evaluation of oxygen transmission rate of packaging...

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.

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 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.

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

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 POTENTIAL WITHOUT PH COMPENSATION...150

LIST OF REFERENCES.................................................................................................152

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

vii

LIST OF TABLES

Table page 2-1 Film description........................................................................................................10

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

2-3 Comparison of measured oxygen transmission 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 media 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 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 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

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






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

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-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





C-1 Redox potential values without compensating for pH 7...........................................151

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 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 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 time in highly reduced media at 23˚C for film types C60 and BDF ..................................................................................................32

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

xi

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 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 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.

xii

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 10-35˚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 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% O2. 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.

1

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).

2

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

3

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.

4

• 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

5

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 7-10˚C is encountered by the

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

6

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 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 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 O2/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

7

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.

8

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

9

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 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 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

10

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).

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 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,

12

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, 35°C.

Oxygen transmission rates were first determined at 23°C 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.

13

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)

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 transmission 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 roll 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.

14

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.

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 5 10 15 20 25 30 35 40

Temperature (degree C)

OT

R(c

c/m

2 /day

)

0% RH

50% RH

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:

=

RTE

kOTR aexp0 2.1

15

where OTR is Oxygen Transmission Rate in cc/m2/day, k0 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 k0

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

100

1000

10000

100000

0.0032 0.00325 0.0033 0.00335 0.0034 0.00345 0.0035 0.00355

1/Temperature (K)

OT

R (c

c/m

2 /day

)

C60

C75

AET

BDF

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

100

1000

10000

100000

0.0032 0.00325 0.0033 0.00335 0.0034 0.00345 0.0035 0.00355

1/Temperature (K)

OT

R(c

c/m

2 /day

)

C60

C75

AET

BDF

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

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 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

0

400

050010001500200025003000350040004500

Wavenumbers

Rel

ativ

e A

bsor

banc

e

Signature Spectrum of LDPE

C60

C75

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

18

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 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 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

19

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 (redox 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

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 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 80˚C 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

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 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 35˚C 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

22

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

Figure 3-2. Rack arrangement inside the chamber

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 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.

24

Oxygen profile was monitored in the high barrier (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 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

Headspace O2 probe

Anaerobic Media

Dissolved O2 probe

Cut Vial

Cap Septum

Sample bag

25

buffer solution (Sensorex, Garden Grove, California). 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 Nernst 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

26

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)

Film Type and

Bag Size

Time in days

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15C60-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 - - - - - 1 1 2 3 4 4 4 4 4 4

B2 - - - - - 2 2 2 2 2 2 2 2 2 2 BDF-S B1 - - - - - 1 1 1 1 1 1 1 1 1 1

B2 - - - - - - 2 2 2 2 2 2 2 2 2 BDF-L B1 - - - - - - - 1 1 1 1 1 1 1 1

B2 - - - - - 1 1 2 2 2 2 2 2 2 2

15

Control - - - - - 1 1 1 1 1 1 1 1 1 1 C60-S B1 - - - - - - - - - - - - - - -

B2 - - - - - 1 1 1 1 1 1 1 1 1 1 C60-L B1 - - - - - - - - - - - - - - -

B2 - - - - - 1 1 1 1 1 1 1 1 1 1 AET-S B1 - - - - - - - - - - - - - - -

B2 - - - - - - - - - - - - - - - AET-L B1 - - - - - - - - - - - - - - -

20

B2 - - - - - - 1 1 1 1 1 1 1 1 1

27

Table 3.2. (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 15BDF-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 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

30

Control - - ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ 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 - - - - - - - - - - - - - - - AET-L B1 - - - - - - - - - - - - - - -

B2 - - - - - - - - - - - - - - - BDF-S B1 - - - - - - - - - - - - - - -

B2 - - - - - - - - - - - - - - - BDF-L B1 - 1 1 1 ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡

B2 - - - - - - - - - - - - - - -

35

Control - • • • • • • • • • • • • • • “*” represents growth greater than or equal to 50 colonies “•” represents growth greater than 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 that there was no growth in all samples at 10˚C for

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

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 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 15˚C.

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.

Figure 3.7. Control plate at 30˚C inside anaerobic box

Table 3-3. Spore outgrowth over time in anaerobic 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 - - - - - - - - - - - - - - -

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

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 germinate. 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 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 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

31

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 30˚C 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.

0

2

4

6

8

10

12

14

16

18

0 1000 2000 3000 4000 5000 6000

Time (mins)

Hea

dspa

ce O

xyge

n (%

)

C60

BDF

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

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 35˚C.

32

0

2

4

6

8

10

0 1000 2000 3000 4000 5000 6000

Time (mins)

Oxy

gen

cont

ent (

mg/

L)

C60

BDF

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

0

2

4

6

8

10

0 500 1000 1500 2000 2500 3000

Time (mins)

Oxy

gen

cont

ent (

mg/

L)

C60

BDF

Figure 3-10. Dissolved oxygen content over time in highly reduced media at 35˚C for

film types C60 and BDF

33

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

follows an irregular pattern due severe growth in media.

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 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 23°C. 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, 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 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.

35

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

15-45˚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 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

37

Table A-1. OTR of packaging films at 0% RH Sample ID Cell Temperature 1/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

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

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

SIZES

40

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

(Days)

Bag 2

1

2

3

4

41

Table B-1. Continued Time

(Days)

Bag 2

5

6

7

8

42


(Days)

Bag 2

9

10

11

12

43


(Days)

Bag 2

13

14

15

44

Table B-2. Growth table for AET 8x8 at 15˚C Anaerobic Media Time (Days)

Bag 2

1

2

3

4

5

6

45

Table B-2. Continued Time (Days)

Bag 2

7

8

9

10

11

46


Bag 2

12

13

14

15

47

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

(Days)

Bag 2

1

2

3

4

48


(Days)

Bag 2

5

6

7

8

49


(Days)

Bag 2

9

10

11

12

50


(Days)

Bag 2

13

14

15

51

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

(Days)

Bag 1 Bag 2

1

2

3

4

52


(Days)

Bag 1 Bag 2

5

6

7

8

53


(Days)

Bag 1 Bag 2

9

10

11

12

54


(Days)

Bag 1 Bag 2

13

14

15

55

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

(Days)

Bag 1 Bag 2

1

2

3

4

56


(Days)

Bag 1 Bag 2

5

6

7

8

57


(Days)

Bag 1 Bag 2

9

10

11

12

58


(Days)

Bag 1 Bag 2

13

14

15

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

60


(Days)

Bag 1 Bag 2

6

7

8

9

10

61


(Days)

Bag 1 Bag 2

11

12

13

14

15

62

Table B-7. Growth table for C60 8X8 at 23˚C Anaerobic Media Time (Days)

Bag 2

1

2

3

4

5

63


Bag 1 Bag 2

1

2

3

4

5

64

Table B-9. Growth table for BDF 8x8 at 23˚C Anaerobic Media Time (Days)

Bag 2

1

2

3

4

5

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

66


Bag 1 Bag 2

1

2

3

4

5

67


Bag 1 Bag 2

1

2

3

4

5

68


Bag 1 Bag 2

1

2

3

4

69


Bag 1 Bag 2

1

2

3

4

70


Bag 1 Bag 2

1

2

3

4

71


Bag 1 Bag 2

1

2

3

4

72


Bag 1 Bag 2

1

2

3

4

73


Bag 1 Bag 2

1

2

3

4

74


Bag 1 Bag 2

1

2

3

75


Bag 1 Bag 2

1

2

3


Bag 1 Bag 2

1

2

3

76


Bag 1 Bag 2

1

2

3


Bag 1 Bag 2

1

2

3

77


Bag 1 Bag 2

1

2

3

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

79


Bag 1 Bag 2

7

8

9

10

11

12

80


Bag 1 Bag 2

13

14

15

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

82


Bag 1 Bag 2

7

8

9

10

11

12

83


Bag 1 Bag 2

13

14

15

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

85


Bag 1 Bag 2

7

8

9

10

11

12

86


Bag 1 Bag 2

13

14

15

87


Bag 1 Bag 2

1

2

3

4

5

6

88

Table 28. Continued Time (Days)

Bag 1 Bag 2

7

8

9

10

11

12

89

Table 28. Continued Time (Days)

Bag 1 Bag 2

13

14

15

90


Bag 1 Bag 2

1

2

3

4

5

6

91


Bag 1 Bag 2

7

8

9

10

11

12

92


Bag 1 Bag 2

13

14

15

93


Bag 1 Bag 2

1

2

3

4

5

6

94


Bag 1 Bag 2

7

8

9

10

11

12

95


Bag 1 Bag 2

13

14

15

96


Bag 1 Bag 2

1

2

3

4

5

6

97


Bag 1 Bag 2

7

8

9

10

11

12

98


Bag 1 Bag 2

13

14

15

99


Bag 1 Bag 2

1

2

3

4

5

6

100


Bag 1 Bag 2

7

8

9

10

11

12

101


Bag 1 Bag 2

13

14

15

102


Bag 1 Bag 2

1

2

3

4

5

6

103


Bag 1 Bag 2

7

8

9

10

11

12

104


Bag 1 Bag 2

13

14

15

105


Bag 1 Bag 2

1

2

3

4

5

6

106


Bag 1 Bag 2

7

8

9

10

11

12

107


Bag 1 Bag 2

13

14

15

108


Bag 1 Bag 2

1

2

3

4

5

6

109


Bag 1 Bag 2

7

8

9

10

11

12

110


Bag 1 Bag 2

13

14

15

111


Bag 1 Bag 2

1

2

3

4

5

6

112


Bag 1 Bag 2

7

8

9

10

11

12

113


Bag 1 Bag 2

13

14

15

114


Bag 1 Bag 2

1

2

3

4

5

6

115


Bag 1 Bag 2

7

8

9

10

11

12

116


Bag 1 Bag 2

13

14

15

117


Bag 1 Bag 2

1

2

3

4

5

6

118


Bag 1 Bag 2

7

8

9

10

11

12

119


Bag 1 Bag 2

13

14

15

120


Bag 1 Bag 2

1

2

3

4

5

121


Bag 1 Bag 2

6

7

8

9

10

122


Bag 1 Bag 2

11

12

13

14

15

123


Bag 1 Bag 2

1

2

3

4

5

6

124


Bag 1 Bag 2

7

8

9

10

11

12

125


Bag 1 Bag 2

13

14

15

126


Bag 1 Bag 2

1

2

3

4

5

6

127


Bag 1 Bag 2

7

8

9

10

11

12

128


Bag 1 Bag 2

13

14

15

129


Bag 1 Bag 2

1

2

3

4

5

6

130


Bag 1 Bag 2

7

8

9

10

11

12

131


Bag 1 Bag 2

13

14

15

132


Bag 1 Bag 2

1

2

3

4

5

6

133


Bag 1 Bag 2

8

9

10

11

12

13

134


Bag 1 Bag 2

14

15

135


Bag 1 Bag 2

1

2

3

4

5

6

136


Bag 1 Bag 2

7

8

9

10

11

12

137


Bag 1 Bag 2

13

14

15

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

139

Table B-45. Continued Time Days

Bag 1 Bag 2

7

8

9

10

11

12

140

Table B-45. Continued Time Days

Bag 1 Bag 2

13

14

15

141


Bag 1 Bag 2

1

2

3

4

5

6

142


Bag 1 Bag 2

7

8

9

10

11

12

143


Bag 1 Bag 2

13

14

15

144


Bag 1 Bag 2

1

2

3

4

5

6

145


Bag 1 Bag 2

7

8

9

10

11

12

146


Bag 1 Bag 2

13

14

15

147


Bag 1 Bag 2

1

2

3

4

5

6

148


Bag 1 Bag 2

7

8

9

10

11

12

149


Bag 1 Bag 2

13

14

15

APPENDIX C OXIDATION REDUCTION POTENTIAL WITHOUT PH COMPENSATION

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

152

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155

BIOGRAPHICAL SKETCH

Jayashree Gnanaraj was born in Madurai, 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 processing and preservation technology from Avinashilingam

University where she was among top three and the student body president of engineering

department 00’- 01‘ which comprised of 600 students. She was also the secretary of Food

Technology Association 99’- 00’. During the last year of under graduation studies she

interned in Indira Gandhi Center for Atomic Research which made her realize her passion

for scientific research.

In August 2001 she was admitted in Agricultural and Biological Engineering

Department at the University of Florida and now feels prepared to get involved with the

food processing field because of the excellent mentoring provided by the faculty.

evaluation of oxygen transmission rate of packaging...

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