aerobic to facultative anaerobe to obligate anaerobic. There is a possibility of C. botulinum producing neurotoxin under favorable conditions which may render foods toxic prior to visible signs of organoleptic spoilage. Potential Control for ROP Fish The National Advisory Committee for Microbiological Criteria for Foods recommended temperature control below 3.3 C as a primary preventive measure against C. botulinum growth. However, temperature abuse of 7-10 C is encountered by the product in retail and distribution chain (NACMCF, 1991). National Food Processors

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6 Association (NFPA) has recommended that ther e be a secondary safety control for foods that are packaged in reduced oxygen atmos pheres and offered at retail (NFPA, 1989). Recently FDA has put forward following control guidelines for ROP seafood. Packaging material has a perm eability of more than 10,000 cc/m2/day at 24 C Water phase salt level is at least 5% Water activity (aW) is below 0.97 pH is 5.0 or less Time temperature integrators (FDA, 2002) Any one hurdle, or a combination of seve ral, may be used to control pathogenic outgrowth. It is important to note that the motiva tion of the recent FDA alerts was not to control toxigenesis, but to ensu re normal rapid aerobic spoila ge so that toxigenesis does not precede organoleptic spoilage. Potential Weakness in FDA’s OTR Specification FDA’s specific interpretation of ROP covers all unfrozen seafood in any hermetically sealed pack age with oxygen transmissi on rate less than 10,000 cc/m2/day. This results in different absolute oxy gen transmission rate s in terms of cc O2/package/day for packages with different films areas. A question arises as to whether an improved regulation based on whole package area (cc/pack age/day) might provide better safety for ROP fish. Such a specification would extend th e flexibility of packaging film selection and allow manufacturers to choose any packaging film, provided that sufficient film area is used to achieve a minimum absolute oxygen transmission rate into packages. Research Hypothesis The hypothesis of this study is that Clostridium sporogenes spores will germinate and grow sooner and more robustly in packages with less film area than those with more

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7 film area for any given film. To test th is hypothesis a design of experiments were conducted in two parts in this project with the following specific objectives : Part I objectives (addressed in Chapter 2) were to Determine oxygen transmission rate (OTR) of commercially available packaging films Study the effect of temperature and relative humidity on OTR. Part II objectives (addressed in Chapter 3) were to measure Time required to observe visible colonies in inoculated regular and anaerobic (highly reduced) seafood simula ting bacterial media when packaged with different areas and incubated at different temperatures. Dynamic oxygen profiles in package h eadspace and media during inoculation. Oxidation reduction potentia l of media samples prior to packaging and when visible colonies were observed.

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8 CHAPTER 2 EFFECT OF TEMPERATURE AND RELATIVE HUMIDITY ON FILM PERMEABILITY Properly designed food packaging systems of fer a means of extending shelf lives of food products. Traditionally, packaging was view ed as a simple physical barrier against contamination or recontamination of containe d food. Plastic films are being increasingly used in food packaging due to advantages in physical, chemical, mechanical and economic properties over other package mate rials such as metals, glass and paper (Rubino et al., 2001). With recent trends towa rds minimally processed foods, packaging must play a greater role in protecting consumers from micr obiological hazards associated with foods (Brody, 2001). Shelf life of products that have not undergone antimicrobial treatment (e.g., sterilization, pasteurization, fr eezing) depends on init ial food quality and design of the package. A package that results in a reduced o xygen level (less than 21%) in a sealed package is often referred to as reduced oxygen packaging (ROP). Even when higher levels of oxygen are used, concentrations can fall below sa fe levels due to microbiological and chemical activity (Cameron et al., 1993) When oxygen levels fall below safe levels, anaerobic conditions develop inside the package. Anaerobic conditions favor growth of Clostridium botulinum while suppressing typi cal aerobic spoilage organisms, which are responsible for the orga noleptic cues of spoilage. Since consumers rely on spoilage indications to make cons umption decisions, anaerobic conditions may allow foods to appear acceptable even though pa thogens and toxins are present. This has

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9 led FDA to restrict the use of ROP for cer tain food products. A recent example involves types of fresh fish and other seafood products. In order to ensure typi cal aerobic spoilage, FDA has set a minimum OTR level of for packag ing material that may be used for fresh fish as one approach for protecting consumers from botulism FDA’s current minimum OTR specification is stated as follows “… packaging that provides an oxygen transmission rate of 10,000 cc/m2/ 24 hrs at 24 C (e.g. 1.5 mil polyethylene) can be regarded as an oxygen-permeable packaging material for fishery products” ( FDA 2002 ) Small errors in permeability can cause signi ficant deviation between the predicted and the actual oxygen levels in packages (Cameron et al., 1995). Since there are very little data published for permeation of gase s through various films (Mapes et al., 1994) direct comparisons between reported permeabili ties can vary widely, and this has led to the need for greater availability of perm eability data, particularly as a function of temperature (Doyon et al., 1991). The aim of this work was to study how OT R varies with temperature and relative humidity for several commercially available p ackaging films that might be considered to be used to package fresh fish. Measurements of OTR are reported for four films obtained from three different packaging film suppliers These films were selected based upon their oxygen transmission rates relative to the FDA specification and were considered as high, medium and low oxygen transmitters. Materials and Method Films tested are identifie d in Table 1. and consisted of C60 and C75 (Dupont Wilmington, Delaware Dupont’s Clysar di vision was purchased by Bemis Corporation on August 1, 2002), AET (Applied Extrusion Tec hnologies, Inc., Atlant a, Georgia) and

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10 BDF (Cryovac-Sealed Air Corporation ,Dun can, South Carolina). The thickness was measured using a micrometer. Table 2-1. Film description Name Type of Film Description C60 High Transmission Clysar 60 HPGF C75 High Transmission Clysar 75 HPGF AET Medium Transmission AET PST2-060 BDF Low Transmission BDF 1000 Oxygen transmission rate (OTR) was m easured using a two-cell Oxtran 2/20 (Mocon Controls Inc, Minneapolis) as shown in Figure 2-1 Figure 2-1. Mocon Oxtran 2/20 The test gas was 96% nitrogen and 4% hydrogen. Oxygen (100%) was applied to the opposite side of the film sample. Films were cut using a razor knife and stainless steel template that provided a film area for testing of 100 cm2 (Figure 2-2).

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11 Figure 2-2. Film cutting template Film samples were loaded onto both the cells of the Oxtran 2/20 apparatus for testing. Before testing, films were conditioned by flushing test gas over both the film surfaces to remove traces of oxygen in the sa mple film. Film samples provided a barrier between oxygen and the N2/H2 gas streams. Oxygen that permeated through the sample was carried by the N2/H2 stream and detected by a coulometric oxygen sensor, which produced an electrical current directly proportional to the flux of oxygen across the film (Figure 2-3). Measurements of OTR were taken at 0% and ap proximately 50% RH,

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12 Figure 2-3. A diagram represen ting gas flow through films inside MOCON instrument. and were expressed as cc/m2/day. Experiments were performed at 10, 15, 23, 30, 35 C Oxygen transmission rates were first determined at 23 C to compare with values given by the suppliers. Films with highest OTRs were identifie d using a Mattson Fourier Transform Infra Red Spectroscopy (FTIR) (Model IR-1000, Madi son, Wisconsin) in order to provide material selection guidance for prospective fresh fish packers.

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13 Results and Discussion Values of OTR for sample films are pr ovided in Table 2-2. The OTR at 0% and 50% RH only were tested because of the limitation of MOCON instrument. Table 2-2. Oxygen transmission rates of different films measured at different temperature and relative humidity Temperature ( C) C 60 (cc/m2/day) C 75 (cc/m2/day) AET (cc/m2/day) BDF (cc/m2/day) 0% RH 50% RH 0% RH 50% RH 0% RH 50% RH 0% RH 50% RH 10 4270 3720 3680 3700 1520 1300 370 370 15 5520 4860 4840 4830 2010 1730 500 480 23 8620 7370 7390 7050 3200 2660 710 720 30 12320 10520 10480 10170 4800 4000 1010 1000 35 16210 13690 13800 13270 6430 5400 1370 1320 Thickness and average OTR at room temperature (23 C) and 0% RH are given in Table 2-3 for each film compared with the values reported by film suppliers. Table 2-3. Comparison of measured oxygen tr ansmission rates with value reported by manufacturer Film Type Manufacturer’s Value Measured value OTR (cc/m2/day) Thickness (gauge) OTR (cc/m2/day) Thickness (gauge) C60 9300 60 8620 65 C75 7750 75 7390 75 AET 3100 60 3200 60 BDF 2227 75 710 163 An apparent significant discrepancy was found between measured values and those supplied with Cryovac’s BDF1000 sample. The ro ll of film was labeled as 75 gauge, for which OTR should have been 2227 cc/m2/day. Repeated trials with the BDF1000 film resulted in an OTR of 705 cc/m2/day. When measured with a digital micrometer, however, thickness was found to be about 163 gauge. Supplier provided OTR values were 2412, 2227, 1474 and 1153 cc/m2/day for 60, 75, 100 and 125 gauge films.

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14 Extrapolating this trend to 163 gauge provide s a value of 716, which matches closely to the measured value. Results show that none of the films tested satisfy the FDA’s film OTR specification for fresh fish packaging (10,000 cc/m2/24 hrs). Additionally, OTR values were not significantly altered by increased relative hum idity at lower temperatures which are normally used for seafood storage as shown in Figure 2-4. 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0510152025303540 Temperature (degree C)OTR (cc/m2/day)0% RH 50% RH Figure 2-4. Comparison of 0% and 50% RH of C60 A plot of the logarithm of OTR versus inve rse absolute temperature gives a straight line suggesting that Arrhenius relationships shown in Figur es 2-5 and 2-6. At 50% RH Film 1 and Film 2 have similar OTR. Oxygen transmission rate increases with temp erature as expected. It is possible to express permeation, OTR, as a function of temperature by the following Arrhenius expression: RT E k OTRaexp0 2.1

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15 where OTR is Oxygen Transmission Rate in cc/m2/day, k0 is the Arrhenius preexponential factor in cc/m2/day, Ea is Arrhenius activation energy in J/mol, R is the Ideal Gas Law constant (8.314 J/mol/K), and T is absolute temperature in Kelvin (K). Equation (1) may be used to estimate OTR at a specific temperature. Values for Ea and k0 are tabulated in Tables 4 and 5 for 0% and 50% RH, respectively. 100 1000 10000 100000 0.00320.003250.00330.003350.00340.003450.00350.00355 1/Temperature (K)OTR (cc/m2/day)C60 C75 AET BDF Figure 2-5. Arrhenius relationship betw een OTR and temperature at 0% RH 100 1000 10000 100000 0.00320.003250.00330.003350.00340.003450.00350.00355 1/Temperature (K)OTR (cc/m2/day)C60 C75 AET BDF Figure 2-6. Arrhenius relationship betw een OTR and temperature at 50% RH

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16 Table 2-4. Ea and k0 values for Arrhenius relationship between OTR and temperature for the packaging films at 0% RH Sample k0 (cc/m2/day) Ea (kJ/mol) R2 C60 6.00E+10 38.70 0.999 C75 4.00E+10 38.20 0.995 AET 8.00E+10 41.90 0.999 BDF 2.00E+10 36.70 0.993 Table 2-5. Ea and k0 values for Arrhenius relationship between OTR and temperature for the packaging films at 50% RH Sample k0 (cc/m2/day) Ea (kJ/mol) R2 C60 3.00E+10 37.60 0.999 C75 2.00E+10 36.70 0.997 AET 5.00E+10 41.10 0.998 BDF 2.00E+09 36.50 0.998 Analysis of FTIR was performed for the Cl ysar films because they were closest to the FDA OTR specification. Identification of these materials should be helpful in selecting the potential candidates for seafood packaging. Results of FTIR showed that these films were essentially thin gauge polyethylene (PE). Sample FTIR spectra are compared with a library of spectra for know n standards (Figure 2-7). Note that, AET PST2-060 is oriented polypropylene film with inner sealable side, and BDF1000 is multilayered co-extruded film with external polypropylene layers.

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17 0 400 0 500 1000 1500 2000 2500 3000 3500 4000 4500 WavenumbersRelative AbsorbanceSignature Spectrum of LDPE C60 C75 Figure 2-7. Comparison of PE, C60 and C75 FTIR spectra

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18 CHAPTER 3 EFFECT OF FILM OTR, PACKAGE AREA AND TEMPERATURE ON CLOSTRIDIUM SPOROGENES SPORE OUTGROWTH In modified atmosphere packaging of fr esh and minimally processed foods, oxygen is often intentionally reduced to decr ease enzymatic, biochemical and aerobic microbiological activities. This method of p ackaging is called reduced oxygen package (ROP) an FDA term for a package that has a potential to result in oxygen levels below 21%. ROP provides an environment that co ntains little or no oxygen, offers unique advantages such as increase in shelf life improved handling and reduced weight lose. However, there may be marked increase in safe ty concerns with some foods, particularly with ROP fresh fish. Studies have dem onstrated that formation of type E botulinum toxin prior to organoleptic spoilage at mildly abus ive temperatures is possible, thus making the seafood product unfit for consumption (Dufresne et al., 2000; Post et al., 1985; Reddy et al., 1996, 1997a, 1997b). To mitigate this problem FDA considers a package that provides an oxygen transmission rate of 10,000 cc/m2/day at 24 C as acceptable for packaging seafood products (FDA, 2002). However, this specification does not take into consideration the design of the package. It is suspected that a regulation that combines film OTR with descriptive parameters of th e package, such as film area, may offer a better regulatory alternative and an ease in choosi ng packaging materi al by the seafood manufacturers. Studies show that residual oxygen plays a key role in food quality and shelf life determination (Tewari et al., 1999). Oxygen pr ofiles indicate change in quality of

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19 products and also the packaging film’s qua lity. Non-destructive monitoring of oxygen profiles inside the package and food produc t has remained a difficult and expensive objective (Johnson, 1997). Optical sensor approach offers a realistic alternative and a number of methods of optical oxygen sensing have been described in recent years (Fitzgerald, 2001). Research shows that C. botulinum is an obligate anaerobe, it remains unclear if a particular concen tration of oxygen is capable of preventing toxigenesis. So luminescence-based oxygen sensor was used for destructive oxygen measurement for this study. Measurement of oxidation reduction (Eh) potential (redox potential) could provide information on how the background redox potenti al might be adjusted by addition of a suitable oxidant or reductant as to make the substrate uncongenial to the likely microbial contaminants while not affecting its palatabil ity and attractiveness as a foodstuff (Brown and Emberger 1980). In anoxic conditions, a marked fall in C. botulinum culture Eh can accompany germination of a large spore inoc ulum, thereby providing conditions suitable for multiplication of the outgr owing vegetative cells (Morris, 2000). Studies have been conducted to see whether there can be a limitin g value of redox potential to prevent the growth of C. botulinum (Lund and Wyatt, 1984; Montvill e and Conway, 1982). But there are very few data available for C. botulinum growth and toxin production where Eh has been used as a variable (Smoot and Pierson, 1979). Clostridium sporogenes is an obligate anaerobe but non pathogenic with similar physiological properties to C. botulinum Therefore, it was used as a surrogate for C. botulinum in this study. The objective of this work was to devel op a scientific rationa le for a new seafood package OTR specification, and to study the re lationships among film OTR, package area

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20 and storage temperature on C.sporogenes spore outgrowth in regular and anaerobic media. Materials and Method Sample Preparation C.sporogenes (PA 3679) spores were purchas ed from National Food Laboratory Inc., (Dublin, California). When spores were received, a stock so lution was prepared by diluting 10 ml of 2x107 CFU/ml into 1000 ml of autocl ave-sterilized, 0.15 M potassium phosphate buffer solution at pH 7. The ini tial concentration fo r all trials was 2x104 CFU/ml. Spore Enumeration Inoculum was treated at 80 C for 20 mins to stimulate germination of the spores and to prevent growth of contaminating or ganisms. Plates were inoculated with concentration of 2x103 CFU/ml by pour plate technique. Regular and highly reduced anaerobic media was used for spore recover y. Regular media was prepared with 24 g of dehydrated brain heart infusion (Fisher Scientific, Springfie ld, New Jersey) and 10 grams of Difco Bacto Agar (Fisher Scientific, Springfield, New Jersey) in 700 ml of 0.15 M potassium phosphate buffer solution to mainta in pH of 7.0. Anaerobic agar was prepared by boiling 40.6 grams of anaerobic agar (Scien tific, Springfield, New Jersey) in 700 ml of distilled water. The media ingredients we re transferred to a Teflon bottle (Nalgene Nunc International, Rochester, New York) a nd autoclave sterilized along with test tubes and pipette tips. Twenty-eight plates were prepared by as eptic pour plate technique for each set of experiments. Film Samples

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21 Bags of two different sizes, 0.083 m2 and 0.325 m2, representing small and large sizes (8x8 and 18x14 inches respectively) were made from the three different film samples mentioned in table 1. Table 3-1. OTR of film used in this study Film Name Film ID Film Type OTR (cc/m2/day) Clysar 60 HPGF C60 High Transmission 8620 AET (PST2-060) AET Medium Transmission 3200 BDF 1000 BDF Low Transmission 710 Duplicate samples were made by placing tw o plates in each bag. The bags were vacuum packed, gas flushed with nitrogen a nd sealed using a vacuum packaging machine (Multivac, Kansa City, Missouri). Specifica lly the machine was programmed to reduce pressure via vacuum from 1 atm to 0.15 atm a nd then return to 0.8 atm with nitrogen gas. The vacuum/gas-flush cycle o ccurred three times .Samples were stored at 10, 15, 20, 30 and 35 C in the state-of-the-art environmental gr owth chambers. Two plates were placed in an anaerobic box (Mitsubish i Gas Chemical Co., Inc, New York, New York) and kept with samples inside the environmental growth chambers. Digital pict ures of the plates were taken using Nikon COOL PIX 5000 every 8-12 hrs unt il visible growth was observed. The time taken to note visible col ony growth in the sample was recorded. Monitoring Oxygen Composition Dynamic oxygen concentration profiles were monitored using a 4-channel FOXY fiber optic oxygen sensor system (Ocean Optic s Inc., Dunedin, Florida). The fiber optic oxygen sensing system incorporates probe s doped at the tip with a compound that fluoresces in response to input light. Fluorescence is quenched by oxygen. Therefore anoxic environments result in significan t fluorescent response, while increasing availability of oxygen results in a reduced response. Calibration ag ainst known conditions

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22 provides a means to measure gaseous and di ssolved oxygen in samples. A FOXY 18-G fiber optic oxygen probe was inserted insi de the bag and dynamic oxygen concentration in packaged media and headsp ace was monitored continuously. Figure 3-1. Sample of bag sizes used for the experiment Figure 3-2. Rack arrangement inside the chamber

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23 Figure 3-3. Back lighted stand used for taking digital pictures Figure 3-4. Fiber optic oxygen sensor system To place the probe in the media a hol e was made using a hot-wire on all petridishes. Vials (40 ml) with septa equi pped with screw caps were cut below the shoulder of the vial. Sample bags were sa ndwiched between caps and open ended vials.

PAGE 36

24 Oxygen profile was monitored in the high barr ier (BDF) and low barrier (C60) small size bags (Figure 3-5). Figure 3-5. Oxygen sampling inside the bag Oxidation Reduction Potential Oxidation Reduction Potential of the anaerobic media was measured in millivolts before and after incubation using Accumet 13-620-81 combination ORP probe (Fisher Scientific, Springfield, New Jersey). The ORP of a sample is measured by comparing the electrical potential between an inert electrode (typically pl atinum) that is in intimate contact with the sample, and a reference elec trode with a known poten tial versus the ideal standard hydrogen electrode (“SHE”). The si lver-silver chloride reference electrode is one of the most commonly used reference electrodes due to its ease of manufacture and its useful temperature range. Th e electrode is a silver wire coated with a thin layer of silver chloride that is depos ited either by electroplating or by dipping the wire in molten silver chloride. The ORP value was measur ed within 24 hrs of visible growth under a nitrogen blanket. Calibration of the ORP probe was performed in pH 4 potassium acid phthalate standard buffer solution and pH 7 potassium and sodium phosphate standard Headspace O2 probe Anaerobic Media Dissolved O2 probe CutVial Cap Septum Sample bag

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25 buffer solution (Sensorex, Garden Grove, Ca lifornia). Both buffer solutions were saturated with quinhydrone at 25 C. Simultaneously, pH of the media at the end of the experiment was also measured. The ORP probe and pH probe were standardized before each set of experiments to ensure accuracy and consistency of the measuring system. Figure 3-6. Equipment used to measure ORP and pH Measured ORP values were adjusted to pH 7.0 to eliminate the effect of pH on Eh by use of equation 3.1 (George et al., 1998). Eh7 = Eobs + Eref + 2.303 (RT/F) (pHX – 7.0) 3.1 Where Eobs is the measured potential of the system, Eref is the reference electrode potential of the internal electrolyte (saturated KCl silver/silver chloride) of the electrode and equals 199 mV, 2.303 (RT/F) is the Ne rnst potential equaled to 59.1 mV at 25 C and pHX is the measured pH of the system. Results and Discussion Time taken for spore outgrowth in regular and highly reduced anaerobic media in different bags over a period of 15 days in temperatures 10, 15, 20, 30 and 35 C is

PAGE 38

26 tabulated in tables 3-2 and 33. Different bag sizes are represented as “S” denoting small bag and “L” denoting large bag of sizes 0.083 m2 and 0.325 m2 respectively. The “B1” and “B2” represent sample duplicates for bag 1 and bag 2, respectively. Table 3-2. Spore outgrowth over time in regula r media for various film types at various temperatures Temperature ( C) Film Type and Bag Size Time in days 1234567891011 12 131415 C60-S B1 B2 C60-L B1 B2 AET-S B1 B2 AET-L B1 B2 BDF-S B1 B2 BDF-L B1 B2 10 Control C60-S B1 B2 C60-L B1 B2 1 1 1 1 1 1 AET-S B1 B2 AET-L B1 11234 4 4 4 4 4 B2 22222 2 2 2 2 2 BDF-S B1 11111 1 1 1 1 1 B2 2222 2 2 2 2 2 BDF-L B1 111 1 1 1 1 1 B2 11222 2 2 2 2 2 15 Control 11111 1 1 1 1 1 C60-S B1 B2 11111 1 1 1 1 1 C60-L B1 B2 11111 1 1 1 1 1 AET-S B1 B2 AET-L B1 20 B2 1111 1 1 1 1 1

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27 Table 3.2. (continued) Temperature ( C) Film Type and Bag Size Time in days 1234567891011 12 131415 BDF-S B1 B2 11 1 1 1 1 1 BDF-L B1 1111 1 1 1 1 1 B2 -Control -* * * * * C60-S B1 -3 3 3 3 3 3 3 3 333 B2 -C60-L B1 -2 2 4 4 4 4 4 4 4 4 444 B2 -AET-S B1 -1 2 2 2 2 2 2 2 2 2 222 B2 -1 1 1 2 2 2 2 2 2 2 222 AET-L B1 -B2 -1 1 1 1 1 1 1 1 1 1 111 BDF-S B1 -3 4 4 4 4 4 4 4 4 4 444 B2 -BDF-L B1 -B2 -1 1 1 1 1 1 1 1 1 1 111 30 Control -‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡‡‡ C60-S B1 -B2 -1 1 1 1 1 1 1 1 1 1 111 C60-L B1 -B2 -AET-S B1 -1 1 1 1 1 1 1 1 1 1 111 B2 -AET-L B1 -B2 -BDF-S B1 -B2 -BDF-L B1 -11 1 ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡‡‡ B2 -35 Control -• • • • • • • • • • • •• • “*” represents growth greater than or equal to 50 colonies “•” represents growth greater th an or equal to 100 colonies “‡” represents growth greater than or equal to 200 colonies “–“represents no growth The integers (1, 2, 3, 4..) represent number of visible colonies noted at that period of time. From the table it can be seen th at there was no growth in all samples at 10 C for a period of 15 days. The small bag of AET ha d growth only at higher temperatures of 30

PAGE 40

28 and 35 C. A maximum number of growth in all the bags were seen at 30 C. At 35 C comparatively lower growth was noticed in a ll bags which is because of increase in OTR at high temperatures. There was growth in al l the control plates in side the anaerobic box at different temperatures. Maxi mum growth in control plates was noted at temperatures higher than 15 C. Two to three days after noticing initial gr owth in the plates, there was no increase in the number of colonies which represents the rise in oxygen level inside the bag that prevents further germination of spores. Figure 3.7. Control plate at 30 C inside anaerobic box Table 3-3. Spore outgrowth over time in an aerobic media for various film types at various temperatures Temperature ( C) Film Type and Bag Size Time in days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C60-S B1 B2 C60-L B1 B2 AET-S B1 10 B2 AET-L B1 B2 BDF-S B1 B2 BDF-L B1 B2

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29 Table 3.3. (continued) Temperature ( C) Film Type and Bag Size Time in days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C60-S B1 B2 C60-L B1 B2 AET-S B1 B2 AET-L B1 B2 BDF-S B1 B2 BDF-L B1 15 B2 C60-S B1 B2 C60-L B1 B2 2 3 AET-S B1 4 4 B2 AET-L B1 ‡ ‡ B2 • • BDF-S B1 ‡ ‡ B2 • • BDF-L B1 ‡ ‡ 23 B2 ‡ ‡ C60-S B1 * B2 4 8 C60-L B1 * B2 6 13 AET-S B1 ‡ ‡ B2 7 11 AET-L B1 ‡ ‡ 30 B2 ‡ ‡ BDF-S B1 ‡ ‡ B2 ‡ ‡ BDF-L B1 ‡ ‡ B2 ‡ ‡ C60-S B1 • • B2 • • C60-L B1 ‡ ‡ 35 B2 ‡ ‡ AET-S B1 ‡ ‡ B2 ‡ ‡ AET-L B1 ‡ ‡ B2 ‡ ‡ BDF-S B1 ‡ ‡ B2 ‡ ‡ BDF-L B1 ‡ ‡ B2 ‡ ‡ “*” represents growth greater than or equal to 100 “•” represents growth greater than or equal to 500 “‡” represents growth greater than or equal to 1000 “–“ represents no growth

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30 Unlike the control plates of regular media, surprisingly there was no growth noticed in control plates of anaerobic media even though they had an ideal anaerobic environment and low ORP value to germinat e. There was no growth noted in 10 and 15 C. Compared to regular media, growth was faster in highly reduced anaerobic media. A gradual increase in the amount of colonies as the temperature increased was obvious. As expected, time taken to observe growth increased as the te mperature decreased. Oxidation reduction potentia ls were measured within 24 hours of growth and values are tabulated below after eliminati on of pH effect (Tab le 3-4). Initial redox potential of anaerobic medi a before inoculation was measured to be 137 mV. Table 3-4. Oxidation reduction potential of highly reduced anaerobic media in bags of various film types at different temperatures Temperature Film Type ORP Ti me taken for Visible Growth ( C) (mV) (Days) S L S L Bag 1 332.8 266.0 C60 Bag 2 322.0 259.4 Bag 1 266.6 262.2 AET Bag 2 300.6 289.8 Bag 1 271.3 257.8 15 BDF Bag 2 260.8 252.2 Bag 1 222.7 239.8 C60 Bag 2 270.4 254.5 4 Bag 1 226.7 253.8 4 4 AET Bag 2 253.0 257.8 4 Bag 1 120.7 78.7 4 4 23 BDF Bag 2 197.1 94.0 4 4 Bag 1 177.9 177.0 3 3 C60 Bag 2 152.9 118.0 3 3 Bag 1 110.4 106.0 3 3 AET Bag 2 216.0 100.6 3 3 Bag 1 224.5 30.6 3 3 30 BDF Bag 2 251.7 20.0 3 3 Bag 1 27.4 39.7 2 2 C60 Bag 2 208.5 120.3 2 2 Bag 1 32.4 48.6 2 2 AET Bag 2 54.8 80.6 2 2 Bag 1 12.3 13.5 2 2 35 BDF Bag 2 19.9 29.7 2 2

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31 There was an increase in redox potential as the temperature decr eased. This is due to more time taken for spore outgrowth at lower temperatures allowing oxygen to permeate inside the bag and increase the ORP of the media. Oxidati on reduction potential of media in C60 bags was lower than that of media in BDF bags regardless of bag sizes. The C60 small bag at 30 C shows a high ORP value which was due to change in probe position or contamination. The headspace oxygen content in high a nd low transmission film was monitored using the FOXY probe at 23 C until growth was observed. Re sults are shown in Figures 3-8, 3-9,3-10. 0 2 4 6 8 10 12 14 16 18 0100020003000400050006000 Time (mins)Headspace Oxygen (%)C60 BDF Figure 3-8. Headspace oxygen content over tim e in film types C60 and BDF at 23 C As seen in Figure 3-8, oxygen partial pre ssure increased rapidly for the high OTR C60 film. Dissolved oxygen content in media was measured at 23 and 35 C.

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32 0 2 4 6 8 10 0100020003000400050006000 Time (mins)Oxygen content (mg/L)C60 BDF Figure 3-9. Dissolved oxygen content ove r time in highly reduced media at 23 C for film types C60 and BDF 0 2 4 6 8 10 050010001500200025003000 Time (mins)Oxygen content (mg/L)C60 BDF Figure 3-10. Dissolved oxygen content ove r time in highly reduced media at 35 C for film types C60 and BDF

PAGE 45

33 The oxygen content of the media gradually increases in C60 whereas in BDF it follows an irregular pattern due severe growth in media.

PAGE 46

34 CHAPTER 4 CONCLUSION AND FUTURE WORK Conclusion Oxygen transmission rate results obtained from MOCON Oxtran 2/20 show that none of the films studied fully sati sfy FDA’s OTR guideline of 10,000 cc/m2/day at room temperature. Clysar 60 (~0.60 mil – polyeth ylene film) provided the highest OTR at around 8600 cc/m2/day at 23 C. These results suggest that it may be difficult for suppliers of packaged fresh fish to sour ce a variety of acceptable packaging films. Arrhenius parameters were provided in or der to allow estimation of OTR values at desired temperatures. The lo west temperature studied, 10 C, represented the limit of capability for the MOCON Oxtran 2/20. However, it is expected that at least for the Clysar (polyethylene) films, Arrhenius relationships will provide suitable OTR estimates throughout the range important for packaged fresh fish (0 – 35 C), because no significant latent thermal transitions exist within this extended lower temperature range (glass transition temperature, Tg, for LDPE occurs at about -175 C). There is not much variati on in growth between small and large bags. The area of the large bag (0.325 m2) represented the maximum size that can be sealed using available vacuum packaging equipment. Although larger package areas might have had a significant effect on spore outgrowth, such si zes would not be of practical value for packaging seafood. So the research hypothe sis of observing a difference in spore outgrowth between lower and larg er film area was not supported.

PAGE 47

35 The difference in package area affected th e oxidation reduction potential of highly reduced anaerobic media at different temp eratures. It was observed that as the temperature decreased, the oxidation reduction potential increased due to the increasing time taken for visible spore outgrowth. As expected, the headspace oxygen and dissolved oxygen content in the media was more in C60 than BDF. Future Work From these experiments it is observed th at redox potential and bag specification plays a vital role in spor e outgrowth. So a package de sign that combines oxidation reduction potential and packaging film permeation to control growth of C. botulinum can be developed. This methodology that combines oxidation reduction potential to dynamic oxygen permeation might prevent C.botulinum hazard and helps in assessing package safety. Similarity in physiological properties between C. sporogenes and C. botulinum has been mentioned earlier. However, C. sporogenes has a different temperature window of 15-45 C for visible spore outgrowth, whereas C. botulinum can grow and produce toxin at temperatures above 3.3 C. The experimental method and data of this project can be used to provide a basis for future experi ments and clues about potential behavior of C. botulinum This data should not be used by FDA for regulations on control of C. botulinum spore outgrowth in ROP packed seafood for the above reasons.

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APPENDIX A OXYGEN TRANSMISSION RATE OF PA CKAGING FILMS AT DIFFERENT TEMPERATURES AND RELATIVE HUMIDITY

PAGE 49

37 Table A-1. OTR of packaging films at 0% RH Sample ID Cell Temperature1/Tabs Measured OTR Ln(OTR)R^2 A 10 0.003534 3736.3 8.2258511 B 10 0.003534 3570.9 8.1805729 A 15 0.003472 4943.45 8.5058187 B 15 0.003472 4731.95 8.4620927 A 23 0.003378 7631.05 8.9399807 B 23 0.003378 7139.7 8.873426 A 30 0.0033 10867.95 9.2935734 B 30 0.0033 10080.65 9.218373 A 35 0.003247 14357.95 9.5720591 Clysar 75HPGF B 35 0.003247 13238.8 9.4909072 0.9952 A 10 0.003534 4263.75 8.3579043 B 10 0.003534 4269.15 8.35917 A 15 0.003472 5543.75 8.6204264 B 15 0.003472 5483.15 8.609435 A 23 0.003378 8636.75 9.0637816 B 23 0.003378 8589.9 9.0583424 A 30 0.0033 12343.35 9.4208727 B 30 0.0033 12280.8 9.4157923 A 35 0.003247 16296.85 9.6987271 0.9993 Clysar 60HPGF B 35 0.003247 16119.35 9.6877757 A 10 0.003534 1514.25 7.3226755 B 10 0.003534 1522.2 7.3279119 A 15 0.003472 1999.3 7.6005524 B 15 0.003472 2017.9 7.6098126 A 23 0.003378 3205 8.0724674 B 23 0.003378 3196.9 8.0699369 A 30 0.0033 4795.8 8.4754958 B 30 0.0033 4790.55 8.4744005 A 35 0.003247 6486.25 8.7774398 AET B 35 0.003247 6371.4 8.7595745 0.9992 A 10 0.003534 386.95 5.9582955 B 10 0.003534 355.05 5.8722586 A 15 0.003472 503.55 6.221683 B 15 0.003472 501.05 6.2167059 A 23 0.003378 717.35 6.5755639 B 23 0.003378 690.95 6.5380675 A 30 0.0033 1013.95 6.9216089 B 30 0.0033 1005.25 6.9129915 A 35 0.003247 1364.45 7.2185067 BDF1000 B 35 0.003247 1366.1 7.2197152 0.9932

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38 Table A-2. OTR of packaging films at 50% RH Sample ID Cell Temperature 1/Tabs Measured OTR Ln(OTR)R^2 A 10 0.003534 3706.45 8.2178298 B 10 0.003534 3736 8.2257708 A 15 0.003472 4844.3 8.485558 B 15 0.003472 4869.1 8.4906644 A 23 0.003378 7372.7 8.9055393 B 23 0.003378 7360.2 8.9038424 A 30 0.0033 10526.55 9.2616559 B 30 0.0033 10504.6 9.2595685 A 35 0.003247 13927.95 9.5416529 Clysar 60HPGF B 35 0.003247 13444 9.5062882 0.999 A 10 0.003534 3636.35 8.1987357 B 10 0.003534 3774.25 8.235957 A 15 0.003472 4744.7 8.4647835 B 15 0.003472 4914.2 8.4998843 A 23 0.003378 6982.7 8.8511909 B 23 0.003378 7111 8.8693982 A 30 0.0033 10044.3 9.2147606 B 30 0.0033 10292.95 9.2392145 A 35 0.003247 13033.2 9.4752552 Clysar 75HPGF B 35 0.003247 13511.3 9.5112817 0.997 A 10 0.003534 1293.3 7.1649524 B 10 0.003534 1303.35 7.1726932 A 15 0.003472 1708.45 7.4433418 B 15 0.003472 1742.25 7.4629327 A 23 0.003378 2649.4 7.8820885 B 23 0.003378 2668.65 7.889328 A 30 0.0033 3983.65 8.2899538 B 30 0.0033 4002.15 8.294587 35 0.003247 5387.2 8.591781 AET 35 0.003247 5406.9 8.5954312 0.998 A 10 0.003534 375.45 5.9281253 B 10 0.003534 364.55 5.8986637 A 15 0.003472 482.45 6.1788773 B 15 0.003472 475.7 6.1647874 A 23 0.003378 728.2 6.5905757 B 23 0.003378 705.35 6.5586941 A 30 0.0033 1008.6 6.9163185 B 30 0.0033 986 6.8936564 A 35 0.003247 1334 7.1959372 BDF1000 B 35 0.003247 1308.75 7.1768278 0.998

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APPENDIX B DIGITAL PICTURES OF SPORE OUTGR OWTH IN DIFFERENT FILMS AND BAG SIZES

PAGE 52

40 Table B-1. Growth table for C60 8X8 at 15 C Anaerobic Media Time (Days) Bag 2 1 2 3 4

PAGE 53

41 Table B-1. Continued Time (Days) Bag 2 5 6 7 8

PAGE 54

42 Table B-1. Continued Time (Days) Bag 2 9 10 11 12

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43 Table B-1. Continued Time (Days) Bag 2 13 14 15

PAGE 56

44 Table B-2. Growth table for AET 8x8 at 15 C Anaerobic Media Time (Days) Bag 2 1 2 3 4 5 6

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45 Table B-2. Continued Time (Days) Bag 2 7 8 9 10 11

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46 Table B-2. Continued Time (Days) Bag 2 12 13 14 15

PAGE 59

47 Table B-3. Growth table for BDF 8x8 at 15 C Anaerobic Media Time (Days) Bag 2 1 2 3 4

PAGE 60

48 Table B-3. Continued Time (Days) Bag 2 5 6 7 8

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49 Table B-3. Continued Time (Days) Bag 2 9 10 11 12

PAGE 62

50 Table B-3. Continued Time (Days) Bag 2 13 14 15

PAGE 63

51 Table B-4. Growth table for C60 18X14 at 15 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 64

52 Table B-4. Continued Time (Days) Bag 1 Bag 2 5 6 7 8

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53 Table B-4. Continued Time (Days) Bag 1 Bag 2 9 10 11 12

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54 Table B-4. Continued Time (Days) Bag 1 Bag 2 13 14 15

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55 Table B-5. Growth table for AET 18X14 at 15 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

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56 Table B-5. Continued Time (Days) Bag 1 Bag 2 5 6 7 8

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57 Table B-5. Continued Time (Days) Bag 1 Bag 2 9 10 11 12

PAGE 70

58 Table B-5. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 71

59 Table B-6. Growth table for BDF 18X14 at 15 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

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60 Table B-6. Continued Time (Days) Bag 1 Bag 2 6 7 8 9 10

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61 Table B-6. Continued Time (Days) Bag 1 Bag 2 11 12 13 14 15

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62 Table B-7. Growth table for C60 8X8 at 23 C Anaerobic Media Time (Days) Bag 2 1 2 3 4 5

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63 Table B-8. Growth table for AET 8x8 at 23 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

PAGE 76

64 Table B-9. Growth table for BDF 8x8 at 23 C Anaerobic Media Time (Days) Bag 2 1 2 3 4 5

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65 Table B-10. Growth table for C60 18x14 at 23 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

PAGE 78

66 Table B-11. Growth table for AET 18x14 at 23 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

PAGE 79

67 Table B-12. Growth table for BDF 18x14 at 23 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

PAGE 80

68 Table B-13. Growth table for C60 8x8 at 30 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 81

69 Table B-14. Growth table for AET 8x8 at 30 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 82

70 Table B-15. Growth table for BDF 8x8 at 30 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

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71 Table B-16. Growth table for C60 18x14 at 30 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 84

72 Table B-17. Growth table for AET 18x14 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 85

73 Table B-18. Growth table for BDF 18x14 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 4

PAGE 86

74 Table B-19. Growth table for C60 8x8 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3

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75 Table B-20. Growth table for AET 8x8 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 Table B-21. Growth table for BDF 8x8 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3

PAGE 88

76 Table B-22. Growth table for C60 18x14 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3 Table B-23. Growth table for AET 18x14 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3

PAGE 89

77 Table B-24. Growth table for BDF 18x14 at 35 C Anaerobic Media Time (Days) Bag 1 Bag 2 1 2 3

PAGE 90

78 Table B-25. Growth table for C60 8x8 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 91

79 Table B-25. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 92

80 Table B-25. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 93

81 Table B-26. Growth table for AET 8x8 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 94

82 Table B-26. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 95

83 Table B-26. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 96

84 Table B-27. Growth table for BDF 8x8 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 97

85 Table B-27. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 98

86 Table B-27. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 99

87 Table B-28. Growth table for C60 18x14 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 100

88 Table 28. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 101

89 Table 28. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 102

90 Table B-29. Growth table for AET 18x14 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 103

91 Table B-29. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 104

92 Table B-29. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 105

93 Table B-30. Growth table for BDF 18x14 at 15 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 106

94 Table B-30. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 107

95 Table B-30. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 108

96 Table B-31. Growth table for C60 8x8 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 109

97 Table B-31. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 110

98 Table B-31. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 111

99 Table B-32. Growth table for AET 8x8 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 112

100 Table B-32. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 113

101 Table B-32. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 114

102 Table B-33. Growth table for BDF 8x8 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 115

103 Table B-33. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 116

104 Table B-33. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 117

105 Table B-34. Growth table for C60 18x14 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 118

106 Table B-34. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 119

107 Table B-34. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 120

108 Table B-35. Growth table for AET 18x14 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 121

109 Table B-35. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 122

110 Table B-35. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 123

111 Table B-36. Growth table for BDF 18x14 at 20 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 124

112 Table B-36. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 125

113 Table B-36. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 126

114 Table B-37. Growth table for C60 8x8 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 127

115 Table B-37. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 128

116 Table B-37. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 129

117 Table B-38. Growth table for AET 8x8 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 130

118 Table B-38. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 131

119 Table B-38. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 132

120 Table B-39. Growth table for BDF 8x8 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5

PAGE 133

121 Table B-39. Continued Time (Days) Bag 1 Bag 2 6 7 8 9 10

PAGE 134

122 Table B-39. Continued Time (Days) Bag 1 Bag 2 11 12 13 14 15

PAGE 135

123 Table B-40. Growth table for C60 18x14 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 136

124 Table B-40. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 137

125 Table B-40. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 138

126 Table B-41. Growth table for AET 18x14 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 139

127 Table B-41. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 140

128 Table B-41. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 141

129 Table B-42. Growth table for BDF 18x14 at 30 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 142

130 Table B-42. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 143

131 Table B-42. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 144

132 Table B-43. Growth table for C60 8x8 at 35 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 145

133 Table B-43. Continued Time (Days) Bag 1 Bag 2 8 9 10 11 12 13

PAGE 146

134 Table B-43. Continued Time (Days) Bag 1 Bag 2 14 15

PAGE 147

135 Table B-44. Growth table for AET 8x8 at 35 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 148

136 Table B-44. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 149

137 Table B-44. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 150

138 Table B-45. Growth table for BDF 8x8 at 35 C Regular Media Time Days Bag 1 Bag 2 1 2 3 4 5 6

PAGE 151

139 Table B-45. Continued Time Days Bag 1 Bag 2 7 8 9 10 11 12

PAGE 152

140 Table B-45. Continued Time Days Bag 1 Bag 2 13 14 15

PAGE 153

141 Table B-46. Growth table for C60 18x14 at 35 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 154

142 Table B-46. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 155

143 Table B-46. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 156

144 Table B-47. Growth table for AET 18x14 at 35 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 157

145 Table B-47. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 158

146 Table B-47. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 159

147 Table B-48. Growth table for BDF 18x14 at 35 C Regular Media Time (Days) Bag 1 Bag 2 1 2 3 4 5 6

PAGE 160

148 Table B-48. Continued Time (Days) Bag 1 Bag 2 7 8 9 10 11 12

PAGE 161

149 Table B-48. Continued Time (Days) Bag 1 Bag 2 13 14 15

PAGE 162

APPENDIX C OXIDATION REDUCTION POTENTIA L WITHOUT PH COMPENSATION

PAGE 163

151 Table C-1 Redox potential values without compensating for pH 7 Temperature Film Type Bag Size S L pH ORP pH ORP Bag 1 7.03 132 7 67 C60 Bag 2 7 123 7.04 58 Bag 1 7.01 67 7.02 62 AET Bag 2 7.01 101 7.03 89 Bag 1 6.92 77 6.98 60 15 BDF Bag 2 6.98 63 6.97 55 Bag 1 6.91 29 6.98 42 C60 Bag 2 6.99 72 7.06 52 Bag 1 6.91 33 6.98 56 AET Bag 2 6.95 57 7.03 57 Bag 1 6.86 -70 6.91 -115 23 BDF Bag 2 6.9 4 6.95 -102 Bag 1 6.27 22 6.78 -9 C60 Bag 2 6.05 10 6.12 -29 Bag 1 6.06 -33 6.07 -38 AET Bag 2 6.12 69 6.13 -47 Bag 1 6.01 84 6.08 -114 30 BDF Bag 2 6.2 100 6.07 -124 Bag 1 6.06 -116 6.25 -115 C60 Bag 2 6.89 16 6.65 -58 Bag 1 6.33 -127 6.3 -109 AET Bag 2 6.32 -104 6.57 -93 Bag 1 6.04 -130 6.18 -137 35 BDF Bag 2 6.17 -130 6.2 -122

PAGE 164

152 LIST OF REFERENCES Brody, A.L. 2001 Packaging to limit micr obiological concerns. Food Technology. 55(12), pp. 74-75. Brown, M.H, Emberger, O. 1980. Oxidation reduc tion potential. In Microbial Ecology of Foods (J.H. Silliker eds.), Academ ic Press, New York. Vol. 1,pp. 112-115. Cameron, A.C, Patterson, B.D, Talasila, P. C and Joles, D.W.1993. Modeling the risk in modified atmosphere packaging: A case for sense and respond packaging. In Proc. Sixth Intl. Controlled Atmosphere Res. Conf, vol. 1. 15-17 June 1993 (G.D. Blanpied, J.A. Bartsch, and J.R. Hi cks eds.), Ithaca, N.Y, pp. 95-102. Cameron, A.C, Talasila, P.C, Joles, D.W. 1995. Predicting film permeability needs for modified atmosphere packaging of ligh tly processed fruits and vegetables. HortScience. Vol. 30(1). pp. 25-34. Dalgaard, P. 2000. Fresh and lightly preserve d seafood. In Shelf-Life Evaluation of Foods. Second edition (C.M.D. Man, A.A. Jones eds.), Aspen publishers, Gaithersburg, Maryland. pp. 110-133. Doyon, G.J, Gagnon, Castaigne, F. 1991. Gas transmission properties of polyvinyl chloride (PVC) films studied under sub ambient and ambient conditions for modified atmospheric packaging app lications. Packaging Technology Science Vol.4. pp. 157-165. Dufrense I, Smith J.P, Liu J.N, Tarte I, Bl anchfield B, Austin J. W. 2000. Effect of films of different OTR on toxin production by C. botulinum Type E in vacuum packaged cold and hot-smoked trout fillets. Journal of Food Safety. Vol. 20. pp. 251-268. Farber, J.M. 1991. Microbiological aspects of modified atmosphere packaging technology –A review. Journal of F ood Protection. Vol. 54(1). pp. 58-70. FDA/CFSAN Bad Bug Book. 1992. http: //www.cfsan.fda.gov/mow/chap2.html Food and Drug Administration (FDA). 1997. Food processing. Food code. Annex 6. Food and Drug Administration (FDA). 2001. Fi sh and Fishery Products Hazards and Controls Guide, 3rd ed. Washington, D.C Food and Drug Administration (FDA). 2002. Import Alert # 16-125.

PAGE 165

153 Fitzgerald, M, Papkovsky, D.M, Smiddy, M, Ke rry, J.P, O’Sullivan, C.K, Buckley, D.J, Guilbault, G.G. 2001. Nondestructive mon itoring of oxygen profiles in packaged foods using phase-fluorimetric oxygen sens or. Journal of Foods Science. Vol. 66(1). pp. 105-110. Gould, G.W. 1996, Methods for preservation and extension of shelf-life. International Journal of Food Microbiology. Vol.33, pp. 51-64. Johnson, B. 1997. Headspace analysis and shelflife. Cereals Foods World. Vol. 42(9). pp. 752-754. Morris, J.G. 2000. The effect of redox potenti al. In The Microbiological Safety and Quality of Food (B.M. Lund, T.C. Bair d-Parker, G.W. Gould eds.), Aspen Publishers, Gaithersburg, Maryland. Vol.1, pp. 235-250. Lund, B.M, Wyatt, G.M. 1984. The effect of redox potential and its interaction with sodium chloride concentration on the probability of growth of C. botulinum type E from spore inocula. Food Microbiology. Vol. 1. pp. 49-65. Mapes, M., Hseuh, H.C., Ji ang, W.S. 1994. Permeation of argon, carbon dioxide, helium, nitrogen and oxygen through Mylar windows. Journal of Vacuum Science and Technology. Vol.12(4). pp. 1699-1704. Montville, T.J and Conway, L.K 1982. Oxidatio n reduction potential o the outgrowth and chemical inhibition of Clostridium botulinum 10755 A spores. Journal of Food Science. Vol.44. pp. 700-705. National Advisory Committee for Microbiological Criteria for Foods. 1991. Vaccum or modified atmosphere packaging for refrigerated raw fishery products. http://www.fsis.usda.gov/OPHS/nacmcf/past/map_fishery.htm NFPA 1989, Guidelines for the Developmen t Production and Handling of Refrigerated Foods. National Food Processors Association, Washington, D.C. Otwell, S. 2002. Proceedings of 6th Joint meeting Seafood Science and Technology Meeting and Atlantic Fisheries Technology Society. Robertson, G.L. 1992. Food Packaging. Marcel Dekker, New York. Rubino, M., Tung, M.A., Yada. S., Britt, I. J. 2001. Permeation of oxygen, water vapor, and limonene through printed and unprinte d biaxially oriented polypropylene films source. J Agric and Food Ch emistry. Vol. 49(6). pp. 3041-3045. Sloan, E.A. 2000. Food Technology. Vol. 54(12). pp. 22. Sumner, S.S, Albrecht, J.A. 1994. Clostridium botulinum Nebraska Cooperative Extension NF94-162. http://www.ia nr.unl.edu/pubs/foods/nf162.htm

PAGE 166

154 Smoot, L.A, Pierson, M.D. 1979. Effect of oxi dation reduction potential on the outgrowth and chemical inhibition of Clostridium botulinum 10755A spores. Journal of Food Science. Vol 44. pp. 700-704. Tewari, G, Jayas, D.S, Holley, R.A. 1999. Cent ralized packaging of retail meat cutsA review. Journal of Food Pr otection. Vol. 62(4). pp. 218-425. Welt, B.A, Sage, D.S, Berger, K.L. 2003. Performance specification of time-temperature integrators designed to protec t against botulism in refrigerated fresh foods. Journal of Food Science. Vol. 68(1). pp. 2-9. Zakour, O.P. 2001. Vacuum packaging and redu ced oxygen packaging of foods. Venture, A Newsletter for the Small Scale Food Entrepreneur Vol. 3(3).

PAGE 167

155 BIOGRAPHICAL SKETCH Jayashree Gnanaraj was born in Madura i, TamilNadu, India, in May 1980 and moved to Coimbatore in 1989 where she completed her elementary, secondary school certification and undergraduate studies. In May 2001 she received her bachelor of engineering degree in food pro cessing and preservation tech nology from Avinashilingam University where she was among top three and the student body president of engineering department 00’01‘ which comprised of 600 st udents. She was also the secretary of Food Technology Association 99’00’. During the last year of under graduation studies she interned in Indira Gandhi Cent er for Atomic Research which made her realize her passion for scientific research. In August 2001 she was admitted in Agri cultural and Biological Engineering Department at the University of Florida and now feels prepared to get involved with the food processing field because of the excel lent mentoring provided by the faculty.


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Material Information

Title: Evaluation of oxygen transmission rate of packaging films on growth of clostridium sporogenes and media oxidation reduction potential in packaged seafood simulating media
Physical Description: Mixed Material
Creator: Gnanaraj, Jay Ashree ( Author, Primary )
Publication Date: 2003
Copyright Date: 2003

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0001087:00001

Permanent Link: http://ufdc.ufl.edu/UFE0001087/00001

Material Information

Title: Evaluation of oxygen transmission rate of packaging films on growth of clostridium sporogenes and media oxidation reduction potential in packaged seafood simulating media
Physical Description: Mixed Material
Creator: Gnanaraj, Jay Ashree ( Author, Primary )
Publication Date: 2003
Copyright Date: 2003

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0001087:00001


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EVALUATION OF OXYGEN TRANSMISSION RATE OF PACKAGING FILMS ON
GROWTH OF CLOSTRIDIUM SPOROGENES AND MEDIA OXIDATION
REDUCTION POTENTIAL IN PACKAGED SEAFOOD SIMULATING MEDIA
















By

JAYASHREE GNANARAJ


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Jayashree Gnanaraj

































This thesis is dedicated to my parents and my brother who have always supported and
encouraged me from near and afar.














ACKNOWLEDGMENTS

I am grateful to Dr. Bruce A. Welt, my advisor, supervisor and mentor who taught

me more than I hoped to learn here at graduate school, without whose support this

research work would not have been possible. His work has been my inspiration. This

work has been a product of his patience and endurance. He has inspired me to be a better

researcher and also a better person. He understood my problems and helped me to

succeed inspite of them. My success is and will be a reflection of his outstanding abilities

as a teacher. Nothing short of this will be adequate to express my gratitude to him.

I would like to thank Dr. Art A. Teixeira and Dr. Hordur G. Kristinsson for

agreeing to serve on my committee, guiding me and always ready to help. I would like to

thank Dr. Steven Otwell for his suggestions. I would like to thank National Fisheries

Institute and Florida Sea Grant for financial assistance without which this project would

not have been completed.

This paper is also result of enduring support and love and cooperation of my

parents, Mrs. and Mr. Gnanaraj. I would like to thank my brother Sriram for being there

for me. My family members have given me strength for what I started. I am indebted to

them for being there as unshakeable pillars of support.

This thesis is incomplete without acknowledging my friends in Gainesville. Special

thanks go to Bob, Billy, Dhuruva, Ralph, Teresa and Vivek. Most of all I would like to

thank the faculty and staff in Department of Agriculture and Biological Engineering.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .................... .. ................................... .... ...... ... ............ .. vii

LIST OF FIGURES ............................... ... ...... ... ................. .x

ABSTRACT ........ .............. ............. ...... .......... .......... xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

FD A A lert .................................... ...................................... ..............
F ood-B orn e B otu lism ..................................................................... ......................2
Significance of Clostridium botulinum ............. ............................................2
Conducive conditions for growth of C. botulinum .......................................3
R educed O oxygen Packaging ............................................... ............. ...............3.
Packaging of Horticultural Products ............. ................................................4
Packaging of Flesh Foods ...................................... ...... ....... ........ 5
Dynamic nature of atmosphere in ROP packaged flesh foods ....................................5
R research H ypothesis............. .......................................................... .................

2 EFFECT OF TEMPERATURE AND RELATIVE HUMIDITY ON FILM
PERM EABILITY .................. ........................................ ................ .8

M materials and M ethod ...................................................... .... .. ........ ..
R results and D discussion ............................................... ........ .. ............ 13

3 EFFECT OF FILM OTR, PACKAGE AREA AND TEMPERATURE ON
CLOSTRIDIUMSPOROGENES SPORE OUTGROWTH...................................... 18

M materials and M ethod ..................................................................... ............... 20
R results and D iscu ssion .............................. ........................ .. ...... .... ...... ...... 25

4 CONCLUSION AND FUTURE WORK ....................................... ............... 34

Conclusion ............... ... ...... ..............................................34
F u tu re W o rk ...................................................... ................ 3 5









APPENDIX

A OXYGEN TRANSMISSION RATE OF PACKAGING FILMS AT DIFFERENT
TEMPERATURES AND RELATIVE HUMIDITY......................................36

B DIGITAL PICTURES OF SPORE OUTGROWTH IN DIFFERENT FILMS
A N D B A G SIZ E S ............ ............................................................................ .. .. .. 39

C OXIDATION REDUCTION POTENTIAL WITHOUT PH COMPENSATION.. 150

L IST O F R E F E R E N C E S ...................................................................... ..................... 152

BIOGRAPHICAL SKETCH ............................................................. ............... 155
















LIST OF TABLES


Table pge

2-1 F ilm description ....... .......................................................................... ....... .. .. 10

2-2 Oxygen transmission rates of different films measured at different temperature
and relative hum idity ....... .......................... ........ ..... ........ .. ........ .... 13

2-3 Comparison of measured oxygen transmission rates with value reported by
manufacturer .......................... .................... .... ........ ....... ....... 13

2-4 Ea and ko values for Arrhenius relationship between OTR and temperature for the
packaging film s at 0% R H ............................................... ............................ 16

2-5 Ea and ko values for Arrhenius relationship between OTR and temperature for the
packaging film s at 50% RH .................................. .....................................16

3-1 OTR of film used in this study ................................................... ..................21

3-2 Spore outgrowth over time in regular media for various film types at various
tem p eratu re s ....................................................... ................ 2 6

3-3 Spore outgrowth over time in anaerobic media for various film types at various
tem p eratu re s ....................................................... ................ 2 8

3-4 Oxidation reduction potential of highly reduced anaerobic media in bags of
various film types at different temperatures......................................................30

A-1 OTR of packaging films at 0% RH ................................ ...................37

A-2 OTR of packaging films at 50% RH ................................ ..................38

B-1 Growth table for C60 8X8 at 15C Anaerobic Media.......................... ...........40

B-2 Growth table for AET 8x8 at 15C Anaerobic Media.........................................44

B-3 Growth table for BDF 8x8 at 15C Anaerobic Media.........................................47

B-4 Growth table for C60 18X14 at 15C Anaerobic Media............... ..................51

B-5 Growth table for AET 18X14 at 15C Anaerobic Media..................................55









B-6 Growth table for BDF 18X14 at 15C Anaerobic Media.................................59

B-7 Growth table for C60 8X8 at 23 C Anaerobic Media.................... .......62

B-8 Growth table for AET 8x8 at 23C Anaerobic Media............... ................63

B-10 Growth table for C60 18x14 at 23C Anaerobic Media..................... .........65

B-11 Growth table for AET 18x14 at 23C Anaerobic Media............. ..............66

B-12 Growth table for BDF 18x14 at 23C Anaerobic Media............. ..............67

B-13 Growth table for C60 8x8 at 30C Anaerobic Media................................... 68

B-14 Growth table for AET 8x8 at 30C Anaerobic Media.........................................69

B-15 Growth table for BDF 8x8 at 30C Anaerobic Media.........................................70

B-16 Growth table for C60 18x14 at 30C Anaerobic Media........................................71

B-17 Growth table for AET 18x14 at 35C Anaerobic Media.......................................72

B-18 Growth table for BDF 18x14 at 35C Anaerobic Media.......................................73

B-19 Growth table for C60 8x8 at 35C Anaerobic Media................................... 74

B-20 Growth table for AET 8x8 at 35C Anaerobic Media......................................75

B-21 Growth table for BDF 8x8 at 35C Anaerobic Media......................................75

B-22 Growth table for C60 18x14 at 35C Anaerobic Media........................................76

B-23 Growth table for AET 18x14 at 35C Anaerobic Media.......................................76

B-24 Growth table for BDF 18x14 at 35C Anaerobic Media.......................................77

B-25 Growth table for C60 8x8 at 15C Regular M edia.............................. ...............78

B-26 Growth table for AET 8x8 at 15C Regular Media............................................81

B-28 Growth table for C60 18x14 at 15C Regular Media ............... ..................87

B-29 Growth table for AET 18x14 at 15C Regular Media................ ................90

B-30 Growth table for BDF 18x14 at 15C Regular Media......................................93

B-31 Growth table for C60 8x8 at 20C Regular M edia...............................................96

B-32 Growth table for AET 8x8 at 20C Regular Media .......................................99









B-33 Growth table for BDF 8x8 at 20C Regular Media .................. .............. 102

B-34 Growth table for C60 18x14 at 20C Regular Media............................................105

B-35 Growth table for AET 18x14 at 20C Regular Media...............................108

B-36 Growth table for BDF 18x14 at 20C Regular Media .......................... .........111

B-37 Growth table for C60 8x8 at 30C Regular Media....... ..................................114

B-38 Growth table for AET 8x8 at 30C Regular Media.................. ..... ... .............117

B-39 Growth table for BDF 8x8 at 30C Regular Media............................120

B-40 Growth table for C60 18x14 at 30C Regular M edia.............................................123

B-41 Growth table for AET 18x14 at 30C Regular Media............. .................126

B-42 Growth table for BDF 18x14 at 30C Regular Media ................ ..................129

B-43 Growth table for C60 8x8 at 35C Regular Media...................................132

B-44 Growth table for AET 8x8 at 35C Regular Media..................... .............135

B-45 Growth table for BDF 8x8 at 35C Regular Media........... ...................138

B-46 Growth table for C60 18x14 at 35C Regular Media............... ...............141

B-47 Growth table for AET 18x14 at 35C Regular Media........................... .........144

B-48 Growth table for BDF 18x14 at 35C Regular Media.................. .............147

C-l Redox potential values without compensating for pH 7....................................151
















LIST OF FIGURES


Figure page

2-1 M ocon Oxtran 2/20 .................................. ... ..... .. .. ............... 10

2-2 F ilm cutting tem plate .................................... .................................... .................... .... 11

2-3 A diagram representing gas flow through films inside MOCON instrument. .........12

2-4 Comparison of 0% and 50% RH of C60 ............ ........................ .. ..............14

2-5 Arrhenius relationship between OTR and temperature at 0% RH.........................15

2-6 Arrhenius relationship between OTR and temperature at 50% RH......................15

2-7 Comparison of PE, C60 and C75 FTIR spectra.................................................17

3-1 Sample of bag sizes used for the experiment ................................ ..................... 22

3-2 Rack arrange ent inside the chamber .......................................... ............... 22

3-3 Back lighted stand used for taking digital pictures ..............................................23

3-4 Fiber optic oxygen sensor system ........................................ ........................ 23

3-5 Oxygen sam pling inside the bag ........................................ ........................ 24

3-6 Equipment used to measure ORP and pH............................. ..........25

3.7 Control plate at 30C inside anaerobic box.................................. .................. ....28

3-8 Headspace oxygen content over time in film types C60 and BDF at 23C ............31

3-9 Dissolved oxygen content over time in highly reduced media at 23C for film
types C 60 and B D F ....................... .. ........................ .. .. ....... ........... 32

3-10 Dissolved oxygen content over time in highly reduced media at 35C for film
types C60 and BD F ............................................................. 32















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 Engineering

EVALUATION OF OXYGEN TRANSMISSION RATE OF PACKAGING FILMS ON
GROWTH OF CLOSTRIDIUM SPOROGENES AND MEDIA OXIDATION
REDUCTION POTENTIAL IN PACKAGED SEAFOOD SIMULATING MEDIA

By

Jayashree Gnanaraj

August 2003

Chair: Dr. Bruce A. Welt
Major Department: Agricultural and Biological Engineering

Studies with packaged fish have shown that obvious spoilage can be delayed by

removing oxygen. However, anaerobic pathogenic Clostridium botulinum may thrive in

reduced oxygen packaging, causing packaged fish to become toxic prior to obvious

spoilage. In an attempt to mitigate development of reduced oxygen atmospheres within

fresh seafood packaging, FDA has specified a minimum oxygen transmission rate (OTR)

for seafood packaging films of 10,000 cc/m2/day at 24C. However, this specification

does not take the actual package design into consideration. It is suspected that a

specification that combines film OTR with descriptive parameters of the package, such as

film area, may offer a better structure for specification. Additionally, while it is generally

accepted that C. botulinum is an obligate anaerobe, it remains unclear if a particular

concentration of oxygen is capable of preventing toxigenesis. Like C. botulinum, C.

sporogenes is an obligate anaerobe but nonpathogenic, so it was used as a surrogate for

C. botulinum in this study.









The objective of this work was to develop a scientific rationale for a new seafood

package OTR specification, and to study the relationships among film OTR, package area

and storage temperature on C.sporogenes spore outgrowth in regular and anaerobic

media.

Commercially available packaging films with a wide range of OTR were used in

the study. OTR as a function of temperature was determined in the range of 10-3 5C at

0% and 50% relative humidity (RH). Films were converted into packages with areas of

8x8 and 18x14 inches. Inoculated petri dishes were sealed in these packages using

multiple vacuum/ nitrogen gas flush cycles. Inoculated packages were incubated at 10,

15, 20, 30 and 35 C. Dynamic oxygen concentrations were measured in packaged media

and package headspace. Oxidation reduction potentials (ORP) of media were measured

before and after incubation.

As expected, oxygen levels in high OTR films increased quickly to an approximate

level of 12% 02. Oxidation reduction potentials tended to become more positive with

rising oxygen levels, suggesting that sample ORP plays an important role in predicting

potential outgrowth of spores.

Results suggest that a critical parameter for inhibiting outgrowth is the time

required to raise oxygen concentration sufficiently to increase ORP above some critical

value. It was found that package area, within a practical range of package dimensions, is

not sufficiently important to provide an avenue for modifying FDAs OTR guideline.

Since film OTR plays a key role in this process, this parameter may continue to offer the

most convenient approach toward ensuring safety of fresh seafood.














CHAPTER 1
INTRODUCTION

Limited availability and increased transportation of raw fish and seafood make it

important to minimize losses. Improved management and food preservation technology

are needed because trends show increased interest in minimally preserved products

(Gould, 1996). Annual landed seafood in Florida was estimated to be over $200 million

(Welt et al., 2003). Fresh pre-prepared seafood items like sushi, raw oysters and clams

and use of fish as a substitute for meat have been instrumental in making fish/seafood an

everyday alternative. Seafood menu mentions for entrees were up 10.2% over previous

year in 2000, growing more than any other center-of-the-plate category, including

chicken and beef (Sloan, 2000). Determination and prediction of shelf life of fresh fish

and lightly preserved seafood has become particularly important to prevent losses due to

spoilage.

FDA Alert

Specifics of Alert

Section 402 (a) (4) of Food, Drug and Cosmetic Act considers refrigerated fresh

fish stored under reduced oxygen conditions such as modified atmosphere packaging

(MAP) and vacuum packaging (VP) as adulterated when no controls for Clostridium

botulinum toxin liberation are employed. FDA issued an import alert which states

"Detention without physical examination of refrigerated products (not frozen) vacuum

packaged or modified atmosphere packaged raw fish and fishery products due to the

potential for C. botulinum toxin production" (FDA, 2002).









This alert affects 4100 U.S seafood processors, most of which are small scale

businesses responsible for processing over 350 species of fish. The alert also affects

foreign seafood processors and U.S seafood importers. Overall financial impacts caused

by these regulations are estimated to be more than $1 million per year (Otwell, 2002).

FDA identifies the following two ways to package unfrozen fish products safely:

* Use of packaging film with a minimum OTR of 10,000 cc/m2/day.

* An indicator can be used in or on the packaging to show that the product has not
been exposed to time and temperature combination that could result in an unsafe
product between the time of packaging and the time of use by the consumer.

Food-Borne Botulism

Food borne botulism is a severe type of food poisoning due to ingestion of foods

containing potent neurotoxin produced by Clostridium botulinum. Intoxication occurs

when toxin enters the body and directly affects bodily functions. Symptoms of this

progressive paralytic disease begin with numbness in the extremities and double vision.

Death is often slow and typically results from suffocation as control of respiration fails.

Though incidence of food borne botulism is low, it remains a considerable food safety

concern because of high mortality rates.

Significance of Clostridium botulinum

C. botulinum is a food pathogen that is common in the natural environment,

particularly in soil and marine and freshwater sediments. This organism is so ubiquitous

that it is not possible to exclude it from foods. C. botulinum is a rod shaped gram

positive, anaerobic bacteria capable of forming heat resistant spores that withstand long

periods of dryness and fairly severe thermal treatments. Seven (A, B, C, D, E, F and G)

strains are recognized based on their antigenic specificity of toxin. Strains causing human

botulism include types (A, B, E and F), while botulism from types C and D occurs in









animals. Given favorable conditions, this organism produces a heat labile neurotoxin that

can be destroyed by boiling for 10 minutes or longer (Sumner et al., 1995). An extremely

small amount of toxin (few nanograms) has been shown to be capable of causing illness.

In 1987, eight cases of type E botulism that occurred due to the consumption of dry salted

whole uneviscerated fish (FDA/CFSAN, 1992).

Conducive Conditions for Growth of C. botulinum

Botulism has been associated with

* Inadequately processed home canned foods.

* Foods with water phase salt concentrations less than 5% (water activity, aw, of
0.97).

* Almost any type of food that is not very acidic (pH above 4.6)

* Sausages, meat products, canned vegetables and seafood products have been the
most frequent vehicles for botulism (FDA/CFSAN, 1992).

Reduced Oxygen Packaging

Altering atmospheres within food packages to extend shelf life is a method of food

preservation. Reduced oxygen packaging (ROP) contains little or no oxygen. FDA

defines ROP as any package that when sealed, has the potential to result in an internal

atmosphere that contains lower concentration of oxygen than standard ambient

conditions. Cook-chill, controlled atmosphere packaging (CAP), modified atmosphere

packaging (MAP), sous vide and vacuum packaging (VP) fall under ROP category.

Advantages of Reduced Oxygen Packaging

Advantages of ROP include

* Prevents growth of aerobic spoilage micro organisms such as pseudomonas,
aerobic yeast and molds which are often responsible for organoleptic spoilage.

* Shelf-life extension.









* Inhibition of oxidative processes that degrade food quality.

* Prevents color deterioration in raw meats during storage and retail display.

* Reduces product shrinkage by preventing water loss (FDA, 1997).

Trends and Rationale for Vacuum Packaging and Modified Atmosphere Packaging

The principle involved in VP is removal of gases from a package. MAP involves

methods to maintain a specific gaseous atmosphere within the package that is different

from standard atmospheric conditions. MAP in conjunction with refrigeration has been

shown to increase shelf life of many types of foods. MAP offers several potential

advantages to the seafood industry, including

* Possibility of centralized production.
* Reduced economic loss by preventing quality degradation.
* Increased distribution efficiency due to standardized packaging.
* Potential shelf-life increases of 50 to 400% (Farber, 1991).

Relationship between packaging film permeability to food safety and quality

Ability to establish and maintain a specific atmosphere in MAP packaging depends

on gas permeation characteristics of the packaging films particularly with respect to

oxygen and carbon dioxide.

Packaging of Horticultural Products

When applying MAP to horticultural products like fruits and vegetables, it is often

desired to maintain low oxygen levels and relatively high carbon dioxide levels

(Robertson, 1992). Such conditions tend to slow product respiration resulting in extended

shelf life. To achieve specific modified atmospheres, a delicate balance between film

permeation and product respiration must be established. When this balance is violated,

either due to improper packaging films or abusive temperatures, anoxic conditions can









develop which results in rapid product quality loss. As a result of these considerations,

highly permeable films are typically used in such applications.

Packaging of Muscle Foods

Important properties to be considered during packaging of muscle foods are

product color and microbial population. Although oxygen may be harmful to red meat

product, it is essential for development of the bright red color that consumer's desire.

Since packaged flesh foods do not respire, MAP of such foods typically involves flushing

packages with a specific atmosphere prior to sealing. Use of high barrier films (low

permeability) are intended to "trap" injected gases in the package. The primary gases

involved are oxygen, carbon dioxide and nitrogen. These packaging techniques typically

utilize high barrier films in an attempt to trap modified atmospheres within package.

Dynamic Nature of Atmosphere in ROP Packaged Muscle Foods

Flesh foods spoil through the combined effects of chemical reaction, biochemical

reactions (enzyme activity) and microbial growth. These reactions typically consume

oxygen, which can lead to anaerobic conditions inside the package. This often leads to

progression of microbial activity from aerobic to facultative anaerobe to obligate

anaerobic. There is a possibility of C. botulinum producing neurotoxin under favorable

conditions which may render foods toxic prior to visible signs of organoleptic spoilage.

Potential Control for ROP Fish

The National Advisory Committee for Microbiological Criteria for Foods

recommended temperature control below 3.3 C as a primary preventive measure against

C. botulinum growth. However, temperature abuse of 7-10C is encountered by the

product in retail and distribution chain (NACMCF, 1991). National Food Processors









Association (NFPA) has recommended that there be a secondary safety control for foods

that are packaged in reduced oxygen atmospheres and offered at retail (NFPA, 1989).

Recently FDA has put forward following control guidelines for ROP seafood.

* Packaging material has a permeability of more than 10,000 cc/m2/day at 24C
* Water phase salt level is at least 5%
* Water activity (aw) is below 0.97
* pH is 5.0 or less
* Time temperature integrators (FDA, 2002)

Any one hurdle, or a combination of several, may be used to control pathogenic

outgrowth.

It is important to note that the motivation of the recent FDA alerts was not to

control toxigenesis, but to ensure normal rapid aerobic spoilage so that toxigenesis does

not precede organoleptic spoilage.

Potential Weakness in FDA's OTR Specification

FDA's specific interpretation of ROP covers all unfrozen seafood in any

hermetically sealed package with oxygen transmission rate less than 10,000 cc/m2/day.

This results in different absolute oxygen transmission rates in terms of cc 02/package/day

for packages with different films areas. A question arises as to whether an improved

regulation based on whole package area (cc/package/day) might provide better safety for

ROP fish. Such a specification would extend the flexibility of packaging film selection

and allow manufacturers to choose any packaging film, provided that sufficient film area

is used to achieve a minimum absolute oxygen transmission rate into packages.

Research Hypothesis

The hypothesis of this study is that Clostridium sporogenes spores will germinate

and grow sooner and more robustly in packages with less film area than those with more









film area for any given film. To test this hypothesis a design of experiments were

conducted in two parts in this project with the following specific objectives :

Part I objectives (addressed in Chapter 2) were to

* Determine oxygen transmission rate (OTR) of commercially available packaging
films

* Study the effect of temperature and relative humidity on OTR.

Part II objectives (addressed in Chapter 3) were to measure

* Time required to observe visible colonies in inoculated regular and anaerobic
(highly reduced) seafood simulating bacterial media when packaged with different
areas and incubated at different temperatures.

* Dynamic oxygen profiles in package headspace and media during inoculation.

* Oxidation reduction potential of media samples prior to packaging and when
visible colonies were observed.














CHAPTER 2
EFFECT OF TEMPERATURE AND RELATIVE HUMIDITY ON FILM
PERMEABILITY

Properly designed food packaging systems offer a means of extending shelf lives of

food products. Traditionally, packaging was viewed as a simple physical barrier against

contamination or recontamination of contained food. Plastic films are being increasingly

used in food packaging due to advantages in physical, chemical, mechanical and

economic properties over other package materials such as metals, glass and paper

(Rubino et al., 2001). With recent trends towards minimally processed foods, packaging

must play a greater role in protecting consumers from microbiological hazards associated

with foods (Brody, 2001). Shelf life of products that have not undergone antimicrobial

treatment (e.g., sterilization, pasteurization, freezing) depends on initial food quality and

design of the package.

A package that results in a reduced oxygen level (less than 21%) in a sealed

package is often referred to as reduced oxygen packaging (ROP). Even when higher

levels of oxygen are used, concentrations can fall below safe levels due to

microbiological and chemical activity (Cameron et al., 1993). When oxygen levels fall

below safe levels, anaerobic conditions develop inside the package. Anaerobic conditions

favor growth of Clostridium botulinum while suppressing typical aerobic spoilage

organisms, which are responsible for the organoleptic cues of spoilage. Since consumers

rely on spoilage indications to make consumption decisions, anaerobic conditions may

allow foods to appear acceptable even though pathogens and toxins are present. This has









led FDA to restrict the use of ROP for certain food products. A recent example involves

types of fresh fish and other seafood products. In order to ensure typical aerobic spoilage,

FDA has set a minimum OTR level of for packaging material that may be used for fresh

fish as one approach for protecting consumers from botulism. FDA's current minimum

OTR specification is stated as follows "... packaging that provides an oxygen

transmission rate of 10,000 cc/m2/ 24 hrs at 24C (e.g. 1.5 mil polyethylene) can be

regarded as an oxygen-permeable packaging material for fishery products" (FDA 2002)

Small errors in permeability can cause significant deviation between the predicted

and the actual oxygen levels in packages (Cameron et al., 1995). Since there are very

little data published for permeation of gases through various films (Mapes et al., 1994)

direct comparisons between reported permeabilities can vary widely, and this has led to

the need for greater availability of permeability data, particularly as a function of

temperature (Doyon et al., 1991).

The aim of this work was to study how OTR varies with temperature and relative

humidity for several commercially available packaging films that might be considered to

be used to package fresh fish. Measurements of OTR are reported for four films obtained

from three different packaging film suppliers. These films were selected based upon their

oxygen transmission rates relative to the FDA specification and were considered as high,

medium and low oxygen transmitters.

Materials and Method

Films tested are identified in Table 1. and consisted of C60 and C75 (Dupont

Wilmington, Delaware Dupont's Clysar division was purchased by Bemis Corporation

on August 1, 2002), AET (Applied Extrusion Technologies, Inc., Atlanta, Georgia) and









BDF (Cryovac-Sealed Air Corporation ,Duncan, South Carolina). The thickness was

measured using a micrometer.

Table 2-1. Film description
Name Type of Film Description
C60 High Transmission Clysar 60 HPGF
C75 High Transmission Clysar 75 HPGF
AET Medium Transmission AET PST2-060
BDF Low Transmission BDF 1000

Oxygen transmission rate (OTR) was measured using a two-cell Oxtran 2/20

(Mocon Controls Inc, Minneapolis) as shown in Figure 2-1


Figure 2-1. Mocon Oxtran 2/20

The test gas was 96% nitrogen and 4% hydrogen. Oxygen (100%) was applied to

the opposite side of the film sample. Films were cut using a razor knife and stainless steel

template that provided a film area for testing of 100 cm2 (Figure 2-2).











R 5.60 -.


7.60


Sampte Area


3.80





3,60

'*: ; ***..IF

.*1


7.60


''*p


Al ol dimensions in cm

Figure 2-2. Film cutting template

Film samples were loaded onto both the cells of the Oxtran 2/20 apparatus for

testing.

Before testing, films were conditioned by flushing test gas over both the film

surfaces to remove traces of oxygen in the sample film. Film samples provided a barrier

between oxygen and the N2/H2 gas streams. Oxygen that permeated through the sample

was carried by the N2/H2 stream and detected by a coulometric oxygen sensor, which

produced an electrical current directly proportional to the flux of oxygen across the film

(Figure 2-3). Measurements of OTR were taken at 0% and approximately 50% RH,










Inner Chamber of Test Cell












Carrier Gas Flow In -)



Carrier Gas Plus Permeated -
Test Gas Flow Out


Outer Chamber of Test Cell


!ing



Film Test Sample





---(---4- Test Gas Flow In




- ->--- Test Gas Flow Out


Figure 2-3. A diagram representing gas flow through films inside MOCON instrument.

and were expressed as cc/m2/day. Experiments were performed at 10, 15, 23, 30, 350C.

Oxygen transmission rates were first determined at 230C to compare with values given by

the suppliers.

Films with highest OTRs were identified using a Mattson Fourier Transform Infra

Red Spectroscopy (FTIR) (Model IR-1000, Madison, Wisconsin) in order to provide

material selection guidance for prospective fresh fish packers.









Results and Discussion

Values of OTR for sample films are provided in Table 2-2. The OTR at 0% and

50% RH only were tested because of the limitation of MOCON instrument.

Table 2-2. Oxygen transmission rates of different films measured at different temperature
and relative humidity
Temperature C 60 C 75 AET BDF
(C) (cc/m2/day) (cc/m2/day) (cc/m2/day) (cc/m2/day)
0% 50% 0% 50% 0% 50% 0% 50%
RH RH RH RH RH RH RH RH
10 4270 3720 3680 3700 1520 1300 370 370
15 5520 4860 4840 4830 2010 1730 500 480
23 8620 7370 7390 7050 3200 2660 710 720
30 12320 10520 10480 10170 4800 4000 1010 1000
35 16210 13690 13800 13270 6430 5400 1370 1320

Thickness and average OTR at room temperature (23 C) and 0% RH are given in

Table 2-3 for each film compared with the values reported by film suppliers.

Table 2-3. Comparison of measured oxygen transmission rates with value reported by
manufacturer
Film Type Manufacturer's Value Measured value
OTR Thickness OTR Thickness
(cc/m2/day) (gauge) (cc/m2/day) (gauge)
C60 9300 60 8620 65
C75 7750 75 7390 75
AET 3100 60 3200 60
BDF 2227 75 710 163

An apparent significant discrepancy was found between measured values and those

supplied with Cryovac's BDF 1000 sample. The roll of film was labeled as 75 gauge, for

which OTR should have been 2227 cc/m2/day. Repeated trials with the BDF 1000 film

resulted in an OTR of 705 cc/m2/day. When measured with a digital micrometer,

however, thickness was found to be about 163 gauge. Supplier provided OTR values

were 2412, 2227, 1474 and 1153 cc/m2/day for 60, 75, 100 and 125 gauge films.











Extrapolating this trend to 163 gauge provides a value of 716, which matches closely to


the measured value.


Results show that none of the films tested satisfy the FDA's film OTR specification


for fresh fish packaging (10,000 cc/m2/24 hrs). Additionally, OTR values were not


significantly altered by increased relative humidity at lower temperatures which are


normally used for seafood storage as shown in Figure 2-4.


18000
0% RH
16000

14000
50% RH
12000

S10000-

8000
1j so o o---------------^ .----------

6000

4000

2000

0
0 5 10 15 20 25 30 35 40
Temperature (degree C)


Figure 2-4. Comparison of 0% and 50% RH of C60

A plot of the logarithm of OTR versus inverse absolute temperature gives a straight


line suggesting that Arrhenius relationships shown in Figures 2-5 and 2-6. At 50% RH


Film 1 and Film 2 have similar OTR.


Oxygen transmission rate increases with temperature as expected. It is possible to


express permeation, OTR, as a function of temperature by the following Arrhenius


expression:



OTR= ko exp{ 2.1
RT












where OTR is Oxygen Transmission Rate in cc/m2/day, ko is the Arrhenius pre-


exponential factor in cc/m2/day, Ea is Arrhenius activation energy in J/mol, R is the Ideal


Gas Law constant (8.314 J/mol/K), and T is absolute temperature in Kelvin (K).


Equation (1) may be used to estimate OTR at a specific temperature. Values for Ea and ko


are tabulated in Tables 4 and 5 for 0% and 50% RH, respectively.



100000



C60
10000 C75
10000




BDF
1000






100
0.0032 0.00325 0.0033 0.00335 0.0034 0.00345 0.0035 0.00355
1/Temperature (K)


Figure 2-5. Arrhenius relationship between OTR and temperature at 0% RH


100000




C60

10000 C75





1000





100
0.0032 0.00325 0.0033 0.00335 0.0034 0.00345 0.0035 0.00355
1/Temperature (K)



Figure 2-6. Arrhenius relationship between OTR and temperature at 50% RH









Table 2-4. Ea and ko values for Arrhenius relationship between OTR and temperature for
the packaging films at 0% RH
Sample ko Ea R
(cc/m2/day) (kJ/mol)

C60 6.00E+10 38.70 0.999

C75 4.00E+10 38.20 0.995

AET 8.00E+10 41.90 0.999

BDF 2.00E+10 36.70 0.993


Table 2-5. Ea and ko values for Arrhenius relationship between OTR and temperature for
the packaging films at 50% RH
Sample ko Ea R2
(cc/m2/day) (kJ/mol)

C60 3.00E+10 37.60 0.999

C75 2.00E+10 36.70 0.997

AET 5.00E+10 41.10 0.998

BDF 2.00E+09 36.50 0.998


Analysis of FTIR was performed for the Clysar films because they were closest to

the FDA OTR specification. Identification of these materials should be helpful in

selecting the potential candidates for seafood packaging. Results of FTIR showed that

these films were essentially thin gauge polyethylene (PE). Sample FTIR spectra are

compared with a library of spectra for known standards (Figure 2-7). Note that, AET

PST2-060 is oriented polypropylene film with inner sealable side, and BDF1000 is

multilayered co-extruded film with external polypropylene layers.








17



400




















4500 4000 3500 3000 2500 2000 1500 1000 500 0
Wavenumbers



Figure 2-7. Comparison of PE, C60 and C75 FTIR spectra














CHAPTER 3
EFFECT OF FILM OTR, PACKAGE AREA AND TEMPERATURE ON
CLOSTRIDIUM SPOROGENES SPORE OUTGROWTH

In modified atmosphere packaging of fresh and minimally processed foods, oxygen

is often intentionally reduced to decrease enzymatic, biochemical and aerobic

microbiological activities. This method of packaging is called reduced oxygen package

(ROP) an FDA term for a package that has a potential to result in oxygen levels below

21%. ROP provides an environment that contains little or no oxygen, offers unique

advantages such as increase in shelf life, improved handling and reduced weight lose.

However, there may be marked increase in safety concerns with some foods, particularly

with ROP fresh fish. Studies have demonstrated that formation of type E botulinum toxin

prior to organoleptic spoilage at mildly abusive temperatures is possible, thus making the

seafood product unfit for consumption (Dufresne et al., 2000; Post et al., 1985; Reddy et

al., 1996, 1997a, 1997b). To mitigate this problem FDA considers a package that

provides an oxygen transmission rate of 10,000 cc/m2/day at 24C as acceptable for

packaging seafood products (FDA, 2002). However, this specification does not take into

consideration the design of the package. It is suspected that a regulation that combines

film OTR with descriptive parameters of the package, such as film area, may offer a

better regulatory alternative and an ease in choosing packaging material by the seafood

manufacturers.

Studies show that residual oxygen plays a key role in food quality and shelf life

determination (Tewari et al., 1999). Oxygen profiles indicate change in quality of









products and also the packaging film's quality. Non-destructive monitoring of oxygen

profiles inside the package and food product has remained a difficult and expensive

objective (Johnson, 1997). Optical sensor approach offers a realistic alternative and a

number of methods of optical oxygen sensing have been described in recent years

(Fitzgerald, 2001). Research shows that C. botulinum is an obligate anaerobe, it remains

unclear if a particular concentration of oxygen is capable of preventing toxigenesis. So

luminescence-based oxygen sensor was used for destructive oxygen measurement for this

study.

Measurement of oxidation reduction (Eh) potential redoxx potential) could provide

information on how the background redox potential might be adjusted by addition of a

suitable oxidant or reductant as to make the substrate uncongenial to the likely microbial

contaminants while not affecting its palatability and attractiveness as a foodstuff (Brown

and Emberger 1980). In anoxic conditions, a marked fall in C. botulinum culture Eh can

accompany germination of a large spore inoculum, thereby providing conditions suitable

for multiplication of the outgrowing vegetative cells (Morris, 2000). Studies have been

conducted to see whether there can be a limiting value of redox potential to prevent the

growth of C. botulinum (Lund and Wyatt, 1984; Montville and Conway, 1982). But there

are very few data available for C. botulinum growth and toxin production where Eh has

been used as a variable (Smoot and Pierson, 1979).

Clostridium sporogenes is an obligate anaerobe but non pathogenic with similar

physiological properties to C. botulinum. Therefore, it was used as a surrogate for C.

botulinum in this study.

The objective of this work was to develop a scientific rationale for a new seafood

package OTR specification, and to study the relationships among film OTR, package area









and storage temperature on C.sporogenes spore outgrowth in regular and anaerobic

media.

Materials and Method

Sample Preparation

C.sporogenes (PA 3679) spores were purchased from National Food Laboratory

Inc., (Dublin, California). When spores were received, a stock solution was prepared by

diluting 10 ml of 2x107 CFU/ml into 1000 ml of autoclave-sterilized, 0.15 M potassium

phosphate buffer solution at pH 7. The initial concentration for all trials was 2x104

CFU/ml.

Spore Enumeration

Inoculum was treated at 80C for 20 mins to stimulate germination of the spores

and to prevent growth of contaminating organisms. Plates were inoculated with

concentration of 2x103 CFU/ml by pour plate technique. Regular and highly reduced

anaerobic media was used for spore recovery. Regular media was prepared with 24 g of

dehydrated brain heart infusion (Fisher Scientific, Springfield, New Jersey) and 10 grams

of Difco Bacto Agar (Fisher Scientific, Springfield, New Jersey) in 700 ml of 0.15 M

potassium phosphate buffer solution to maintain pH of 7.0. Anaerobic agar was prepared

by boiling 40.6 grams of anaerobic agar (Scientific, Springfield, New Jersey) in 700 ml

of distilled water. The media ingredients were transferred to a Teflon bottle (Nalgene

Nunc International, Rochester, New York) and autoclave sterilized along with test tubes

and pipette tips. Twenty-eight plates were prepared by aseptic pour plate technique for

each set of experiments.

Film Samples









Bags of two different sizes, 0.083 m2 and 0.325 m2, representing small and large

sizes (8x8 and 18x14 inches respectively) were made from the three different film

samples mentioned in table 1.

Table 3-1. OTR of film used in this study
Film Name Film ID Film Type OTR
(cc/m2/day)
Clysar 60 HPGF C60 High Transmission 8620
AET (PST2-060) AET Medium Transmission 3200
BDF 1000 BDF Low Transmission 710

Duplicate samples were made by placing two plates in each bag. The bags were

vacuum packed, gas flushed with nitrogen and sealed using a vacuum packaging machine

(Multivac, Kansa City, Missouri). Specifically the machine was programmed to reduce

pressure via vacuum from 1 atm to 0.15 atm and then return to 0.8 atm with nitrogen gas.

The vacuum/gas-flush cycle occurred three times .Samples were stored at 10, 15, 20, 30

and 35C in the state-of-the-art environmental growth chambers. Two plates were placed

in an anaerobic box (Mitsubishi Gas Chemical Co., Inc, New York, New York) and kept

with samples inside the environmental growth chambers. Digital pictures of the plates

were taken using Nikon COOLPIX 5000 every 8-12 hrs until visible growth was

observed. The time taken to note visible colony growth in the sample was recorded.

Monitoring Oxygen Composition

Dynamic oxygen concentration profiles were monitored using a 4-channel FOXY

fiber optic oxygen sensor system (Ocean Optics Inc., Dunedin, Florida). The fiber optic

oxygen sensing system incorporates probes doped at the tip with a compound that

fluoresces in response to input light. Fluorescence is quenched by oxygen. Therefore

anoxic environments result in significant fluorescent response, while increasing

availability of oxygen results in a reduced response. Calibration against known conditions








provides a means to measure gaseous and dissolved oxygen in samples. A FOXY 18-G

fiber optic oxygen probe was inserted inside the bag and dynamic oxygen concentration

in packaged media and headspace was monitored continuously.


Figure 3-1. Sample of bag sizes used for the experiment


:if } --- .:i a


Figure 3-2. Rack arrangement inside the chamber




















Figure 3-3. Back lighted stand used for taking digital pictures

\ ZIRW PM,


Figure 3-4. Fiber optic oxygen sensor system
To place the probe in the media a hole was made using a hot-wire on all
petridishes. Vials (40 ml) with septa equipped with screw caps were cut below the
shoulder of the vial. Sample bags were sandwiched between caps and open ended vials.









Oxygen profile was monitored in the high barrier (BDF) and low barrier (C60) small size

bags (Figure 3-5).



SSeptum
Sample __ Cap
bag 1 Headspace
02 probe
Cut Vial
SDissolved 02
Anaerobic probe
Media




Figure 3-5. Oxygen sampling inside the bag

Oxidation Reduction Potential

Oxidation Reduction Potential of the anaerobic media was measured in millivolts

before and after incubation using Accumet 13-620-81 combination ORP probe (Fisher

Scientific, Springfield, New Jersey). The ORP of a sample is measured by comparing the

electrical potential between an inert electrode (typically platinum) that is in intimate

contact with the sample, and a reference electrode with a known potential versus the ideal

standard hydrogen electrode ("SHE"). The silver-silver chloride reference electrode is

one of the most commonly used reference electrodes due to its ease of manufacture and

its useful temperature range. The electrode is a silver wire coated with a thin layer of

silver chloride that is deposited either by electroplating or by dipping the wire in molten

silver chloride. The ORP value was measured within 24 hrs of visible growth under a

nitrogen blanket. Calibration of the ORP probe was performed in pH 4 potassium acid

phthalate standard buffer solution and pH 7 potassium and sodium phosphate standard









buffer solution (Sensorex, Garden Grove, California). Both buffer solutions were

saturated with quinhydrone at 25C. Simultaneously, pH of the media at the end of the

experiment was also measured. The ORP probe and pH probe were standardized before

each set of experiments to ensure accuracy and consistency of the measuring system.





















Figure 3-6. Equipment used to measure ORP and pH

Measured ORP values were adjusted to pH 7.0 to eliminate the effect of pH on Eh

by use of equation 3.1 (George et al., 1998).

Eh7 = Eobs + Eref + 2.303 (RT/F) (pHX 7.0) 3.1

Where Eobs is the measured potential of the system, Eref is the reference electrode

potential of the internal electrolyte (saturated KC1 silver/silver chloride) of the electrode

and equals 199 mV, 2.303 (RT/F) is the Nernst potential equaled to 59.1 mV at 25C and

pHX is the measured pH of the system.

Results and Discussion

Time taken for spore outgrowth in regular and highly reduced anaerobic media in

different bags over a period of 15 days in temperatures 10, 15, 20, 30 and 35C is










tabulated in tables 3-2 and 3-3. Different bag sizes are represented as "S" denoting small

bag and "L" denoting large bag of sizes 0.083 m2 and 0.325 m2 respectively. The "B1"

and "B2" represent sample duplicates for bag 1 and bag 2, respectively.

Table 3-2. Spore outgrowth over time in regular media for various film types at various
temperatures


Temperature
(C)


Time in days


Film Type
and
Bag Size
1
C60-S B1 -
B2-
C60-L B1 -
B2-
AET-S B1 -
B2-
AET-L B1 -
B2-
BDF-S B1 -
B2-
BDF-L B1 -
B2-
Control
C60-S B1 -
B2-
C60-L B1 -
B2-
AET-S B1 -
B2-
AET-L B1 -
B2-
BDF-S B1 -
B2-
BDF-L B1 -
B2-
Control
C60-S B1 -
B2-
C60-L B1 -
B2-
AET-S B1 -
B2 -
AET-L B1 -
B2-


14 15


2 3 4 5


6 7 8 9























1 1 2 3
222 2

-2 2 2
222
-- 1 1










- 1 1 1


-
-










Table 3.2. (continued)
Temperature Film Type Time in days
(C) and
Bag Size
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
BDF-S B1- ----- -
B2 - -1 1 1 1 1 1 1
BDF-L B1 - 1 1 1 1 1 1 1 1 1
B2 -
Control -- --* *
C60-S B1 3 3 3 3 3 3 3 3 3 3 3
B2 -
C60-L B1 2 2 4 4 4 4 4 4 4 4 4 4 4
B2 -
AET-S B1 1 2 2 2 2 2 2 2 2 2 2 2 2
B2 1 1 1 2 2 2 2 2 2 2 2 2 2
30 AET-L B1- ------ --
B2 1 1 1 1 1 1 1 1 1 1 1 1 1
BDF-S B1 3 4 4 4 4 4 4 4 4 4 4 4 4
B2 -
BDF-L B1- ------ --
B2 -1 1 1 1 1 1 1 1 1 1 1 1 1
Control t t t t t t t t t
C60-S B1- ------ ---
B2 1 1 1 1 1 1 1 1 1 1 1 1 1
C60-L B1- ------ ---
B2 -
AET-S B1 1 1 1 1 1 1 1 1 1 1 1 1 1
B2 -
35 AET-L B1- ------ --
B2 -
BDF-S B1- ------ --
B2 -
BDF-L B1 1 1 1 t t t t t t t
B2 -
Control *
"*" represents growth greater than or equal to 50 colonies
"*" represents growth greater than or equal to 100 colonies
"8" represents growth greater than or equal to 200 colonies
"-"represents no growth

The integers (1, 2, 3, 4..) represent number of visible colonies noted at that period

of time. From the table it can be seen that there was no growth in all samples at 10C for

a period of 15 days. The small bag of AET had growth only at higher temperatures of 30










and 35C. A maximum number of growth in all the bags were seen at 30C. At 35C

comparatively lower growth was noticed in all bags which is because of increase in OTR

at high temperatures. There was growth in all the control plates inside the anaerobic box

at different temperatures. Maximum growth in control plates was noted at temperatures

higher than 15C.

Two to three days after noticing initial growth in the plates, there was no increase

in the number of colonies which represents the rise in oxygen level inside the bag that

prevents further germination of spores.



/A,
















Figure 3.7. Control plate at 30C inside anaerobic box

Table 3-3. Spore outgrowth over time in anaerobic media for various film types at
various temperatures
Temperature Film Type and Time in days
(C) Bag Size
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
C60-S B1 --- ---- -
B2 -
C60-L B1------- -
10
B2 -
AET-S B1 --- ---- -
B2 -
AET-L B1 --- ---- -
B2 -
BDF-S B1 --- ---- -
B2 -
BDF-L B1------- -
B2- -------- -











Table 3.3. (continued)
Temperature Film Type and
(C) Bag Size


Time in days


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
C60-S B1 - -
B2 -- -
C60-L B1 - -
B2 -- -
AET-S B1 - -
B2 ----- -
15
AET-L B1 - -
B2 -- -
BDF-S B1 - -
B2 -- -
BDF-L B1 - -
B2 -- -
C60-S B1 - -
B2 -
C60-L B1 - -
B2 2 3
AET-S B1 4 4
B2- -
23
AET-L B1 -
B2 -
BDF-S B1 -
B2 -
BDF-L B1 -
B2 -
C60-S B1 *
B2 4 8
C60-L B1 *
B2 6 13
30
AET-S B1 -
B2 7 11
AET-L B1 -
B2 -
BDF-S B1 -
B2 -
BDF-L B1 -
B2 -
C60-S B1 *
B2 *
35
C60-L B1 -
B2 -
AET-S B1 -
B2 -
AET-L B1 -
B2 -
BDF-S B1 -
B2 -
BDF-L B1 -
B2 -
"*" represents growth greater than or equal to 100
"*" represents growth greater than or equal to 500
":" represents growth greater than or equal to 1000
-" represents no growth










Unlike the control plates of regular media, surprisingly there was no growth noticed

in control plates of anaerobic media even though they had an ideal anaerobic

environment and low ORP value to germinate. There was no growth noted in 10 and

15C. Compared to regular media, growth was faster in highly reduced anaerobic media.

A gradual increase in the amount of colonies as the temperature increased was obvious.

As expected, time taken to observe growth increased as the temperature decreased.

Oxidation reduction potentials were measured within 24 hours of growth and

values are tabulated below after elimination of pH effect (Table 3-4). Initial redox

potential of anaerobic media before inoculation was measured to be 137 mV.

Table 3-4. Oxidation reduction potential of highly reduced anaerobic media in bags of
various film types at different temperatures
Temperature Film Type ORP Time taken for Visible Growth
(C) (mV) (Days)
S L S L
C60 Bag 1 332.8 266.0
Bag 2 322.0 259.4
15 AET Bag 1 266.6 262.2
Bag 2 300.6 289.8
BDF Bag 1 271.3 257.8
Bag 2 260.8 252.2
C60 Bag 1 222.7 239.8
Bag 2 270.4 254.5 4
23 AET Bag 1 226.7 253.8 4 4
Bag 2 253.0 257.8 4
BDF Bag 1 120.7 78.7 4 4
Bag 2 197.1 94.0 4 4
C60 Bag 1 177.9 177.0 3 3
Bag 2 152.9 118.0 3 3
30 AET Bag 1 110.4 106.0 3 3
Bag 2 216.0 100.6 3 3
BDF Bag 1 224.5 30.6 3 3
Bag 2 251.7 20.0 3 3
C60 Bag 1 27.4 39.7 2 2
Bag 2 208.5 120.3 2 2
35 AET Bag 1 32.4 48.6 2 2
Bag 2 54.8 80.6 2 2
BDF Bag 1 12.3 13.5 2 2
Bag 2 19.9 29.7 2 2










There was an increase in redox potential as the temperature decreased. This is due

to more time taken for spore outgrowth at lower temperatures allowing oxygen to

permeate inside the bag and increase the ORP of the media. Oxidation reduction potential

of media in C60 bags was lower than that of media in BDF bags regardless of bag sizes.

The C60 small bag at 30C shows a high ORP value which was due to change in probe

position or contamination.

The headspace oxygen content in high and low transmission film was monitored

using the FOXY probe at 23 C until growth was observed. Results are shown in Figures

3-8, 3-9,3-10.


18 1


0 1000 2000 3000 4000 5000 6000
Time (mins)


Figure 3-8. Headspace oxygen content over time in film types C60 and BDF at 23C

As seen in Figure 3-8, oxygen partial pressure increased rapidly for the high OTR

C60 film. Dissolved oxygen content in media was measured at 23 and 35C.























v C60


4



BDF
2




0
0 1000 2000 3000 4000 5000 6000
Time (mins)



Figure 3-9. Dissolved oxygen content over time in highly reduced media at 23 C for film
types C60 and BDF


10




8


S6



0
ta 4




2




0


2000


Time (mins)


Figure 3-10. Dissolved oxygen content over time in highly reduced media at 35C for
film types C60 and BDF


3000









The oxygen content of the media gradually increases in C60 whereas in BDF it

follows an irregular pattern due severe growth in media.














CHAPTER 4
CONCLUSION AND FUTURE WORK

Conclusion

Oxygen transmission rate results obtained from MOCON Oxtran 2/20 show that

none of the films studied fully satisfy FDA's OTR guideline of 10,000 cc/m2/day at room

temperature. Clysar 60 (-0.60 mil polyethylene film) provided the highest OTR at

around 8600 cc/m2/day at 230C. These results suggest that it may be difficult for

suppliers of packaged fresh fish to source a variety of acceptable packaging films.

Arrhenius parameters were provided in order to allow estimation of OTR values at

desired temperatures. The lowest temperature studied, 100C, represented the limit of

capability for the MOCON Oxtran 2/20. However, it is expected that at least for the

Clysar (polyethylene) films, Arrhenius relationships will provide suitable OTR estimates

throughout the range important for packaged fresh fish (0 350C), because no significant

latent thermal transitions exist within this extended lower temperature range (glass

transition temperature, Tg, for LDPE occurs at about -1750C).

There is not much variation in growth between small and large bags. The area of

the large bag (0.325 m2) represented the maximum size that can be sealed using available

vacuum packaging equipment. Although larger package areas might have had a

significant effect on spore outgrowth, such sizes would not be of practical value for

packaging seafood. So the research hypothesis of observing a difference in spore

outgrowth between lower and larger film area was not supported.









The difference in package area affected the oxidation reduction potential of highly

reduced anaerobic media at different temperatures. It was observed that as the

temperature decreased, the oxidation reduction potential increased due to the increasing

time taken for visible spore outgrowth. As expected, the headspace oxygen and dissolved

oxygen content in the media was more in C60 than BDF.

Future Work

From these experiments it is observed that redox potential and bag specification

plays a vital role in spore outgrowth. So a package design that combines oxidation

reduction potential and packaging film permeation to control growth of C. botulinum can

be developed. This methodology that combines oxidation reduction potential to dynamic

oxygen permeation might prevent C. botulinum hazard and helps in assessing package

safety.

Similarity in physiological properties between C. sporogenes and C. botulinum has

been mentioned earlier. However, C. sporogenes has a different temperature window of

15-45C for visible spore outgrowth, whereas C. botulinum can grow and produce toxin

at temperatures above 3.3 C. The experimental method and data of this project can be

used to provide a basis for future experiments and clues about potential behavior of C.

botulinum.

This data should not be used by FDA for regulations on control of C. botulinum

spore outgrowth in ROP packed seafood for the above reasons.















APPENDIX A
OXYGEN TRANSMISSION RATE OF PACKAGING FILMS AT DIFFERENT
TEMPERATURES AND RELATIVE HUMIDITY











Table A-1. OTR of packaging films at 0% RH
Sample ID Cell Temperature 1/Tabs
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
A 23 0.003378
Clysar 75HPGF A 23 0003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
Clysr A 23 0.003378
Clysar 60HPGF
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
AETA 23 0.003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
BDF1000 A 23 0.003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247


Measured OTR
3736.3
3570.9
4943.45
4731.95
7631.05
7139.7
10867.95
10080.65
14357.95
13238.8
4263.75
4269.15
5543.75
5483.15
8636.75
8589.9
12343.35
12280.8
16296.85
16119.35
1514.25
1522.2
1999.3
2017.9
3205
3196.9
4795.8
4790.55
6486.25
6371.4
386.95
355.05
503.55
501.05
717.35
690.95
1013.95
1005.25
1364.45
1366.1


R^2


Ln(OTR)
8.2258511
8.1805729
8.5058187
8.4620927
8.9399807
8.873426
9.2935734
9.218373
9.5720591
9.4909072
8.3579043
8.35917
8.6204264
8.609435
9.0637816
9.0583424
9.4208727
9.4157923
9.6987271
9.6877757
7.3226755
7.3279119
7.6005524
7.6098126
8.0724674
8.0699369
8.4754958
8.4744005
8.7774398
8.7595745
5.9582955
5.8722586
6.221683
6.2167059
6.5755639
6.5380675
6.9216089
6.9129915
7.2185067
7.2197152


0.9952











0.9993











0.9992












0.9932











Table A-2. OTR of packaging films at 50% RH
Sample ID Cell Temperature 1/Tabs
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
A 23 0.003378
Clysar 60HPGF
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
A 23 0.003378
Clysar 75HPGF A 23 0003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
AET A 23 0.003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
35 0.003247
35 0.003247
A 10 0.003534
B 10 0.003534
A 15 0.003472
B 15 0.003472
BDF1000 A 23 0.003378
B 23 0.003378
A 30 0.0033
B 30 0.0033
A 35 0.003247
B 35 0.003247


Measured OTR
3706.45
3736
4844.3
4869.1
7372.7
7360.2
10526.55
10504.6
13927.95
13444
3636.35
3774.25
4744.7
4914.2
6982.7
7111
10044.3
10292.95
13033.2
13511.3
1293.3
1303.35
1708.45
1742.25
2649.4
2668.65
3983.65
4002.15
5387.2
5406.9
375.45
364.55
482.45
475.7
728.2
705.35
1008.6
986
1334
1308.75


R^2


Ln(OTR)
8.2178298
8.2257708
8.485558
8.4906644
8.9055393
8.9038424
9.2616559
9.2595685
9.5416529
9.5062882
8.1987357
8.235957
8.4647835
8.4998843
8.8511909
8.8693982
9.2147606
9.2392145
9.4752552
9.5112817
7.1649524
7.1726932
7.4433418
7.4629327
7.8820885
7.889328
8.2899538
8.294587
8.591781
8.5954312
5.9281253
5.8986637
6.1788773
6.1647874
6.5905757
6.5586941
6.9163185
6.8936564
7.1959372
7.1768278


0.999











0.997











0.998











0.998















APPENDIX B
DIGITAL PICTURES OF SPORE OUTGROWTH IN DIFFERENT FILMS AND BAG
SIZES









Table B-1. Growth table for C60 8X8 at 15C Anaerobic Media
Time Bag 2

(Days)

1









2









3









4






41


Table B-1. Continued
Time Bag 2









Table B-1. Continued
Time Bag 2









Table B-1. Continued
Time Bag 2







Table B-2. Growth table for AET 8x8 at 15C
Time Bag 2


* ii j)i..


)ic Media









Table B-2. Continued
Time Bag 2









Table B-2. Continued
Time Bag 2







47



Table B-3. Growth table for BDF 8x8 at 15C Anaerobic Media
Time Bag 2

(Days)

1










2


4


I






48


Table B-3. Continued
Time Bag 2









Table B-3. Continued
Time Bag 2









Table B-3. Continued
Time Bag 2





Table B-4. Growth table for C60 18X14 at 15C Anaerobic Media


Bag 1


Bag 2


C
c'..


911






52


Table B-4. Continued
Time 1 Bag 1


Bag 2






53


Table B-4. Continued
Time Bag 1 Bag 2

(Days)

9









10









11


12









Table B-4. Continued
Time Bag 1 Bag 2

(Days)

13









14


15









Table B-5. Growth table for AET 18X14 at 15C Anaerobic Media
Time Bag 1 Bag 2

(Days)

1








2


4






56


Table B-5. Continued
Time Bag 1 Bag 2

(Days)

5









6










7


8









Table B-5. Continued
Time Bag 1 Bag 2

(Days)

9









10









11


12









Table B-5. Continued
Time Bag 1 Bag 2

(Days)

13








14


15






59


Table B-6. Growth table for BDF 18X14 at 15C Anaerobic Media
Time Bag 1 Bag 2

(Days)

1




























4


5









Table B-6. Continued
Time Bag 1 Bag 2

(Days)

6









7









8








9


10









Table B-6. Continued
Time Bag 1 Bag 2

(Days)

11








12








13








14


15







Table B-7. Growth table for C60 8X8 at 23C
Time Bag 2


U

~rn::-~i-~ii


)






63



Table B-8. Growth table for AET 8x8 at 23 C Anaerobic Media
Time Bag 1 Bag 2
(Days)
1


2


4


5











Table B-9. Growth table for BDF 8x8 at 23C
Time Bag 2


Nl"' a


)ic Media


ii


*r;;;; r
*t
";~3~
'* i Y
~*
r*
12
r.?
..
.. S


~Pti;







Table B-10. Growth table for C60 18x14 at 23C Anaerobic Media


Bag 1


Bag 2


iH
M| .;


aC


~E~
._Y~
~~~.;;;;;;;;;;;i, _


Ix~


$ ''








Bag 2


2.. "J


: *-"a :


Bag 1


P'









Table B-12. Growth table for BDF 18x14 at 23C Anaerobic Media


Bag 1


Csr 0)
SjHfr j


F/


'K


Bag 2









Table B-13. Growth table for C60 8x8 at 30C
Time Bag 1


Wii i!~"


\'


jill


:':~' ~c~
~j~Llii~l









Table B-14. Growth table for AET 8x8 at 30C
Time Bag 1


anaerobic Media
Bag 2






































.. '


:..;d;: ~;dY




































N'


L-5


A


/


-" ~~
--;ii~;;;iiiiiiiiiiiii


:^.
'?**






72



Table B-17. Growth table for AET 18x14 at 35C Anaerobic Media
Time Bag 1 Bag 2
(Days)
1


4





























N


7


I ;
* *


:tii u
'~'F- ~~F~W
ii r.


~-.:~;**r~






74



Table B-19. Growth table for C60 8x8 at 35C Anaerobic Media
Time Bag 1 Bag 2
(Days)
1






2











Table B-20. Growth table for AET 8x8 at 35C


Table B-21. Growth table for BDF 8x8 at 35C Anaerobic Media


Time Bag 1
(Days)
1


2


Bag 2


i.
'i*
.,e'' 'ii
''C ;'?*~Y~









Table B-22. Growth table for C60 18x14 at 35C Anaerobic Media


Table B-23. Growth table for AET 18x14 at 35C Anaerobic Media






77


Table B-24. Growth table for BDF 18x14 at 35C Anaerobic Media
Time Bag 1 Bag 2
(Days)
1







78



Table B-25. Growth table for C60 8x8 at 15C Regular Media
Time Bag 1 Bag 2
(Days)








2






.... ... ........ ....







4







5


6









Table B-25. Continued
Time Bag 1


Bag 2









Table B-25. Continued
Time Bag 1


Bag 2








Table B-26. Growth table for AET 8x8 at 15C
Time Bag 1


s~/^


Media


Bag 2


SI


i./. .J


N


,N


%QC,









Table B-26. Continued
Time Bag 1


s~/^


Bag 2


SI


u


.Y









Table B-26. Continued
Time Bag 1


Bag 2












Table B-27. Growth table for BDF 8x8 at 15C
Time Bag 1


_z; ,./ -.^


Media


Bag 2


H ~


~~,;-


" ,N!H', "".' /i..










Table B-27. Continued
Time Bag 1


Bag 2


... .... ..:."_


EiN!iiii':'i:" E : :" EP:"i~ll
..ii c ... ",ii:.. .'.,2
-;I:~f q .yma
i r~* ~~4:
f


/... i ..i....:i
*2iZ- : ~












Table B-27. Continued


Bag 2


C>!5"".


Time


Bag 1


*C;~i~-~;L~?L


I : ..........


;r .:'Ri;"""'r~BW?
t








Table B-28. Growth table for C60 18x14 at 15
Time Bag 1


s~/^


rK 2


Media


Bag 2


s~f /-


i./. .J


\;?~~


%QC,








Table 28. Continued
Time Bag 1


c1k


Bag 2


S-,,..
)ol


..... I ,
~'*,.


j


\;?~~


.iii .... .... .iN 1i
' -.".I


.. ...