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Effect of Filtered Wood Smoke Processing on Spoilage Bacteria, Pathogenic Bacteria, and Sensory Characteristics of Yello...

Permanent Link: http://ufdc.ufl.edu/UFE0021762/00001

Material Information

Title: Effect of Filtered Wood Smoke Processing on Spoilage Bacteria, Pathogenic Bacteria, and Sensory Characteristics of Yellowfin Tuna
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Crynen, Stefan Karl
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: cmvs, co, color, filteredsmoke, fish, quality, salmonella, tuna
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Smoking is a very old technique for preserving fish, meat and dairy products and for enhancing their flavor. Currently the preservation aspect of ?smoking fish? is often ignored since other more effective preservation methods, like freezing and refrigeration, have been developed. Today, most smoking applications target mainly the enhanced flavor aspect of smoking, rather then the increased shelf life of food products. Filtered smoke processing is a new method that uses the preservation effect of smoking on fish and fish products without major changes in their sensory characteristics, like flavor or texture. The goal of this project was to study the effects of filtered smoke processing on spoilage and pathogenic bacteria, quality aspects of warm water fish species and to optimize the smoke treatment method. Unlike most preservation techniques like freezing or refrigeration, filtered smoke also enhances the appearance of red muscle products such as tuna or mahi. This work also showed that color of the warm water fish species can be enhanced through filtered smoke treatment, especially in conjunction with refrigeration and freezing. Fresh tuna steaks were treated with filtered and artificial smoke for 24 and 48 hours and then analyzed for 14 days for Salmonella spp. growth, total aerobic bacteria growth and changes in color, especially the redness of the samples. A similar study was conducted where the samples were frozen and stored at -20?C for 30 days prior to analysis. The first two studies showed that there was nearly no affect of filtered or artificial wood smoke processing on the growth of Salmonella spp. for either the fresh or frozen stored samples. However, there appeared to be an inhibitory effect of both the filtered and the artificial smoke treatment on the growth of aerobic spoilage bacteria during the first 4 days of observation. This effect seemed to be enhanced in the samples that were stored at -20 Degree Celsius for 30 days. However, these effects suggest no improvement in the shelf life of tuna as caused by any of the treatments. The color analysis confirmed the effectiveness of filtered smoke processing on the preservation of the color properties and appearance of the samples, especially after frozen storage. No significant differences could be seen between the filtered smoke and the artificial smoke treatments in any of the mentioned studies. Moreover in a sensorial taste panel no significant differences could be found based on odor and appearance between filtered smoke and artificial smoke treated tuna. To identify whether a product was treated with filtered or artificial smoke for the purpose of quality assurance, a rapid gas chromatography identification method was developed to quantify the amount of residual carbon monoxide in products and at the same time verify whether a product was treated with filtered smoke or the artificial counterpart by a chromatographic fingerprint.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Stefan Karl Crynen.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kristinsson, Hordur G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021762:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021762/00001

Material Information

Title: Effect of Filtered Wood Smoke Processing on Spoilage Bacteria, Pathogenic Bacteria, and Sensory Characteristics of Yellowfin Tuna
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Crynen, Stefan Karl
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: cmvs, co, color, filteredsmoke, fish, quality, salmonella, tuna
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Smoking is a very old technique for preserving fish, meat and dairy products and for enhancing their flavor. Currently the preservation aspect of ?smoking fish? is often ignored since other more effective preservation methods, like freezing and refrigeration, have been developed. Today, most smoking applications target mainly the enhanced flavor aspect of smoking, rather then the increased shelf life of food products. Filtered smoke processing is a new method that uses the preservation effect of smoking on fish and fish products without major changes in their sensory characteristics, like flavor or texture. The goal of this project was to study the effects of filtered smoke processing on spoilage and pathogenic bacteria, quality aspects of warm water fish species and to optimize the smoke treatment method. Unlike most preservation techniques like freezing or refrigeration, filtered smoke also enhances the appearance of red muscle products such as tuna or mahi. This work also showed that color of the warm water fish species can be enhanced through filtered smoke treatment, especially in conjunction with refrigeration and freezing. Fresh tuna steaks were treated with filtered and artificial smoke for 24 and 48 hours and then analyzed for 14 days for Salmonella spp. growth, total aerobic bacteria growth and changes in color, especially the redness of the samples. A similar study was conducted where the samples were frozen and stored at -20?C for 30 days prior to analysis. The first two studies showed that there was nearly no affect of filtered or artificial wood smoke processing on the growth of Salmonella spp. for either the fresh or frozen stored samples. However, there appeared to be an inhibitory effect of both the filtered and the artificial smoke treatment on the growth of aerobic spoilage bacteria during the first 4 days of observation. This effect seemed to be enhanced in the samples that were stored at -20 Degree Celsius for 30 days. However, these effects suggest no improvement in the shelf life of tuna as caused by any of the treatments. The color analysis confirmed the effectiveness of filtered smoke processing on the preservation of the color properties and appearance of the samples, especially after frozen storage. No significant differences could be seen between the filtered smoke and the artificial smoke treatments in any of the mentioned studies. Moreover in a sensorial taste panel no significant differences could be found based on odor and appearance between filtered smoke and artificial smoke treated tuna. To identify whether a product was treated with filtered or artificial smoke for the purpose of quality assurance, a rapid gas chromatography identification method was developed to quantify the amount of residual carbon monoxide in products and at the same time verify whether a product was treated with filtered smoke or the artificial counterpart by a chromatographic fingerprint.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Stefan Karl Crynen.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kristinsson, Hordur G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021762:00001


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EFFECT OF FILTERED WOOD SMOKE PROCESSING ON SPOILAGE BACTERIA,
PATHOGENIC BACTERIA AND SENSORY CHARACTERISTICS OF YELLOWFIN TUNA





















By

STEFAN CRYNEN


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

UNIVERSITY OF FLORIDA

2007


































2007 Stefan Crynen


































To my loving parents, my brother and especially my fiancee Gogce.









ACKNOWLEDGMENTS

I sincerely want to express my deep gratitude towards my major advisor, Dr. Hordur G.

Kristinsson, who helped me to complete this study with his advice guidance and support. I would

also like to thank my committee members Dr. Murat Balaban and Dr. Bruce Welt for their

suggestions, advice and help in the completion of this research.

I would like to thank Mr. Blane Olson. His generosity and support made this project

possible.

I would also like to thank my family for their love and support and especially my fiancee

Gogce who believed in me and supported with guidance and understanding thru this entire

project.

Finally I would like to thank Dr. Charles Sims, Yavuz Yagiz and Lorenzo Puentes and his

Team for their support with the Taste Panel experiments, Sibel Damar and Sara Aldaous for her

friendship and support and all my fellow class and lab mates at the University of Florida.










TABLE OF CONTENTS


page

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

LIST OF TABLES .................... ........................ ............... ........7

LIST OF FIGURES ................................... ............ .. .............................8

LIST OF ABBREVATIONS ................ ........ ...... ................

A B STR A C T .................. .......... .............. ....................................................... 12

CHAPTER

1 INTRODUCTION ............... ...................... .............................. .............. 14

2 LITERATURE REVIEW ..................................................................... ........ 16

Sm oking of Fish and Seafood Products........................................................................ ...... 16
Filtered Sm oke..................... .......... ........... .....................17
Bacteria and Other Microorganisms in Fish and Seafood.............................................19
Listeria Monocytogenes... .................. ........... .......................... 20
Clostridium Botulinum............ .................................................20
Salmonella Spp. ................................................21
Biogenic Amines ................................................21
Parasites........................ .... .............. .................21
Sensory Characteristics of Fresh and Smoked Seafood .......... ........... ...........22

3 OBJECTIVES ......................................................25

4 PRELIMINARY STUDIES........................... .....................26

Tuna Microbiology Study..................................................26
Tuna Color Study...................................... .................................................28
Identification of Filtered Smoke Treated Products..........................................................29

5 MATERIAL AND METHODS..............................................36

Fresh and Frozen Storage ........................................ ........36
Sample Preparation and Treatments ..................................... ......... ........37
Salmonella Study .............................. ..............................................38
Total Aerobic Plate Count Study............... ........................40
Color Analysis ...................................................... ........41
Sensory Taste Panel Analysis.......................... ..........................................41
Rapid Gas Chromatography Identification Method .......................................... .....42
Statistical Analysis................................................ ........44


..............................................3 8
Total Aerobic Plate Count Study ................................ .......................... ........40
C olor A n aly sis ................................................................4 1
Sensory Taste Panel A nalysis............................................41
Rapid Gas Chromatography Identification Method ........................................ .....42
Statistical A nalysis................................................... 44









6 RE SU LTS AN D D ISCU SSION ............................................................................. ............47

Salm onella R esults........... ................................................................................ ...... ......47
F resh Storage Study ...... ...... ....................................................................48
Frozen Storage Study .................................... .. .. ........ .. ............49
T total A erobic P late C ount ........... ......... ...... ......... .............................. ............................52
F resh Storage Study ...... ...... .................................................... ................52
Frozen Storage Study .................................. .. .. ........ .. ............54
C o lo r A n aly sis .................................................................5 7
F resh Storag e Stu dy .................................... ...... .. ..... ......................................5 8
Frozen Storage Study ....................... ........................................... ..... 60
Sensory Taste Panel ............................... ... ........... .......... .... ................. 68
G C -A analysis .................. ................ .. ............... ........... .. ............... 69
Identification of Gaseous Components in Filtered and Artificial Smoke .....................71
A analysis of Treated Sam ples .............. .......................... .................................. 75

7 SUMMARY AND CONCLUSIONS ..............................................................................80

APPENDIX

A TASTE PANEL DEMOGRAPHICS AND COMMENTS .......................................... 83

B COLOR STU D Y PICTU RES....................................... ............................ ............... 89

LIST O F R EFER EN CE S .............. ....................................................... ...................93

B IO G R A PH IC A L SK E T C H .............................................................................. .....................97

























6









LIST OF TABLES


Table page

5-1 Treatment combinations used in all studies of this project.............................................. 38

5-2 N ikon D 200 Settings .......................................................... ........... ............ .. 45

5-3 GC-Settings for the rapid identification m ethod.......................................................... 45

6-1 ANOVA results for Salmonella spp. for the fresh storage study.................................. 50

6-2 ANOVA results for Salmonella spp. for the frozen storage study. ................................. 51

6-3 ANOVA results for aerobic plate count for the fresh storage study............................ 56

6-4 ANOVA results for aerobic plate count for the frozen storage study............................ 57

6-5 ANOVA results for the average a*-values for the fresh storage study......................... 62

6-6 ANOVA results for the average a*-values for the frozen storage study ...................... 63

6-7 Results of the four taste panel triangle tests................................................................... 69

6-8 Percentage of gaseous components in the treatment gasses. ......................................... 72

A-i Comments from the Panelists for the Fresh Odor Triangle Test ................................... 83

A-2 Comments from the Panelists for the Frozen Odor Triangle Test ............... ............... 84

A-3 Comments from the Panelists for the Fresh Color Triangle Test .................................. 85

A-4 Comments from the Panelists for the Frozen Color Triangle Test............... .............. 87

A-5 Demographic Variance of the Odor Triangle Tests ................................................ 88

A-6 Demographic Variance of the Color Triangle Tests .............................................. 88









LIST OF FIGURES


Figure pe


3-1 Tuna treated for 16 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of
18% CO with 21% CO2 balanced with nitrogen.............. ........................................ 30

3-2 Tuna treated for 32 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of
18% CO with 21% CO2 balanced with nitrogen...................................................... 31

3-3 Tuna treated for 48 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of
18% CO with 21% CO2 balanced with nitrogen...................................................... 31

3-4 Tuna treated for 16 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a
mixture of 18% CO with 21% CO2 balanced with nitrogen ........................................ 32

3-5 Tuna treated for 32 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a
mixture of 18% CO with 21% CO2 balanced with nitrogen ........................................ 32

3-6 Tuna treated for 48 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a
mixture of 18% CO with 21% CO2 balanced with nitrogen ........................................ 33

3-7 Chromatogram of the analysis of ClearsmokeTM........... ............................................ 33

3-8 Chromatogram of the analysis of industrial carbon monoxide (-100% CO) ............... 34

3-9 Chromatogram of the analysis of atmospheric air. ...................................... ............... 34

3-10 Chromatogram of the analysis of nitrogen (N2)....................................................... 34

3-11 Chromatogram of the analysis of carbon dioxide (C02) ................................................. 35

5-1 Flow diagram of all studies conducted during this project............................................. 46

6-1 Average amount of CFU/lOg of tuna for Salmonella spp. for the fresh storage study..... 50

6-2 Average amount of CFU/lOg of tuna for Salmonella spp. for the frozen storage study. 51

6-3 Average amount of CFU/lOg of tuna for aerobic plate count for the fresh storage study.55

6-4 Average amount of CFU/lOg of tuna for aerobic plate count for the fresh storage study.56

6-5 Average a*-values of the color of tuna samples over 14 days for the frozen storage study.
....................................... ........................... ..................... 62

6-6 Average a*-values of the color of tuna samples over 14 days for the fresh storage study.
....................................... ........................... ..................... 63










6-7 Average L*-values of the color of tuna samples over 14 days for the fresh storage study.
.................................................................... ................... 64

6-8 Average L*-values of the color of tuna samples over 14 days for the frozen storage study.
.................................................................... ................... 65

6-9 Average b*-values of the color of tuna samples over 14 days for the fresh storage study.
.................................................................... ................... 66

6-10 Average b*-values of the color of tuna samples over 14 days for the frozen storage study.
.................................................................... ................... 67

6-11 Tuna steaks after 30 days of frozen storage at -20C............... ....................... 67

6-12 Carbon monoxide standard curve. ................... ......... ........... ............ 72

6-13 M ethane standard curve. ................................................................................................... 73

6-14 Carbon dioxide standard curve. .......... ....................................... ............... 73

6-15 E thylene standard curve .............................. ............................ .................................... 74

6 -16 E th an e stan d ard cu rv e. ...................................................................................................... 74

6-17 Chromatogram of the headspace analysis over a sample treated with artificial smoke.... 76

6-18 Chromatogram of the headspace analysis over a sample treated with filtered smoke...... 76

6-19 Chromatogram of the headspace analysis over an untreated control sample ............... 77

6-20 Chromatogram of the headspace analysis over a sample, purchased at a local ethnic store.
.................................................................... ................... 77

6-21 Chromatogram of the injection of 50 [l of pure filtered smoke.................................. 78

6-22 Chromatogram of the injection of 50 [l of pure filtered smoke "B"............................ 78

6-23 Chromatogram of the injection of 50 [l of pure artificial smoke.............................. 79

6-24 Chromatogram of the injection of 50 [l of air sampled from the surrounding lab
environm ent. .............................................................................. 79

B-1 Control group images for the frozen storage study captured by the CMVS.................. 89

B-2 Filtered smoke (24h-treatment) group images for the frozen storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 89

B-3 Filtered smoke (48h-treatment) group images for the frozen storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 89









B-4 Artificial smoke (24h-treatment) group images for the frozen storage study captured by
the C M V S. ................................................................ ........... ..... 90

B-5 Artificial smoke (48h-treatment) group images for the frozen storage study captured by
the C M V S. ................................................................ ........... ..... 90

B-6 Control group images for the fresh storage study captured by the CMVS....................... 90

B-7 Filtered smoke (24h-treatment) group images for the fresh storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 91

B-8 Filtered smoke (48h-treatment) group images for the fresh storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 91

B-9 Artificial smoke (24h-treatment) group images for the fresh storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 91

B-10 Artificial smoke (48h-treatment) group images for the fresh storage study captured by the
C M V S ..... ........................... ........................ ........ ......... ...... 92









LIST OF ABBREVATIONS

ANOVA Analysis of Variance

AS Artificial Smoke (A mixture of 21 % carbon monoxide, 18 % carbon
dioxide, 1.1% oxygen and a balance of nitrogen)

CMVS Color Machine Vision System

Ctrl Control Group

FID Flame Ionization Detector

FS Filtered Smoke

FSHN Food Science and Human Nutrition Department
University of Florida

GC Gas Chromatograph

SAS Statistical Analysis Software









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECT OF FILTERED WOOD SMOKE PROCESSING ON SPOILAGE BACTERIA,
PATHOGENIC BACTERIA AND SENSORY CHARACTERISTICS OF TUNA FISH

By

Stefan Crynen

December 2007

Chair: Hordur G Kristinsson
Major: Food Science and Human Nutrition


Smoking is a very old technique for preserving fish, meat and dairy products and for

enhancing their flavor. Currently the preservation aspect of "smoking fish" is often ignored since

other more effective preservation methods, like freezing and refrigeration, have been developed.

Today, most smoking applications target mainly the enhanced flavor aspect of smoking, rather

then the increased shelf life of food products.

Filtered smoke processing is a new method that uses the preservation effect of smoking on

fish and fish products without major changes in their sensory characteristics, like flavor or

texture. The goal of this project was to study the effects of filtered smoke processing on spoilage

and pathogenic bacteria, quality aspects of warm water fish species and to optimize the smoke

treatment method.

Unlike most preservation techniques like freezing or refrigeration, filtered smoke also

enhances the appearance of red muscle products such as tuna or mahi. This work also showed

that color of the warm water fish species can be enhanced through filtered smoke treatment,

especially in conjunction with refrigeration and freezing.









Fresh tuna steaks were treated with filtered and artificial smoke for 24 and 48 hours and

then analyzed for 14 days for Salmonella spp. growth, total aerobic bacteria growth and changes

in color, especially the redness of the samples. A similar study was conducted where the samples

were frozen and stored at -200C for 30 days prior to analysis. The first two studies showed that

there was nearly no affect of filtered or artificial wood smoke processing on the growth of

Salmonella spp. for either the fresh or frozen stored samples. However, there appeared to be an

inhibitory effect of both the filtered and the artificial smoke treatment on the growth of aerobic

spoilage bacteria during the first 4 days of observation. This effect seemed to be enhanced in the

samples that were stored at -200C for 30 days. However, these effects suggest no improvement in

the shelf life of tuna as caused by any of the treatments. The color analysis confirmed the

effectiveness of filtered smoke processing on the preservation of the color properties and

appearance of the samples, especially after frozen storage. No significant differences could be

seen between the filtered smoke and the artificial smoke treatments in any of the mentioned

studies. Moreover in a sensorial taste panel no significant differences could be found based on

odor and appearance between filtered smoke and artificial smoke treated tuna. To identify

whether a product was treated with filtered or artificial smoke for the purpose of quality

assurance, a rapid gas chromatography identification method was developed to quantify the

amount of residual carbon monoxide in products and at the same time verify whether a product

was treated with filtered smoke or the artificial counterpart by a chromatographic fingerprint.









CHAPTER 1
INTRODUCTION

Smoking fish, meat and dairy products is a very old technique that has been used as a

method of long term preservation for over 600 years (Cutting 1961). Until other methods like

refrigeration and freezing were developed and established, smoking fish and meat products was

one of the most used preservation methods. The advantages of smoking food products are

increased shelf life, enhanced flavor, and prevention of insect infestation. Although traditional

fish smoking can still be found in practice today, the distinct boundaries of its use have vanished

(Dillon and others 1994). In industrial countries, smoking of fish and meat products is used today

mainly to enhance flavor and texture (Robinson 1983). Modern forms of smoke processing

provide sometimes only little protection against microbial spoilage and especially cold smoked

products can spoil as readily as non-smoked foods (Hsu and others 1979).

A new technique that uses filtered and compressed wood smoke to treat fish products by

using a low temperature treatment process, attempts to use the preserving aspect of wood smoke

on food products without changing their flavor or texture. Although most smoking applications

today aim to change and enhance the flavor of a food product where it is accepted by the

consumer, generally smoke flavor is not widely accepted. As with other flavor and taste

expressions like spiciness, sweetness, rancidity or others, the range of likeability goes all the way

from no smoke flavor over mild taste to very strong taste for smoked food products. With filtered

smoke applications it is now possible to adjust the level of smoke flavor of a food product while

at the same time increasing the antimicrobial effect by concentrating effective components.

This project aims to determine the antimicrobial effects of filtered wood smoke processing

on fish products while assessing changes that occur in color, texture, taste and overall likeability.

This study will determine the effects of filtered wood smoke treatment on the growth, inhibition









and reduction of Salmonella spp. The effects were studied on the natural fauna present when the

fish was delivered and on inoculated.

Since the filtered smoke treatment is expected to maintain and enhance color properties,

especially related to the red muscle parts, it is important to determine whether a product was

treated with filtered smoke or just with industrial carbon monoxide, which is already proven to

enhance the color of red muscle products. A part of this study was to develop and improve a

rapid gas chromatography identification method, designated to determine whether a product was

treated with filtered smoke or carbon monoxide. The method is based on a gas chromatography

profile, which shows a combination of several natural gases, like methane, ethane, butane and

pentanes, in different concentrations that produce chromatographic fingerprint for smoke treated

products.









CHAPTER 2
LITERATURE REVIEW

Smoking of Fish and Seafood Products

Smoking fish and meat products is a very old food processing technique and has been used

over a long time as a method of long term preservation of foods (Burgess and Bannerman 1963).

Smoked foods are generally better preserved and protected against insects infestation than

untreated products. Smoking fish and fish products can be divided into two distinct types, a hot

smoking process and a cold smoking process.

Hot smoking involves cooking products during or immediately after the smoke treatment,

usually in the same chamber, at temperatures around 700 to 800C. Traditional hot smoking is

actually a three step process. The product is first smoked between 30 and 60 minutes at 300C to

dry and toughen the skin. In general the more dry the surface of the products the more smoke

flavor is absorbed into the product. The second step is the actual smoking step where the

temperature is increased to about 500C and the amount of smoke blown into the oven is raised.

This is considered the smoking step. The last step is the cooking step where the product is

cooked at temperatures between 700 and 900C for about 1 to 2 hours (Dillon and others 1994).

For cold smoke processing, the difference to hot smoking is the temperature at which the

product is smoked and finished. The purpose of cold smoking is to apply a smoky flavor to the

product without cooking, so the product may be consumed raw, such as cold smoked salmon, or

prepared as desired by the consumer. The products are smoked at temperatures below 300C with

an initial heavy smoke deposit into the chamber that is tapered off toward the end. For some

species, like herring and mackerel, the temperature is increased after the smoking step to 400C so

that natural oils can come to the surface to provide the final product with a glossy appearance

(Dillon and others 1994).









Filtered Smoke

Filtered Smoke treatment is a new technique to preserve meats and seafood by utilizing the

preserving aspects of wood smoke and at the same time minimizing the introduction of flavor

and odor giving compounds, which are filtered out of the smoke before it is used in the

processing of the food product. The idea to use filtered smokes to preserve color and spoilage of

seafood products started when modified atmospheric packaging became commercially available,

and carbon monoxide, a main component in natural and filtered wood smoke, was used to

enhance and preserve the fresh appearance of seafood (Otwell 2006).

Although the use of industrial carbon monoxide to treat meat and seafood products is still

debated in many countries, the use of wood smoke has been acknowledged and accepted as a

preservation method by competent health authorities in every nation of the world (Olson 2006).

Filtered smoke is the next step in this long history of preservation that utilizes the preserving

aspects of wood smoke (e.g. carbon monoxide and carbon dioxide) and minimizes or even

eliminates unwanted changes in flavor, texture and taste to the product.

Compared to the traditional hot and cold smoking, with treatment temperature from 300C

(for cold smoking) up to 800C (for hot smoking), the filtered smoke is applied to the product at

0-50C. The whole process, as described by Olson (2006), involves the generation of wood smoke

by burning wood chips in a smoke chamber without air intake, to ensure the incomplete

combustion of the wood, leading to smoke, then run this smoke to a series of filters to remove

particles such as tar, ash and soot and finally remove most of the odor and flavor compounds

with and active carbon filter. This filtered smoke can be used immediately or stored in

compressed gas cylinders for later use. The food products are then prepared for the filtered

smoke treatment by filleting and speaking to increase the surface area, and then placed on racks

and put into the smoking chamber. The chamber is then evacuated from the remaining air and the

17









filtered smoke is introduced into the chamber and replaced several times during the treatment,

which can take from 2 to 48 hours.

The filtered wood smoked products show great advantages in their retail value, compared

to untreated or naturally smoked products, due to the enhancement of their color, texture, odor

and taste properties. The color of food products is one of the major aspects most consumers are

interested. If the color of a piece of tuna is brown or grey instead of red or pink, as expected from

a fresh product, the consumer will most likely reject the product. The carbon monoxide, present

in filtered smoke, will bind to muscle heme proteins, which determine the color of the muscle

based on their chemical state. Freezing or other long term storage and exposure to air (oxygen)

will change the conformation of the heme protein, which results in brown to gray colors of the

product. Carbon monoxide will bind to the heme protein and stabilizes it so that the color change

due to long term storage and freezing can be prevented and the product looks appealing red and

pink (Olson 2006).

The same mechanism also affects odor and taste of the product due to oxidative rancidity

of the lipids in the fish muscle. Lipid degradation and the reactions of these degradation products

lead to undesirable off-odors and flavors. It is believed that hemoglobin and myoglobin are key

prooxidants that cause the oxidation and degradation of lipids that lead to off flavors and odors.

By stabilizing the heme proteins with carbon monoxide, the oxidation of lipids and therefore the

formation of off flavors and odors can be reduced. As a result, the fish product smells and tastes

fresh longer (Kristinsson and others 2006b).

Some research has been done on the effect of carbon monoxide, one of the main

components of filtered smoke, on the growth of microorganisms. Several studies have been

conducted with various levels of carbon monoxide on red meat and seafood to investigate how









these treatments inhibit the growth of spoilage bacteria. Some recent studies compared the

growth of total aerobic bacteria with different treatments, including a control group (no

treatment), a 4% CO treatment, a treatment with 100% nitrogen to test the exclusion of oxygen, a

treatment with 100% CO, a treatment with filtered smoke and a treatment with 18% CO. The

results showed that the greatest reduction in total aerobic bacteria was noticed for the filtered

smoke and the 18% CO treatment together with the 100% CO treatment (Kristinsson and others

2007). The 100% nitrogen treatment showed some bacterial count reduction, while the bacterial

counts for the 4% CO treatment and the control group were increased. In a long term freezing

study, the 18% CO and filtered smoke treatments showed an even higher bacterial count

reduction (Kristinsson and others 2006b). Nevertheless, data on how filtered smoke and carbon

monoxide affect the microbial flora of meat and seafood during fresh and frozen storage is

limited and this area needs to be more investigated.

Bacteria and Other Microorganisms in Fish and Seafood

All fresh fish and fish products contain a natural variety of bacteria and spores (Jahncke

and Herman 2001). These bacteria and spores are partially responsible for the spoilage of fresh

fish and processed fish products (Gram and Huss 1996). Some might also be a potential hazard

for human health because of their pathogenic properties (Jahncke and Herman 2001). The visible

evidence of spoilage is usually the growth of molds and slimy bacterial colonies, but spoilage

can also change the sensory characteristics of a food product, such as smell (off-odors) and taste

(off-flavor) (Gram and Huss 1996). Cold-smoked fish products are usually ready-to-eat food

products that have not received sanitizing or stabilizing heat treatment (Gram 200 1b). Therefore

pathogens and biological hazards are of particular concern for these products. Some potential

hazards associated with cold-smoked fish products are: Listeria monocytogenes, Clostridium

botulinum type E, Salmonella spp., biogenic amines, and parasites (Jahncke and Herman 2001).

19









Listeria Monocytogenes

Listeria monocytogenes is a widely distributed Gram-positive, food borne pathogen that

naturally occurs in many raw food products. It can grow between 1-450C and between 0 and

10% NaC1. High levels ofL. monocytogenes in unheated ready-to-eat food products have been

associated with listerioses (McLauchlin 1997). Since cold-smoked fish is a ready-to-eat product

it has been linked to sporadic cases of listerioses (Gram 2001b). The traditional way of isolating

these organisms involves the use of selective media such as PALCAM agar, blood agar with

nalidixic acid agar, Oxford Listeria selective agar (LSA) and enrichment and pre-enrichment

broths with incubation at 300C for 48 h. Since the growth ofL. monocytogenes may be hindered

by other microorganisms, PALCAM agar and Oxford LSA have advantages over other media,

because of their ability to reduce the presence of contaminating micro-organisms (Neamatallah

and others 2003). To eliminate or control the growth ofL. monocytogenes a variety of different

treatments of raw and finished product have already been approved. Two treatments involve: (1)

washing the raw fish with water that contains chlorine, and (2) treatment of the raw fish with

calcium hydroxide solution (pH 12). Other treatment options include washing the raw fish with

acidified sodium chloride solutions, ozone treatment, steam surface pasteurization and

electrochemical brine tank treatments. For the finished product, freezing and addition of

approved microbial growth inhibitors are options to stop or control growth ofL. monocytogenes

(Jahncke and others 2004).

Clostridium Botulinum

Clostridium botulinum is the name of a range of Gram-positive, anaerobic, spore forming

bacteria that produce the botulinum neurotoxin. These bacteria and their specific neurotoxins

have been divided into 7 types (A, B, C, D, E, F and G) based on their antigenic properties. The

disease caused by these neurotoxins is called botulism. Generally botulism occurs rarely today.

20










However, the neurotoxins from Clostridium botulinum are still some of the most potent toxins

known. For cold-smoked fish products, the Group II of C. botulinum and particular strains that

produce the type E neurotoxin are a major concern (Gram 200 l1a).

Salmonella Spp.

Salmonella spp. can be carried by fish and shellfish which show no signs of disease. The

contamination of this organism derives from terrestrial sources and fish may serve as a vector for

Salmonella spp. (Novotny and others 2004) Especially shellfish from sewage-polluted waters

seems to be a major problem. Although outbreaks of Salmonella spp. have occurred, processed

seafood products are usually considered to present a lower risk (Heinitz and Johnson 1998).

Biogenic Amines

Biogenic amines are nitrogen compounds that can be expected in nearly all foods that

contain proteins or free amino acids (Shalaby 1996). In particular, histamine and tyramine have

been associated with some toxicological characteristics and outbreaks of food poisoning. The

presence of biogenic amines above a certain level in non-fermented foods indicates the presence

of undesired microbial activity and could therefore be used as an indicator of microbial spoilage.

While not all biogenic amines correlate with the growth of spoilage organisms, histamine,

putrescine and cadaverine levels usually increase during spoilage of fish and fish products

(Santos 1996). Biogenic amines and particularly histamine have been also implicated as the

causative agent in a number of scombroid food poisonings. It is also a concern that in cold-

smoked products secondary amines such as putrescine and cadaverine can react with nitrite and

form carcinogens (Flick and others 2001).

Parasites

Cold-smoked or cold-smoked and dried fish and fish products may contain human

pathogenic parasites such as Anisakis spp. (a nematode or roundworm), Diphyllobothrium spp. (a

21









cestode or tapeworm) and Nanophyetus salmincola. The harvest of parasite free fish in the wild

is very difficult. Some aquacultured fish, however, are considered free of parasites, since their

diet and environment may be controlled. The most effective way to ensure that viable parasites

are not present in cold smoked fish products is to freeze the raw fish prior to the smoking step for

a prescribed time that assures destruction of all viable parasites in that fish (Bledsoe and Oria

2001).

Sensory Characteristics of Fresh and Smoked Seafood

For most people, fish and seafood is associated with the typical fishy smell that also

surrounds fishing piers and fish markets. However, only few consumers know that this "fishy"

smell is already a sign of degradation of the fish and seafood products. Most fresh fish and

seafood products have no distinct smell or odor at all when caught and processed very fresh. Fish

that has been stored for a couple of days or previously been frozen develops a slight fishy smell

due to the oxidation of lipids (Olson 2006). Further degradation of fish and seafood then

ultimately leads to very unpleasant fishy and spoiled smell that will tell the consumer that this

product is spoiled and no longer consumable.

Odor and flavor is only one of the sensory characteristics consumers rely on when making

a decision to buy or prepare fish and seafood. In fact, the most important characteristic is the

color of the fish or seafood, since most consumers will make eye contact with the product the

first time through a glass display, where the color and overall appearance is the only factor they

can base their decision. A fresh red and pink color for most seafood products rich in dark muscle

is preferred over the brown and gray color of long stored, untreated products. The third sensory

characteristic is the texture and mouth-feel of fresh and processed seafood that determines if a

customer is satisfied with the product.









All these sensory characteristics change dramatically when seafood products are processed

using the hot or cold smoking technique with natural wood smoke. Hot and cold smoking

introduces substantial amounts of flavor compounds into the seafood, which are identified by

most consumers as smoky flavor and taste. While a long time ago smoking was discovered to be

a good preservation method that also introduces flavor and taste to the products, today mostly the

flavor effect is desired since other better preservation techniques are available. Hot smoking of

fish and seafood results in a fully cooked product with intense smoky flavor and taste. Some

forms of hot smoking also involve an intense drying step which removes so much water out of

the product that it becomes shelf stable at room temperature without further preservation. Cold

smoking is a less intense form of smoking and allows the smoky flavor and taste to be introduced

into the product without cooking it. While the hot smoking process changes the color and texture

of the product the cold smoking process leaves the color and texture of the seafood nearly

unchanged if done at the right temperature (Burt 1988).

Compared to the traditional smoking processes, filtered smoke and carbon monoxide

treatments are performed at refrigeration temperatures and have only minimal impact on the

texture of the product. Also the taste and smell are nearly unchanged due to the treatment since

carbon monoxide is tasteless and most of the taste and odor giving compounds have been

removed from the filtered smoke. However, the treatments may have a long term preservation

effect that exceeds the traditional smoking techniques. The taste and smell are not only

unchanged, but further preserved for an extended period, since the carbon monoxide, which is

also present in filtered smoke inhibits the lipid oxidation which causes off odors and tastes

(Kristinsson and others 2006b). The color of fish and seafood products treated with filtered









smoke and carbon monoxide is enhanced and preserved during fresh and frozen storage, which

increases the consumer acceptance and likeability (Olson 2006).









CHAPTER 3
OBJECTIVES

The hypothesis for this study is filtered wood smoke processing increases the shelf life and

the quality of Yellowfin tuna by inhibiting pathogenic and spoilage bacteria without altering the

sensory characteristics of the product. The aim of this experiment was to treat Yellowfin tuna

with filtered wood smoke to determine whether spoilage and pathogenic bacteria are inhibited by

this treatment, and to observe any significant correlation between the inhibition of spoilage

bacteria and the growth and inhibition of pathogenic bacteria.

In the first part of this experiment Yellowfin tuna steaks were treated with filtered wood

smoke at 0C and for time periods of 24 and 48 hours. The growth or inhibition of spoilage and

pathogenic bacteria was measured before and after treatment, on frozen storage, and also every

second day of refrigerated storage up to a total storage time of two weeks. The second part of

this experiment aimed to determine the significant changes in odor and color of the filtered

smoke treated products compared to the artificial smoke treated products.

A final objective of this study was to design and validate a quick gas chromatography

method to identify whether a product was treated with filtered smoke or just with carbon

monoxide. The use of carbon monoxide is currently forbidden in Europe and strictly regulated in

many other countries.









CHAPTER 4
PRELIMINARY STUDIES

Filtered wood smoke processing is a recently developed processing technique that is

gaining widespread use throughout the globe. Only a few companies produce filtered smoked

fish products so far and the market has not been fully developed yet. Since the filtered wood

smoke processing is so new, not many studies have been done on this particular subject. Ongoing

studies in our laboratory involve the determination of polycyclic aromatic hydrocarbons (PAH)

in filtered wood smoke and treated products, developing a rapid analysis to identify products

treated with filtered wood smoke and general identification of the major components of filtered

wood smoke and their relevance to the process. We have also done work on the effect of filtered

smoke on the quality of fish muscle, but limited to few treatment times and only a few properties

of fish muscle.

Tuna Microbiology Study

Overall these results indicate that filtered smoke and artificial smoke seem to improve the

shelf life of tuna, but more conclusive data over a longer period is required to confirm the

hypothesis.

Figure 3-1 shows that colony forming units (CFU) count decreased immediately for all

treatments, even for the control group, which might be an indication for a reduction in CFU/g

due to the stress of handling the samples. After the treatment the control group, the 100% carbon

monoxide and the 100% nitrogen group showed a larger increase in CFU/g, followed by the

filtered smoke and the artificial filtered smoke treatments. The artificial smoke treatment showed

overall the lowest bacterial count until day 6, while the filtered smoke treatment increased

significantly in CFU/g after day 3. While conducting this study cross contamination might have

occurred for one of the three monitored samples for the filtered smoke treatment, which might









explain this high value. All other treatments, 100% carbon monoxide, 100% nitrogen and control

showed higher bacterial load until day 6 compared to the artificial smoke.

Figure 3-2 shows a similar initial effect for all treatments as seen in Figure 1. The CFU

count decreased for all treatments, including the control group, after the treatments ended. Over

the next six day the control group and the 100% nitrogen group increased the most in bacterial

count. The filtered smoke treatment showed the smallest increase in bacterial load over the next

six days, followed by the artificial smoke group and the 100% carbon monoxide group.

Figure 3-3 shows the CFU count for all treatments after 48 hours. The graph shows that

there is no initial decrease in CFU count for any of the treatments, which could be because the

treatment time of 48 hours allowed the microorganisms to leave the lag phase already during the

treatment and so they started growing again. Still the control group, the 100% nitrogen group and

the 100% carbon monoxide group showed initial increases in bacterial count followed by the

filtered smoke and artificial smoke treatments. After six days all treatments reached a similar

bacterial load and showed no difference.

A study was conducted to investigate the effect of carbon monoxide, carbon dioxide, nitrogen

and filtered smoke on the overall bacterial growth on tuna steaks over one week. Tuna steaks

were cut from a fresh whole tuna loin and tumbled for a minute in a sterile container to ensure

equal bacterial load on the surface. Two steaks for each treatment were then chosen and placed

into a decontaminated gas tight container. The following treatments were applied to the tuna

steaks: control (no treatment), carbon monoxide (-100%), 100% nitrogen, filtered smoke, and

"artificial filtered smoke" (18% CO, 21% CO2 and 61% nitrogen). The steaks were held at 40C

for 16, 32 and 48 hours inside the decontaminated containers, which were filled with each gas,

Samples were taken from each steak before and immediately after the treatments, as well as 1, 3









and 6 days after the end of the treatments. Each sample was then analyzed for the total bacterial

count using "total plate count" Petrifilms from 3M Corp. Figures 3-1 to 3-3 show the average

total bacterial count per gram for all treatments after 16, 32 and 48 hours of treatment.

Tuna Color Study

Tuna steaks were cut from one fresh tuna loin and placed into a gastight container where

they were treated with carbon monoxide (-100%), 100% nitrogen, filtered smoke and "artificial

filtered smoke" (18% CO, 21% CO2 and balanced with nitrogen) for 16, 32 and 48 hours. The

control group was exposed to the surrounding air. The tuna steaks were analyzed with a color

vision machine for their color value, according to the L*a*b color system. Pictures were taken

before (day -1), after treatment (day 0) and on day 1, 3 and 6 after the treatment and the average

color value was determined and calculated by using the color vision machine and color vision

software. The average L*-, a*- and b*- values were recorded for each sample. Since the major

interest of this study is in the maintenance and enhancement of the red color of the tuna muscle,

the a* value representing the redness of the sample as a positive number (negative represents

greenness), were compared to determine how the redness of the tuna changes after the

treatments. Figures 3-4 to 3-6 show the average a*- values (redness) of the untreated and treated

samples over a time period of 6 days after treatment.

As shown above for 16, 24 and 48 hours the control (CTRL) as well as the filtered smoke

(FS), the 100% carbon monoxide (100% CO) and the 18% carbon monoxide (18% CO) have

increased a*-values immediate after the treatment. Only the sample treated with pure nitrogen

(100% N2) showed an immediate decline in the a*-values after treatment and then continued to

decline over the period of 6 day, except for the 48 hour treatment where a slight increase in a*-

value can be seen on day 1. Overall the nitrogen treatment seems to have a negative affect on the

a*-values, which can be seen immediately after treatment. The control groups show for the 16

28









and 48 hour treatments increases in a*-values and unchanged a*-values for the 24 hour

treatment. From day 1 to day 6 after the treatment the a*-values from the control group declined

rapidly for the 16, 32 and 48 hour treatment. The filtered smoke, 100% CO and 18% CO

treatments showed a slight increase in a*-values for the 16 and 32 hour treatment and a large

increase in a*-values for the 48 hour treatment at day 0. These three treatments show

stabilization in a*-values for the 16 hour treatment until day 1 and then a slow decline until day

6. The 32 and 48 hour treatment show stable a*-values until day 3 and then a decline at day 6.

Overall the 100% CO treatment showed the highest improvement and stabilization in a*-values

after the treatment and over the period of 6 days. The filtered smoke and the 18% CO treatment

seemed to stabilize the a*-values nearly as good as the 100% CO treatment, especially over the

extended period of 6 days. The lack of data points between these measurements makes it clear

that more frequent measurements are needed to describe the color changes due to filtered smoke

and carbon monoxide treatments.

Identification of Filtered Smoke Treated Products

A study was conducted to identify gas compounds that were typical for filtered wood

smoke. A gas chromatograph from Agilent (6890N) was equipped with a packed column and a

nickel catalyst tube to convert carbon monoxide and carbon dioxide into methane. The nickel

catalyst was needed to detect carbon monoxide and carbon dioxide with the FID (Flame

ionization detector), which are otherwise not detectable. 100% carbon monoxide, filtered wood

smoke and unfiltered fresh generated wood smoke were directly injected into the gas

chromatograph (GC) and analyzed. The chromatograms showed a large number of peaks for the

smoke samples and only one peak for the carbon monoxide sample as expected. Natural gas and

refinery gas standards from Scott Specialty Gasses were then analyzed in the GC with the same

method as the smoke samples before.










The previous results led to the idea to identify smoke treated products by analyzing gas

compounds that are evaporated from smoked products. An experiment was conducted where

tuna steaks were treated with 100% carbon monoxide, filtered smoke and natural wood smoke. A

portion of 10 g of sample from each treatment was transferred into a 60 ml vial with a gas tight

lid with septa inlet. The samples were heated in a water bath at 1000C for 5 minutes. After the

heat treatment a sample of 100 il was taken from the headspace of the vials with a gas tight

syringe and injected into the GC. The results show that smoke treated products show two

significant peaks that were not present in products that were untreated or treated with carbon

monoxide, nitrogen and carbon dioxide. Note that carbon monoxide and carbon dioxide can not

be detected directly by using the FID (Flame Ionization Detector), therefore these components

will be methanized (transformed into methane) after separation on the column, which can then be

detected by the FID. Figures 3-7 to 3-11 show the chromatograms collected from this study.



1.00E+07


--No Treatment 16 hrs
1.00E+06 -100 % CO 16 hrs
-- Filtered Smoke 16 hrs
-x--- 100 % Nitrogen 16 hrs
.. --18 % CO 16 hrs
3 1.00E+05



1.OE+04 30 days of frozen storage

-2 0 2 Days at 44C 6 8
Figure 3-1. Tuna treated for 16 hours with 100% CO, filtered smoke, 100% nitrogen and a
mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was
exposed to the surrounding air only. The graph shows the measurement of colony
forming units (CFU) at day -1 (before treatment), day 0 (right after treatment) and
then day 1, 3 and 6 after treatment.


then day 1, 3 and 6 after treatment.












1.00E+09

1.00E+08


30 days of frozen storage


1.U00+0U -No Treatment 32 hrs
100 % CO 32 hrs
'- 1.00E+06 -
A- Filtered Smoke 32 hrs
.- 1.00E+05 / 100 % Nitrogen 32 hrs
LL 18 % CO 32 hrs
1.00E+04

1.00E+03
-2 0 2 4 6 8
Days at 40C
Figure 3-2. Tuna treated for 32 hours with 100% CO, filtered smoke, 100% nitrogen and a
mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was
exposed to the surrounding air only. The graph shows the measurement of colony
forming units (CFU) at day -1 (before treatment), day 0 (right after treatment) and
then day 1, 3 and 6 after treatment.


1.00E+09

1.00E+08

1.00E+07

S 1.00E+06

- 1.00E+05
LL
1.00E+04

1.00E+03


- No Treatment 32 hrs
-100 % CO 32 hrs
A Filtered Smoke 32 hrs
*--100 % Nitrogen 32 hrs
*--18 % CO 32 hrs


30 days of frozen storage


-2 0 2 4 6 8
Days at 40C
Figure 3-3. Tuna treated for 48 hours with 100% CO, filtered smoke, 100% nitrogen and a
mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was
exposed to the surrounding air only. The graph shows the measurement of colony
forming units (CFU) at day -1 (before treatment), day 0 (right after treatment) and
then day 1, 3 and 6 after treatment.















1I


day 0 day 1


day 3


* N2 16 hr
* FS 16 hr
* 100% CO 16 hr
* 18 % CO 16 hr
* CTRL 16hr


day 6


Figure 3-4. Tuna treated for 16 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2)
and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group
(CTRL) was exposed to the surrounding air only. The graph shows the measurement
of average a-value at Day -1 (before treatment), Day 0 (right after treatment) and then
Day 1, 3 and 6 after treatment.



30 -.


iTTT


day -1


ITT


I TT


day


day 1


day 3


* N2 32 hr
* FS 32 hr
* 100 % CO 32 hr
* 18 % CO 32 hr
SCTRL 32 hr


day 6


Figure 3-5. Tuna treated for 32 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2)
and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group
(CTRL) was exposed to the surrounding air only. The graph shows the measurement
of average a-value at Day -1 (before treatment), Day 0 (right after treatment) and then
Day 1, 3 and 6 after treatment.


I TI


day -1


16 iLT













45
40
35
30
25
20
15
10
5
0
day -1


day 0


day 1


day 3
day 3


day 6


* N2
*FS
* 100% CO
* 18% CO
* CTRL48 hr


Figure 3-6. Tuna treated for 48 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2)
and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group
(CTRL) was exposed to the surrounding air only. The graph shows the measurement
of average a-value at Day -1 (before treatment), Day 0 (right after treatment) and then
Day 1, 3 and 6 after treatment.


Figure 3-7. Chromatogram of the analysis of ClearsmokeTM. The first peak represents carbon
monoxide at around 0.7 minutes, the second peak represents methane at around 2.5
minutes and the last peak represents carbon dioxide at around 3.7 minutes


TIT


i\













pA
28
28-
24
22-
20
18-
16-
14
12


Figure 3-8. Chromatogram of the analysis of industrial carbon monoxide (-100% CO). This
peak represents carbon monoxide at around 0.7 minutes. The sample was diluted to
0.1% carbon monoxide in air v/v.


Figure 3-9. Chromatogram of the analysis of atmospheric air. The shown peak represents a
trace amount of carbon monoxide residual in the air. Nitrogen and oxygen are not
detectable by the FID.



Figure 3-10. Chromatogram of the analysis of nitrogen (N2). This peak also represents trace
amounts of carbon monoxide. Nitrogen and oxygen are not detectable by the FID.


*-- ~LL--- --Lu~-l. .-2L,-r--url-----Y*l III--hYI--Y---L.-UYLI~Y-~YYI~-L-~LY- _I)





















Figure 3-11. Chromatogram of the analysis of carbon dioxide (C02) The first peak again show a
small amount of carbon monoxide sample and the second peak represents carbon
dioxide.









CHAPTER 5
MATERIAL AND METHODS

Fresh and Frozen Storage

This project is mainly divided into two parts, a fresh storage and a frozen storage study.

Figure 5-1 shows a scheme for the project plan. For the frozen storage study the samples were

prepared as described in "Sample Preparation and Treatments" and each sample was then

vacuum sealed in a Foodsaver Bag, placed in the freezer and then stored for 30 days at -200C.

Samples for the frozen storage study were thawed out two days prior to the start of the

experiments at 40C in the cold room. The frozen storage samples were not analyzed during the

frozen storage, but rather after the frozen storage in the same manner as the samples for the fresh

storage study.

For the fresh storage study the samples were prepared as described in "Sample Preparation

and Treatments" and every sample was then transferred into a ZipLoc bag and stored in the

cold room at 40C for the remainder of the experiment.

Fresh and frozen storage studies were timed in a manner that fresh and frozen samples

could be analyzed together at the end of the frozen storage period. For the Salmonella, Total

Aerobic Plate Count and the Color-Study, the samples were analyzed before the treatment (day -

1), immediately after the treatment (day 0) and then at day 1, day 2 and every other day after that

for a total of 14 days. For the frozen storage studies Day 1 represents the day when the thaw out

process was completed, while it represents the actual first day after treatment for the fresh

storage studies. For the taste panel and GC- experiments only samples representing day 1 after

treatment or thaw out were chosen and analyzed.









Sample Preparation and Treatments

For all studies except the taste panel study samples were prepared in duplicates per

treatment. For all studies fresh yellowfin tuna was ordered from Save-On Seafood (Tampa, Fl)

and was processed and treated immediately upon delivery. For all studies the workplace was

cleaned and sanitized with 70% ethanol v/v in water to minimize microbial cross contamination.

The fully trimmed tuna loins were cut into 2.5 cm thick steaks, which were again trimmed and

then sorted for uniformity, especially for the color and taste panel studies. All samples were then

transferred into the prepared Foodsaver bags. From a roll 23 by 28 cm bags were cut and sealed

on three sites. The bags were then equipped with a silicon septum valve recovered from PVF gas

sampling bags from LabPure Instruments. Each bag filled with one tuna steak was individually

vacuum sealed and then stored on ice until treatment. For the Salmonella study the samples were

inoculated with Salmonella enteriditis as described in the chapter "Salmonella study". Each bag

was then filled with three liters of the designated treatment atmosphere except for the control

which was kept vacuum sealed. The treatment gases were injected directly from the gas cylinders

via a hose, equipped with a pressure gun and a needle, thru the septa valve into the sample bags.

The amount of gas introduced to each bag was controlled with a gas flow meter from "Alicat

Scientific" Model M-50SLPM-D which was set to Air mode since this is the closest

representation of the treatment gases in terms of density. The actual volume of each treatment

gas was not too important in these experiments, but rather the fact that each bag was filled with

the same amount of treatment gas to ensure uniformity among the treatments.

The filtered smoke was delivered in compressed gas cylinders and ready to use for the

treatments. The filtered smoke was delivered with a certified analysis stating that it contained

21% carbon monoxide, 18 % carbon dioxide, 55% nitrogen and is balanced with oxygen and

other gases. Trace components from the incomplete combustion of wood chips that were not

37










filtered out were not analyzed or determined. According to the typical treatment times used in the

industry it was chosen to determine the effect of a 24 and a 48 hour treatment of filtered smoke

on tuna. The second treatment gas that was used in these experiments was a pure mixture of 21

% carbon monoxide, 18 % carbon dioxide, 1.1% oxygen and a balance of nitrogen. This mixture

was chosen since it represents the exact composition of the filtered smoke without the trace

components that are possibly found in filtered wood smoke. This mixture was called artificial

smoke and will be abbreviated as AS. The original filtered smoke will be abbreviated as FS. To

compare AS to FS equal treatment times (24 and 48 hours) were chosen for the AS treatments as

well. The control group for all experiments consists of tuna steaks which were vacuum-packed

and sealed and then kept on ice for 24 hours prior to analysis. Table 4-1 shows an overview over

the treatment combinations that were applied to all studies.

Table 5-1. Treatment combinations used in all studies of this project
Label Treatment Gas Treatment Time Storage Conditions
FS24F Filtered smoke (FS) 24 hours 30 days at -20C
FS48F Filtered smoke (FS) 48 hours 30 days at -20C
AS24F Artificial smoke (AS) 24 hours 30 days at -20C
AS48F Artificial smoke (AS) 48 hours 30 days at -20C
CtrlF No gas treatment 24 hours 30 days at -20C
FS24 Filtered smoke (FS) 24 hours 1 day at 4C
FS48 Filtered smoke (FS) 48 hours 1 day at 4C
AS24 Artificial smoke (AS) 24 hours 1 day at 4C
AS48 Artificial smoke (AS) 48 hours 1 day at 4C
Ctrl No gas treatment 24 hours 1 day at 4C

Salmonella Study

To study the effect of filtered and artificial smoke on the inhibition or growth of

Salmonella spp. tuna steaks were prepared as described in "Sample preparations and treatments"

with 4 steaks per treatment. Two steaks per treatment were inoculated with Salmonella

enteriditis, which was obtained from the American Type Culture Collection (ATCC #13076).

The culture was prepared and enriched according to the instructions given by the ATCC in









Nutrient Broth. For each steak 1 ml of culture was spread evenly over the entire steak and the

bag was shaken for 1 minute to ensure an even distribution of the culture over the tuna steak. For

the Salmonella analysis a cube of 10 g was cut from the each inoculated tuna steak in a manner

that the cube consists of three surfaces exposed and three internal exposed sites to ensure an even

distribution of microorganisms each time the steaks were sampled. The two other steaks were

kept non-inoculated to see the effect of the treatments on the naturally existing Salmonella spp.

flora. These steaks were sampled in the same manner as the inoculated samples. The non-

inoculated samples were sampled at day -1, day 0, day 1 and day 2 from that point it was decided

to abandon the non-inoculated study, since no Salmonella growth could be identified. These

samples were analyzed a last time at day 14 to ensure that there was actually no Salmonella

growth and the decision to abandon this study was correct. The inoculated steaks were sampled

right after the inoculation but before the treatment (Day -1), directly after the treatment (Day 0)

and at Day 1, 2 and then every other day until day 14. During this time the steaks were stored in

Ziploc Bags at 40C in a cold room.

Each 10 g samples for each analysis were placed into a sterile Nasco Whirlpack bag and

90 ml of Hardy Diagnostics Dilu-LOK II Phosphate buffer with magnesium chloride (buffer

solution) added to the sample. The sample was squeezed and mixed by hand inside the bag with

the buffer solution. Three Dilutions were prepared by pipetting 10 ml of the sample solution into

90 ml of sterile buffer solution. More Dilutions were prepared if the results from the previous

day indicated that the first three dilutions would have to numerous to count microorganisms.

Then 0.2 ml of each dilution were plated onto two preplated XLD-Agar Plates from Biomerieux

Industries (0.1 ml per plate) and spread with a Lazy L spreader over the entire plate to ensure

uniform growth. Always the last three dilutions were plated and counted. All plates were









incubated at 350C for 24 hours. To determine the amount of Colony Forming Units (CFU) per ml

of solution all yellow colonies with black centers were counted by hand for each plate. Which

dilutions were prepared and plated was decided on the basis of the results from the previous day.

Most of the Salmonella study was performed under a sterile hood.



Total Aerobic Plate Count Study

To study the effect of filtered and artificial smoke on the inhibition or growth of all

naturally present aerobic bacteria, tuna steaks were prepared as described in Sample

preparations and methods" with 2 steaks per treatment. During preparation and treatment all

samples were handled aseptically to avoid cross contamination. All sample steaks were stored in

Ziploc Bags at 40C during the entirety of the 14 day shelf life study. For the fresh storage study

the steaks were sampled before treatment (Day -1), directly after treatment (Day 0) and at Day 1,

2 and then every other day until day 14. For the frozen storage study the steaks were sampled

before treatment (Day -1), directly after treatment (Day 0) and then after the two day defrosting

process (Day 1) and at day 2 and every other day until day 14. To determine the amount of CFU

per g of tuna at any given sampling time, a 10 g cube consisting of three surface-exposed and

three inside-exposed sites was cut from each steak and placed aseptically into a sterile Nasco

Whirlpack bag. The bag was then filled with 90 ml of sterile premade Dilu-LOK II Butterfields

buffer from Hardy Diagnostics and the sample cube was squeezed and mixed inside the bag

with the buffer by hand for 1 minute. The sample solution in the bag is then considered the first

dilution. Further dilutions were prepared with the same buffer solution by transferring 9 ml of the

sterile buffer solution and 1 ml of the sample solution aseptically into a sterilized culture tube.

More Dilutions were prepared if the results from the previous day indicated that the first three

dilutions would have to numerous to count microorganisms. For each dilution and sample 2 ml

40









were plated onto two PetrifilmTM Plates for "Aerobic Plate Count" from 3M Microbiology

Products with 1 ml of solution per plate. Always the last three dilutions were plated and counted.

All plates were then incubated at 34.50C for 48 hours. Each plate was counted by hand to

determine the amount of CFU per gram of sample. Which dilutions were prepared and plated

was determined based on the results from the previous day.

Color Analysis

The effect of filtered and artificial smoke on the color of tuna during 14 days of storage at

4C was determined by a digital Color Machine Vision System (CMVS). The CMVS was used

according to the procedure described by Balaban and Luzuriaga (2001). The CMVS can report

the average L*-(lightness), a*-(redness) and b*-(yellowness) values for each sample. For this

experiment the main focus was on the changes in the average a*- values since the redness of tuna

is one of the main quality criterion for the industry and especially the consumer. Pictures were

taken before treatment (Day -1), direct after treatment (Day 0) and at day 1 (after thaw out period

for the frozen storage study), day 2 and then every second day until day 14. The samples were

placed in a light box and top lighting with two fluorescent lights each to simulate illumination by

noonday summer sun (D65 illumination). The door remained closed while images were

capturedto assure uniformity of light inside and to minimize the effect of outside light. Images

were captured using a camera (Nikon D200 Digital Camera, Nikon Corp., Japan) located inside

the chamber mounted to face the bottom of the light box. The Nikon D200 Settings used are

described in Table 5-2. A red reference tile was laid into each picture to compensate for changes

in light and camera settings in the computer analysis of the pictures.

Sensory Taste Panel Analysis

To determine if there were any human detectable differences between a filtered and an

artificial smoked tuna steak in appearance and smell, a sensory taste panel was conducted with

41









60 random untrained panelists. The taste panel analysis was conducted on two different days,

with the first day focusing on the odor differences among the samples and on the second day the

color differences were analyzed. Only the 48 hour treatments with filtered smoke and artificial

smoke for the frozen and the fresh storage study were compared, since it was known that these

treatments would give the most odor and color differences. Four triangle tests were conducted to

determine if there were any detectable differences in odor or color between 48 hour treated

filtered and artificial smoked tuna after frozen (30 days at -200C) and fresh (1 day at 40C)

storage. The design, test and analysis of this study were conducted in the FSHN taste panel

facility with the use of the Compusense software program. The detailed design for each

triangle test can be found in Appendix A "Taste Panel Design Sheets". Each panelist who

identified the odd sample as the different sample was asked to write down any comments he

might have to describe the difference. Tables B-1, B-2, B-3 and B-4 show a summery of all

comments given by the panelists.

Rapid Gas Chromatography Identification Method

The main components of filtered and artificial smoke are carbon monoxide, carbon dioxide

and nitrogen, as well as a small amount of oxygen. However, filtered smoke contains more then

just these components. A GC method which was mainly developed to quantify the amount of

carbon monoxide in red muscle food might also have the potential to determine whether a

product was treated with real filtered wood smoke or just a combination of 4 industrial gases that

we call artificial smoke. The first step was to determine which components can be identified in

filtered smoke that are not present in artificial smoke with the simple GC method developed for

carbon monoxide quantification. An Agilent Technologies 6890N Network GC System,

equipped with a Flame Ionization Detector (FID), a Supelco 80/100 Porapak Q Column (1.82 m

long) and a hydrogen aided Nickel Catalyst (to convert carbon monoxide and carbon dioxide into

42









methane and make it detectable for the FID) was used for this study. The settings for the rapid

identification procedure are shown in Table 5-3. PVF Gas sampling bags from LabPure

Laboratory Instruments were used to collect the pure gasses and I-Chem Economy 100 series 60

ml glass vials with a Teflon-fluorocarbon-resin/silicone septa lid were used for gas dilutions and

sampling. The glass vials were flushed with nitrogen prior to sampling to minimize any

contamination from the surrounding air. To identify the gas components in filtered smoke its

chromatogram was compared with the chromatogram of the "Refinery Gas Test Sample" from

Agilent Technologies. After the all peaks from the filtered smoke chromatogram had been

identified all these gases were ordered in high purities to create standard curves (for

quantification) for each of them. Ethylene (99.5% purity) and ethane (99.0% purity) were

obtained from Scott Specialty Gasses. Methane (Ultra High Purity Grade), carbon monoxide

(CP-Grade) and carbon dioxide (CP-Grade) were obtained from Airgas. Standard curves for

each of these gasses were produced by diluting each gas several times and injecting different

amounts of each dilution into the GC. The areas under the corresponding peaks were then plotted

against the actual quantity of gas injected. After a linear regression analysis an equation was

obtained for each gas that allows their quantification by peak area.

Two different samples of filtered smoke, the artificial smoke and a filtered smoke, which

was obtained from another source (filtered smoke B), were analyzed with the GC for their

content of each of the individual gasses.

The final step was to identify whether a piece of tuna was treated with filtered or artificial

wood smoke and to quantify the amount of carbon monoxide per gram of muscle tissue. For this

analysis 10 g of sample from each treatment and each storage study were minced and transferred

quickly into the 60 ml glass vials and the vials were sealed. The samples were then heated at









100C for five minutes and then cooled down to room temperature for another five minutes. A

quantity of 100 [il of the headspace atmosphere from each vial was then injected into the GC and

analyzed for the previously mentioned gas components. Since the artificial smoke does not

contain any methane, ethylene or ethane, it would be unlikely for the tuna to absorb these gases

and release them into the headspace of the vial as it would do with a sample treated with filtered

smoke. Therefore the presence of these gases in the headspace over a sample would indicate a

treatment with filtered smoke.



Statistical Analysis

For the Salmonella, total aerobic plate count and color-studies the data for the fresh and the

frozen storage was analyzed separately. Analysis of Variance (ANOVA) was used to determine

significant differences between all treatments at all sampling times. When significant differences

among the sample means were detected Tukey's Studentized Range (HSD) test was used to do a

pair wise comparison between these sample means. The level of significance for all of the

ANOVA and Tukey's test were set to 5 % or 0.05.

Simple linear regression analysis was used to determine the standard curve equations for

the GC analysis of carbon monoxide, carbon dioxide, methane, ethylene and ethane. The

Statistical Analysis Software (SAS) and Microsoft Excel were used for the analysis of the data.









Table 5-2. Nikon D200 Settings
Setting
Device
Lens
Focal length
Sensitivity
Optimize image
High ISO NR
Exposure mode
Metering mode
Shutter speed and aperture
Exposure compensation (in camera)
Focus mode
Long exposure NR
Exposure compensation (by capture NX)
Sharpening
Tone compensation
Color mode
Saturation
Hue adjustment
White balance


Specification
Nikon D200
VR 18-200 mm F 3.5-5.6 G
36 mm
ISO 100
Custom
Off
Manual
Multi-pattern
1/3s-F/11
0EV
AF-S
Off
0EV
Auto
Auto
Model
Normal
0
Direct sunlight


Table 5-3. GC-Settings for the rapid identification method
Parameters Settings
Injection Temperature 100C
Carrier Gas and Flow rate Helium at 26.9 ml/min (splitless)
Nickel Catalyst Temperature 375C
Oven Temperature 300C isotherm
Runtime 5 minutes
Detector Temperature 250C
Column 80/100 Porapak Q packed column (1.82 m)




















































Taste Panel Experiment


Gas Chramatography Development of rapid Verification of rapid gas
Analysis of filtered I gas chromatography 0 chromatography
smoke identification method identification method

Figure 5-1. Flow diagram of all studies conducted during this project









CHAPTER 6
RESULTS AND DISCUSSION

Salmonella Results

The first part of this project was to investigate what effect a filtered smoke and artificial

smoke treatment will have on the growth of Salmonella enteriditis on Yellowfin tuna after fresh

and frozen storage. It should be noted at this point that it was planned to follow the Salmonella

growth over a period of 14 days for inoculated and non-inoculated samples. Salmonella spp. is

the leading cause of several food poisoning related diseases on humans (Scott 1996) and

Salmonella enteriditis is one of the most aggressive species (Butt and others 2004). Salmonella

enteriditis was therefore obtained from the American Type Culture Collection (ATCC). The non-

inoculated samples showed absolutely no Salmonella growth during the first 4 days of the study

for all samples and all treatments. It was therefore decided to abandon the analysis of the non-

inoculated samples and keep them under the same conditions as the inoculated samples to

analyze them a last time at day 14. When no growth of Salmonella was detected on the non-

inoculated samples at day 14, it was decided not to include any collected data on the non-

inoculated samples since there was obviously nothing to report. Although no Salmonella spp.

were found on the non-inoculated samples, several studies still report incidences with Salmonella

spp. in raw and cold smoked seafood, where the chance of a Salmonella incident is usually

higher in imported seafood (Heinitz and Johnson 1998; Heinitz and others 2000).

The focus was laid on the analysis of the growth of Salmonella spp. on the inoculated

samples after filtered smoke and artificial smoke treatments for the fresh and frozen studies,

since Salmonella species were detected in several smoked fish samples from different countries

(Heinitz and Johnson 1998; Fell and others 2000). Figures 6-1 and 6-2 show the average amount

of Colony Forming Units (CFU) for each treatment at any measured time point for the fresh and









frozen studies, respectively. For both the fresh and the frozen study, Analysis of Variance

(ANOVA) tests were conducted for every observed day to detect any significant differences

among the average CFU count for all treatments. Tables 6-1 and 6-2 show the results of the

ANOVA tests as well as the results of the Tukey pair wise comparisons, which were conducted

when a significant difference was detected by an ANOVA test.

Fresh Storage Study

As Table 6-1 for the fresh study shows, a significant difference among the sample means

could only be detected at day 4 of the study. However, the data for Tukey's test shows that there

is no significant difference among all treatments except for the 48 hour artificial smoke

treatment, which is significantly different from all other treatments except the 24 hour filtered

smoke treatment. It is very likely that this difference is based on a random effect and an

experimental error than an effect of the 48 hour artificial smoke treatment. It should be noted

here that to conserve time and resources for all experiments duplicate samples were prepared and

analyzed. All statistical analysis is based on the analysis of duplicate samples. Nevertheless it

can be clearly seen that none of the applied treatments seems to have a major effect on the

growth or inhibition of growth of Salmonella spp. It is of course well observed in Figure 6-1 that

from the day of inoculation during the complete time of observation, the growth of Salmonella

spp. was inhibited and the count of Salmonella CFU decreased. A possible explanation for this

effect might be the competition of the Salmonella bacteria with the natural existing microbial

flora present on the tuna samples (Revolledo and others 2003; Liao 2007). Therefore it is

suggested that not so much any specific treatment, artificial or filtered smoke, inhibited the

growth of the Salmonella spp. but rather the growth of natural existing microorganisms present

on the tuna samples. Another observation supporting these findings is the fact that no Salmonella

growth was detected in the non-inoculated samples. Heinitz and others (2000) reported a 10%

48









chance of Salmonella spp on raw imported seafood and only a 2.8% chance on raw domestic

seafood based on samples taken by USDA field laboratories over a period of 9 years.

Frozen Storage Study

The results for the frozen storage study were quite similar to the fresh storage study, as

shown in Figure 6-2. There were no significant differences among the treatment means for all

observed days as shown in Table 6-2, which shows the p-values of the ANOVA tests that were

conducted to detect any significant differences among the sample means for each observed day.

It seems that in Figure 6-2 compared to the fresh storage study there was some growth of

Salmonella spp. observed between the inoculation and the end of the treatments, but this can be

explained by a minor experimental flaw that was eliminated immediately after the first

measurement. When the steaks were inoculated with Salmonella enteriditis, the culture was kept

in a buffer solution and 1 ml of this buffer solution was spread over the entire surface of the

sample steak as evenly as possible. A sample for analysis was then taken immediately, as

described in "Materials and Methods", before the sample was vacuum packed for the treatment.

It was later discovered that the vacuum packing actually contributes to the distribution of the

culture on the sample through the force of atmospheric pressure on the outside of the packaging

material. For the fresh storage study all samples were vacuum packed after the inoculation and

then reopened and sampled before the treatment to improve the even distribution of the culture.

Since in fact all samples were vacuum packed before any treatment was applied to the samples

there should have been no further impacts on the outcome of this analysis.

It can therefore be concluded that filtered smoke and artificial smoke processing have no

immediate effect on the growth or inhibition of Salmonella spp. whether the sample were stored

frozen or kept at 40C.










Although it was not observed in this experiment, the inhibition or growth of Salmonella

spp. can be highly influenced by the natural microbial flora present on the product (Liao 2007).

Table 6-1. ANOVA results for Salmonella spp. for the fresh storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value 0.79 0.05 0.09 0.14 0.01 0.51 0.46 0.30 0.73 0.47
Ctrl a a a a a a a a a a
FS24 a a a a ab a a a a a
FS48 a a a a a a a a a a
AS24 a a a a a a a a a a
AS48 a a a a b a a a a a
P-Values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other


--Ctrl ---FS24 A--FS48 -X-AS24 -K-AS48


1.OOE+05




1.OOE+04


1.00E+03 I i




1.00E+02 ....
-2 0 2 4 6 8 10 12 14 16
Days

Figure 6-1. Average amount of CFU/1Og of tuna for Salmonella spp. for the fresh storage study.
The graph shows the average CFU/lOg of tuna at day -1 (before any treatment was
applied), day 0 (right after the treatment) and day 1 though day 14. The samples were
kept at 4C during the entire time of observation. All samples were inoculated with
Salmonella enteriditis before the day -1 measurement. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.










--Ctrl ---FS24 ---FS48 -X-AS24 --AS48


1.00E+05




1.00E+04




1.00E+03



<- 30 days frozen storage
1.00E+02 .........
-2 0 2 4 6 8 10 12 14 16
Days

Figure 6-2. Average amount of CFU/1Og of tuna for Salmonella spp. for the frozen storage
study. The graph shows the average CFU/lOg of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were frozen for 30 days at -200C between day 0 and day 1.The samples were then
kept at 4C during the remaining time of observation. All samples were inoculated
with Salmonella enteriditis before the day -1 measurement. The samples were treated
with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for
24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.

Table 6-2. ANOVA results for Salmonella spp. for the frozen storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value 0.05 0.78 0.20 0.75 0.72 0.16 0.28 0.41 0.75 0.81
Ctrl a a a a a a a a a a
FS24 a a a a a a a a a a
FS48 a a a a a a a a a a
AS24 a a a a a a a a a a
AS48 a a a a a a a a a a
P-values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other









Total Aerobic Plate Count

In the early days of natural wood smoke processing the main purpose was to preserve

meats and seafood for a prolonged period. Although other factors like the heat treatment and the

dehydration of the product (Burt 1988), which accompanied the smoke treatment, are affecting

the microbial flora present on the product, it was suggested that certain components, present in

natural wood smoke might inhibit the growth of spoilage bacteria and enhance their shelf life

(Kristinsson and others 2006b). This study therefore focuses on the effect of filtered wood smoke

on the growth or inhibition of aerobic bacteria naturally present on Yellowfin tuna. Earlier

studies already suggested that filtered wood smoke processing inhibits the growth of aerobic

bacteria during the first 6 days of storage (Danyali 2004). In this study the effect of filtered wood

smoke and artificial wood smoke processing for 24 and 48 hours on fresh and frozen tuna was

analyzed over a period of 14 days of storage at 40C. The frozen tuna was treated prior to

subjecting it to 30 days of frozen storage. Figures 6-3 and 6-4 show the results of the average

CFU count for all treatments at all observed time points for the fresh and frozen storage studies,

respectively. For each single time point, ANOVA tests were conducted to detect any significant

differences among the sample means of the treated and untreated products. When significant

differences were detected Tukey's pair wise comparison tests were conducted to determine

exactly which treatments are significantly different at this time point. Table 6-3 and 6-4 show the

p-values of the ANOVA tests for each time point as well as the results of the pair wise

comparison for the fresh and frozen studies respectively.

Fresh Storage Study

Results of the fresh storage study show that no significant differences could be detected for

most of the observed days, except day 0 (immediately after treatment) and day 2. The p-value of

the ANOVA tests for day 1 shows that no significant difference could be detected at a

52









significance level of 0.05, but there was a significant difference at a level of 0.09 and higher. As

described before, all experiments were conducted in duplicates. Therefore one outlier can

influence the outcome. Still it has to be observed that at day 1 the control group showed a much

higher CFU count than all other groups. These findings are similar to the results described by

Kristinsson and others (2006b). It can be said that all treatments seem to suppress the growth of

aerobic bacteria during the first 4 days of observation. Total aerobic bacterial count then

suddenly stopped increasing at day 4 and reached an equal level for all treatments, including the

control group. From day 4 to day 14 there were no significant differences detectable between the

control group and any of the treatments. The reduced CFU count at day 4 represents the end of

the lag phase of a typical bacterial growth curve (Creager and others 1990), followed by the log

or exponential phase (Tortora and others 1992) from day 4 to day 6 and the stationary phase

from day 8 to day 14. The end of the stationary phase can not be seen here since no

measurements were taken after day 14. Although no significant differences could be detected

after day 4 it seems like there is a small second lag phase for the sample treated with filtered and

artificial smoke at day 8 compared to the control group, however the lack of significance and of

data surrounding this event makes these findings inconclusive. Overall it can be said that the

filtered and artificial smoke treated samples appear to be better protected against aerobic bacteria

growth during the first three days of storage at 40C after the treatment. However no significant

improvement on shelf life can be reported after the exponential growth of the organisms was

initiated. There is also no evidence in this study suggesting any significant difference between

the filtered and artificial smoke treatments or a prolonged 48 hour treatment compared to the 24

hour treatment for both treatment gasses.









Frozen Storage Study

The results for the frozen storage study showed that there are no significant differences

among the sample means for day -1 (before treatment), day 0 (after treatment) and day 1, day 10,

day 12 and ay 14. At day 1, this is the first sampling day after the 30 day frozen storage period,

all samples seem to have the same CFU count regardless of their treatment. This can be

explained by the fact that so shortly after the thaw out process only few viable cells could be

sampled and survived the stress of freezing and thawing (Speck and Ray 1977; Bhaduri and

Cottrell 2004). However, already at day 2 it can be observed that CFU count for the control

group is significantly higher then the CFU count for all other treatments. It should be noted that,

although not statistically significant, the CFU count for the control group seems already to be

higher directly after the treatment and before the samples were placed into frozen storage at day

0. Furthermore should be noted that at day 0 it seems that the 48 hour filtered smoke and

artificial smoke treatments inhibit the growth of aerobic bacteria more effectively then the 24

hour treatments, respectively. From day 4 to day 6 the same behavior is observed as compared to

the fresh storage study, where day 4 marks the end of the lag phase of the bacterial growth and

the beginning of the log or exponential phase. From day 6 to day 14 a more or less stable

stationary phase is observed. The slight increase in CFU count for all samples during the last 8

days could suggest that there are still microorganisms recovering from the 30 day frozen storage

(Speck and Ray 1977). Nevertheless the control group, although not longer statistically

significant, has higher CFU counts than the treatment groups at all times from day 6 to 14. The

statistical analysis shows that for most of the time points no significant difference could be

detected between filtered and artificial smoke processed samples, whether they were treated for

24 or 48 hours. These findings again suggest that a 48 hour treatment does not improve the

overall results compared to a 24 hour treatment. Also, the artificial smoke treatment seems to

54










affect the growth of aerobic bacteria in a similar way as filtered smoke treatment contrasted to

the results from other studies which suggest an advantage of the filtered smoke treatment over

the artificial smoke treatment (Danyali 2004; Kristinsson and others 2007).

Overall, both the filtered and the artificial smoke treatment, show an effect on the growth

of aerobic bacteria on tuna, especially before the exponential growth phase is initiated.

Furthermore the combined effect of freezing and filtered or artificial smoke seems to prolong the

shelf life of tuna fish after thawing by inhibiting aerobic bacterial growth during the first 6 days

after thawing. Similar findings have been reported by Rawles and others (1996) and Demir and

others (2004)


-*-Ctrl ---FS24 ---FS48 ---AS24 -)--AS48
1.00E+08

1.00E+07 -

1.00E+06

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.OOE+01
-2 0 2 4 6 8 10 12 14 16
Days

Figure 6-3. Average amount of CFU/lOg of tuna for aerobic plate count for the fresh storage
study. The graph shows the average CFU/lOg of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were kept at 40C during the entire time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.


ntreated.










-*-Ctrl ---FS24 ---FS48 -X-AS24 --AS48


1.00E+08

1.00E+07 -

1.OOE+06 -

1.OOE+05

u. 1.00E+04

1.00E+03

1.00E+02

1.OOE+01 30 days frozen storage

1.00E+00
-2 0 2 4 6 8 10 12 14 16

Days

Figure 6-4. Average amount of CFU/10g of tuna for aerobic plate count for the fresh storage
study. The graph shows the average CFU/lOg of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were frozen for 30 days at -200C between day 0 and day 1.The samples were then
kept at 4C during the remaining time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.

Table 6-3. ANOVA results for aerobic plate count for the fresh storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value 0.85 >0.01 0.08 >0.01 0.86 0.47 0.52 0.52 0.39 0.11
Ctrl a a a a a a a a a a
FS24 a b a b a a a a a a
FS48 a b a c a a a a a a
AS24 a b a bc a a a a a a
AS48 a b a c a a a a a a
P-values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other


ther









Table 6-4. ANOVA results for aerobic plate count for the frozen storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value 0.72 0.28 0.49 >0.01 0.02 0.01 >0.01 0.29 0.06 0.12
Ctrl a a a a a a a a a a
FS24 a a a b b b b a a a
FS48 a a a ab b b c a a a
AS 24 a a a b b b c a a a
AS48 a a a b b b c a a a
P-values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other
Color Analysis

The appearance and color of fresh and frozen seafood is one of the major characteristics

consumers use to judge the quality of a product. Most of the time the consumer is not able to

taste or smell the actual product since it is either vacuum packed and frozen or it is laid out on a

tray behind the counter where the consumer can only visually examine the product. Therefore a

fresh color and appearance are of major importance. For fresh Yellowfin tuna a bright red to dark

red color is desired for consumer acceptance. Carbon monoxide processing and modified

atmospheric packaging have been known and used for a while to preserve and enhance the color

properties and especially the redness of fish and seafood products (Mancini and Hunt 2005;

Sorheim and others 2006). For this experiment a Color Machine Vision System was used to

analyze the color and color changes of the samples during this study. Since the degree of redness

(a*-value) is one of the most important quality indicators in red muscle seafood, such as tuna, the

average a*-value of each sample was determined for each treatment at any time point. Figures 6-

5 and 6-6 show the average a*-values for each treatment measured over a period of 14 days for

the fresh and frozen study, respectively. ANOVA tests were conducted to detect any significant

differences among the treatment means for each observed day of the fresh and frozen storage

studies. Table 6-5 and 6-6 show the results of the ANOVA tests as well as the results of the

Tukey's pair wise comparisons, which were conducted to determine which sample means where









different from each other. Reading the table column by column, same letters assigned to sample

means show there are no significant differences among these means. The L*-values (lightness)

and b*-values yellownesss) were also analyzed and are shown in Figures 6-7 to 6-10. Kristinsson

and others (2006a) reported that in several studies the redness of the fish muscle was highly

influenced by carbon monoxide but not the lightness (L*-values) and the yellowness (b*-values).

Fresh Storage Study

Table 6-5 shows that the ANOVA tests detected significant differences among the sample

means for all observed days of the fresh storage study. It can also be seen that for all observed

days, except the first day before any treatment were applied to the samples, the control group

means are always significantly different from the other treatment means, according to Tukey's

pairwise comparison. Similar to the frozen storage study there are significant differences among

the samples before any treatment were applied, which again can be explained by the way sample

steaks were visually sorted and assigned to the treatments, as shown in Figure 6-5. In comparison

to the frozen storage study however we were starting clearly at the same a*-value level. It also

seems that there is a difference between the 24 and 48 hour treatments at this point that can not

be explained by the treatments since no treatments were applied at this time. At day 0, directly

after the treatments all samples showed an improvement in a*-values, even the control group,

which could be explained that all samples were transferred directly into the open Ziploc bags

after the treatment (the control group too) where the are immediately exposed to the oxygen in

the air, compared to the frozen study where the samples were kept in the vacuum bags for the

frozen storage. Similar to the frozen storage study, the 48 hour treatments for filtered and

artificial smoke showed a greater improvement in redness than the 24 hour treatment or the

control group. At day 1 after the treatment, the filtered smoke treated samples showed a great

improvement in the redness, similar to the frozen storage study, while the artificial smoke treated

58









samples show no improvement in redness from this point until day 6. Actually, the redness in the

artificial smoke treated sample decreased slightly from day 4 to day 6. The a*-values of the

filtered smoke treated samples follow nearly the same pattern as recorded for the frozen storage

study. The control group also follows a similar pattern in the development of the a*-values as

compared to the frozen study, where there can be seen a decline in redness until day 6 and then a

slight increase until day 14. The artificial smoke treated samples seem to follow now a similar

pattern where a decline in redness can be from day 0 until day 6 and then a slight increase until

day 14. Increased overall variation among the sample means, indicated by increased standard

deviation values, suggests, similar to the frozen storage study, that more factors than just the

treatments start to influence the color development of the samples starting from day 6. So far, no

reasonable explanation could be found why the artificial smoke treatments does not show the

same effect as the filtered smoke treatment in the fresh storage study. Still, the improvement in

redness among the artificial smoke treated samples exceeds the redness of the control group by

far and results in fresh and red looking tuna steaks, as shown in Figure 6-6.

The results from the analysis of L*-values (brightness) and b*-values yellownesss) for the

fresh storage study were quite similar to the findings of the frozen storage study (Figures 6-7 and

6-9), where the L*- and b*- values follow the pattern of the a*-values and show large increases

in brightness and yellowness for all treatments during the first 6 days of observation while the

control group stays behind. These findings again confirm that there are changes in the L*- and

b*- values affected by the filtered smoke and artificial smoke treatment, contrary to the reports of

several other studies (Danyali 2004; Garner 2004; Balaban and others 2005), where little effects

on L*- and b*- values were reported.









It should be noted at this point that overall no differences were observed whether a sample

was treated for 24 or 48 hours, except when analyzed right after the treatment. It is therefore

suggested that a 24 hour treatment of either filtered or artificial smoke should be sufficient to

improve the color properties of tuna for fresh or frozen storage.

Frozen Storage Study

As Table 6-6 indicates, for every observed day, except the last day of the frozen storage

study there are significant differences among the treatment means. Furthermore Tukey's test

results show that for every observed day (except day 14) the control group is significantly

different from all other treatments. Surprisingly the control group means are even significantly

different from all other treatment means before any treatment was applied to any of the samples.

This could be explained by a minor flaw in the way the sample steaks were sorted and assigned

to the treatments visually prior to the beginning of this experiment. However, Figure 6-6 shows

clearly that in the overall development of redness (a*-values) of the samples the initial

measurements are very close to each other with relatively small standard deviations resulting in a

highly detectable difference among the samples for the ANOVA test. It is also clearly visible

that the value of the standard deviations increased over time, when other factors like microbial

spoilage, oxidation and degradation influence the appearance of each sample individually.

Therefore, at day 14 no significant differences can be detected among the treatments and the

control samples. Immediately after treatment Figure 6-6 indicates that the 48 hour treatment with

filtered and artificial smoke results in higher a*-values then the 24 hour treatment, which is

confirmed significant by Tukey's test (Table 6-6). This can be explained by the fact that the 48

hour treated samples spent 24 more hours in treatment prior to the analysis and the carbon

monoxide had therefore more time to penetrate into the muscle and form the carboxy-myoglobin

complex, which is responsible for the redness of the sample. It is also clearly visible that there

60









are no significant differences between the filtered and artificial smoke treatments. Day 1

represents the first day of analysis after the 30 day frozen storage period, and it is clearly visible

that the a*-values, and therefore the redness, of all treatments greatly improved while the control

group lacks significantly behind. A small increase can be seen in the control group, possibly to

the effect of oxygen exposure of the sample, when the bags where opened for analysis after the

thaw-out process. The a*-values for all treatments seem to be stable for a period of about 4 days

of storage at 40C, from which point they slowly decline. At the end of the 14 day study the

differences among the samples are so big, indicated by increased standard deviations, that no

significant differences can be detected among any treatment and the control group. The control

groups shows a decrease in their a*-value for the first 4 days of the analysis and then a very slow

increase, which could be a result of increased spoilage and oxidation, but they never reach the

redness as resulted by the treatments. Figure 6-11 shows visual comparison of untreated tuna

steaks and filtered smoke treated tuna steaks after 30 days of frozen storage right after thawing.

The differences in color and overall appearance are clearly visible. The control group has a dark

grayish appearance whereas the filtered smoke treated samples look nice red and fresh. The L*-

values (Figure 6-8) show a similar pattern as described for the a*-values above, an effect that

was also reported by Balaban and others (2006). The lightness of the 24 hour treated samples

(filtered and artificial smoke) increased dramatically after the frozen storage, while the 24 hour

treatments increased only half as much in lightness and the control groups stoud unchanged.

Also the b*-values (Figure 6-10) show a increase in yellowness for the 48 hour treated samples

compared to the 24 hour treated samples and the control group. These findings are quite

interesting since Kristinsson and others (2006a) reported that no significant changes have been

found in several studies among the L*- and b*-values for carbon monoxide treated fish.










Table 6-5. ANOVA results for the average a*-values for the fresh storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01
Ctrl a a a a a a a a a a
FS24 b c c c d c bc b b b
FS48 a b b b b b b bc b b
AS24 b d d c d c c c b b
AS 48 a b b n c b b bc b b
P-values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other


-4-Ctrl -4-FS24 -*-FS48 -+-AS 24 --AS 48


60.00


50.00


40.00


30.00


20.00


10.00


0.00


-2 0 2 4 6 8 10 12 14 16

Days

Figure 6-5. Average a*-values of the color of tuna samples over 14 days for the frozen storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were kept at 40C during the entire time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.












-4-Ctrl -4-FS24 -*-FS48 -X-AS24 -*-AS48


60.00 -


50.00


40.00 -


30.00 -


20.00


10.00


0.00


<- 30 days frozen storage


-2 0 2 4 6 8 10 12 14 16
Days


Figure 6-6. Average a*-values of the color of tuna samples over 14 days for the fresh storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were frozen for 30 days at -200C between day 0 and day 1.The samples were then
kept at 4oC during the remaining time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.

Table 6-6. ANOVA results for the average a*-values for the frozen storage study.
Days -1 0 1 2 4 6 8 10 12 14
p-Value >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 0.05 0.12
Ctrl a a a a a a a a a a
FS24 b b b b b b b ab ab a
FS48 b b b b b b b bc ab a
AS24 b c b b b b c bc b a
AS48 b c b b b b bc c ab a
P-values smaller then 0.05 indicate a significant difference among the average CFU counts for
the specific treatments. Treatment means with the same letter are not significantly different from
each other











-*-Ctrl ---FS24 +--FS48 -X-AS24 -*-AS48


50.00



45.00


40.00



35.00


30.00- 1 1



25.00 i
-2 0 2 4 6 8 10 12


14 16


Days


Figure 6-7. Average L*-values of the color of tuna samples over 14 days for the fresh storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were kept at 40C during the entire time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.











-*-Ctrl ---FS24 +--FS48 -X-AS24 -*-AS48


50.00



45.00


40.00



35.00


30.00



25.00


<- 30 days frozen storage


2 0


2 4 6 8


14 16


Days


Figure 6-8. Average L*-values of the color of tuna samples over 14 days for the frozen storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were frozen for 30 days at -200C between day 0 and day 1.The samples were then
kept at 4C during the remaining time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.











-*-Ctrl ---FS24 +--FS48 -X-AS24 -*-AS48


25.00



20.00


15.00



10.00


5.00 -



0.00 ... 1-
-2 0 2 4 6 8 10 12


14 16


Days


Figure 6-9. Average b*-values of the color of tuna samples over 14 days for the fresh storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were kept at 40C during the entire time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.










FS24 -*-FS48 -X-AS24 -)*AS48


25.00



20.00


15.00



10.00


5.00


-> 30 days frozen storage
0.00 -.. .....
-2 0 2 4 6 8 10 12 14 16
Days

Figure 6-10. Average b*-values of the color of tuna samples over 14 days for the frozen storage
study. The graph shows the average a*values of tuna at day -1 (before any treatment
( was applied), day 0 (right after the treatment) and day 1 though day 14. The samples
were frozen for 30 days at -200C between day 0 and day 1.The samples were then
kept at 4C during the remaining time of observation. The samples were treated with
filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24
(AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated.


A


Figure 6-11. Tuna steaks after 30 days of frozen storage at -200C. A) Shows the untreated
control sample. B) Shows the filtered smoke treated sample. C) Shows the artificial
smoke treated sample


SCtrl


ted sample


* Ctrl









Sensory Taste Panel

The sensory characteristics of a product such as appearance, smell and taste are very

important factors for consumers to make their decision to buy a product. Since the use of filtered

and artificial smoke had similar results in their effect on color and appearance, as well as

microbial growth of the samples, the task was now to verify if there are any human detectable

differences in smell and appearance among filtered and artificial smoke treated samples. Four

triangle tests were conducted and 60 random untrained panelists per test were asked if they could

identify the odd sample by odor (first and second triangle tests) and by appearance (thirds and

fourth triangle tests). The design, execution and analysis of these triangle tests were conducted

with the Compusense Software. Table 6-7 shows the results of all four triangle tests, where the

actual number of correct answers is compared to the number of correct answers necessary to

establish a level of significance at 5%. This means that a lower number of correct answers

indicate that there are no significant differences detectable in a human sensory panel between the

treatments. It can be seen that for all four tests no significant differences have been detected

between filtered and artificial smoke treated samples in odor and appearance for the fresh and

frozen storage studies. All samples were prepared in the same manner and presented to the

panelists immediately after treatment or after thawing. All samples were treated for 48 hours

since a longer treatment suggested a greater effect on color and odor from preliminary

experiments.

To gain additional information, each panelist who identified the odd sample correctly was

asked to note any comments he or she might have about the differences among the samples that

led to their decision of choosing the odd sample. Tables A-5 to A-8 in Appendix A show all

comments given by the panelists for all four tests. For the color analysis, most of the panelists

described the filtered smoke sample to appear darker and more red then the artificial smoke

68









sample for the fresh storage study, while nearly equal number of panelists describe either the

artificial smoked or the filtered smoked sample as lighter in color for the frozen storage study.

This confirms in part the findings of the color study, where for the frozen storage study no

significant differences were found at day 1 between the filtered and artificial smoke treated

samples. The samples for the color studies and the taste panel analysis were obtained separately

from each other at different time points and also treated separately and at different times.

Comments regarding the odor triangle tests show that an equal amount of people noticed a

stronger smell in either the filtered smoke or the artificial smoke treated samples for both the

fresh and frozen storage studies. It is therefore inconclusive whether a particular smell or odor

was responsible for these panelists to decide which the odd sample was. It should be noted at this

point that only the panelists who identified the odd sample correctly were asked to comment

their decision and that there is a possibility that some of these panelists found the odd sample by

chance and not based on a real difference. The Compusense analysis software includes the

possibility of a right answer by guessing in their statistical analysis. Also the demographical

variety was limited to students from the University of Florida between the ages of 18 to 24 years.

Table 6-7. Results of the four taste panel triangle tests.
Tests 1 2 3 4
Total number of panelists 60 60 60 60
Number of correct answers needed 27 27 27 27
Actual Number of correct answers 23 26 19 19
Significance (p-Value) 0.244 0.068 0.654 0.654
Number 1 represents the odor test with frozen samples, number 2 the odor test with fresh
samples, number 3 the color test with frozen samples and number 4 the color test with fresh
samples.
GC-Analysis

Carbon monoxide is known to preserve and enhance the color properties of red muscle

foods and is used in several applications today. Small quantities of carbon monoxide (> 0.5%)

are used today in modified atmospheric packaging to preserve the redness of fresh meats, such as









ground beef (Sorheim and others 1999; Kusmider and others 2002; Hunt and others 2004) and

pork sausages (Sorheim and others 2001; Martinez and others 2005; Sorheim and others 2006).

A short term treatment with higher percentages of carbon monoxide (up to 100%) is used in the

seafood industry to retain the color of certain seafoods prior to frozen storage. However, carbon

monoxide is not a brand new component in the process of preserving meats and seafood. Natural

wood smoke, produced by the incomplete combustion of wood chips, always contains a certain

amount of carbon monoxide, which is applied to the product during the smoking process. During

cold smoking, a process where the product is kept at a low temperature (usually below 15C)

while the smoke is applied, the product is not cooked and therefore retains its color and texture

properties on the inside. The outside of the product usually turns into a slight brown to grey color

because of solid particles of the smoke that condense on the product during smoking. Filtered

wood smoke is a relatively new invention where these particles and most of the odor and flavor

components are filtered out and therefore don't condense onto the product. According to the

analysis of the manufacturer the main components of the filtered smoke used in this experiment

are carbon monoxide (21%), carbon dioxide (18%), oxygen (1.1%) and a balance of nitrogen.

However, filtered smoke contains many more components that have not yet been identified.

Since the production of filtered wood smoke can be a complicated and costly process, a cheaper

alternative is a treatment where the mentioned gas components are mixed together from

commercially available gasses and used in the same way as the filtered smoke. For this reason

we called this mixture artificial smoke (AS). As the previous studies showed it had a similar

effect on the color properties, sensory characteristics and microbial growth as the original filtered

smoke. To differentiate whether a product was treated with the original filtered smoke or the

artificial smoke mixture, a rapid gas chromatography method was developed that focuses mainly









on the identification and quantification of other gaseous components which are present in filtered

smoke but not in artificial smoke.

Identification of Gaseous Components in Filtered and Artificial Smoke

The fist step in the development was to identify the gaseous components in filtered smoke

and artificial smoke and develop a standard curve for each of these components with the GC-

Method mentioned in the chapter "Materials and Methods". Filtered smoke was injected into the

GC and 5 peaks could be verified with retention times less than five minutes. Two of these peaks

(retention times around 0.49 minutes and 1.52 minutes) were known already from previous

experiments as carbon monoxide and carbon dioxide respectively. The other three peaks were

not identified yet. It was also known from previous experiments that only low molecular

hydrocarbons will be eluted from the GC column in less than five minutes. It was therefore

decided to use refinery gas standard from Agilent technologies, which is a mixture of low

molecular weight hydrocarbons (like methane, ethylene, ethane, propane and so on), to identify

the three unknown components found in filtered smoke.

It should be noted at this point that there are most likely many more gaseous components

contained in filtered smoke, but with retention times much longer than 30 minutes in this

particular GC setup and therefore do not contribute to a "rapid" identification method.

With a comparison of the filtered smoke and the gas standard chromatograms the three

unknown components could be identified as methane, ethylene and ethane respectively in the

order of their elution.

The second step was now to obtain these five gases found in filtered smoke in the highest

purities available and produce standard curves for each of them to quantify them in the filtered

smoke, artificial smoke and the treated samples. Figures 6-12 to 6-16 show these standard curves

and display the resulting equations that were used to quantify the amount of each gas, based on

71










and injection volume of 100 pl and their respective peak area. Now the five components found in

filtered smoke with this method could be identified and quantified. As a comparison the artificial

smoke and a filtered smoke (FS-B), which was obtained from a different company, were

analyzed with this method. Table 6-8 shows the percentage of each of these five gasses found in

filtered smoke (FS), filtered smoke B (FS-B) and artificial smoke (AS). Since it is was clear that

the artificial smoke lacks any methane, ethylene and ethane, the next step was to experiment

whether any of these five gasses can also be found absorbed in the actual products.

Table 6-8. Percentage of gaseous components in the treatment gasses.
Treatment Gas Filtered smoke A Filtered smoke B Artificial Smoke
Carbon Monoxide 22.53 11.00 22.20
Methane 9.84 2.85 0.00
Carbon Dioxide 14.32 12.55 19.43
Ethylene 0.94 0.14 0.00
Ethane 0.17 0.20 0.00


1.200
y = 0.0002002x + 0.0003965
1.000 R= 0.9999182

0.800

o
0.600
c0
.o 0.400

S0.200

0.000
0 1000 2000 3000 4000 5000 6000
Peak Area [pA]

Figure 6-12. Carbon monoxide standard curve. This graph shows the average peak area for the
injection of different quantities of carbon monoxide. The displayed equation results
from the linear regression of the displayed data can be used to calculate the amount of
carbon monoxide in a 100pl sample based on the peak area











1.200

1.000

0.800

0.600

0.400


y = 0.0003414x 0.0006153
R2 = 0.9999588


0.200

0 .000 <---------------------------
0 500 1000 1500 2000 2500 3000 3500

Peak Area [pA]

Figure 6-13. Methane standard curve. This graph shows the average peak area for the injection
of different quantities of methane. The displayed equation results from the linear
regression of the displayed data can be used to calculate the amount of methane in a
100.l sample based on the peak area


1.200

1.000

0.800

0.600

0.400

0.200

0.000


y = 0.0001941x + 0.0003750
R2 = 0.9999415


0 1000 2000 3000 4000 5000


6000


Peak Area [pA]

Figure 6-14. Carbon dioxide standard curve. This graph shows the average peak area for the
injection of different quantities of carbon dioxide. The displayed equation results
from the linear regression of the displayed data can be used to calculate the amount of
carbon dioxide in a 100ll sample based on the peak area











1.200

1.000

0.800

0.600

0.400


y = 0.0000994x + 0.0030171
R2 = 0.9999063


0.200 -

0.000 .....-1-1
0 2000 4000 6000 8000 10000 12000

Peak Area [pA]

Figure 6-15. Ethylene standard curve. This graph shows the average peak area for the injection
of different quantities of ethylene. The displayed equation results from the linear
regression of the displayed data can be used to calculate the amount of ethylene in a
100pl sample based on the peak area


1.200

1.000

0.800

0.600

0.400

0.200

0.000


y = 0.0000995x + 0.0020152
R2 = 0.9997862


0 2000 4000 6000 8000 10000


12000


Peak Area [pA]

Figure 6-16. Ethane standard curve. This graph shows the average peak area for the injection of
different quantities of ethane. The displayed equation results from the linear
regression of the displayed data can be used to calculate the amount of ethane in a
100pl sample based on the peak area









Analysis of Treated Samples

The last step in the development of the rapid GC identification method was to find a way

to actually determine whether a product was treated with filtered smoke or just a mixture of

carbon monoxide and carbon dioxide, called artificial smoke. Previous experiments showed that

when a certain amount of treated muscle tissue is heated at 1000C for about 5 minutes in a sealed

vial, hydrocarbons that were absorbed into the tissue during the treatment will get released into

the headspace of the vial where the can be sampled with a syringe and then analyzed with the

GC. Samples were taken from each treatment at day 2 after treatment and analyzed with the

procedure described in "Material and Methods". Figures 6-17 to 6-24 display the chromatograms

obtained by this analysis that represent the filtered smoke and artificial smoke treated samples as

well as well as the control samples. Figure 6-21 shows the chromatogram of pure filtered smoke,

directly analyzed with the GC. By comparing these figures it is obvious that in the headspace

from artificial smoke treated samples the peaks for methane, ethylene and ethane are missing, as

well as in the control samples. The filtered smoke treated samples show the peaks for carbon

monoxide, methane, carbon dioxide and ethylene in the order they were eluted from the column

respectively. Only the ethane peak is missing from the filtered smoke treated samples as well. A

possible explanation for the missing ethane peak in the filtered smoke treated sample could be

that ethane is not as well absorbed as other hydrocarbons or not as easily released by the sample.

Nevertheless all chromatograms from the artificial smoke treated samples showed the same

results as well as did the filtered smoke treated among each other and the control samples.

Finally filtered smoke treated frozen tuna was obtained from a local ethnic grocery store and

tested the same way. The chromatogram of this sample is shown in figure 6-20. Note that for all

treatments several samples were obtained and analyzed with the GC method. For every sample

two chromatograms were obtained and analyzed to ensure the method works accurately.

75
























Figure 6-17. Chromatogram of the headspace analysis over a sample treated with artificial
smoke. The first peak from left represents carbon monoxide and the second peak
represents carbon dioxide. The area under a peak is proportional to the quantity of the
representing compound.

pA j


Figure 6-18. Chromatogram of the headspace analysis over a sample treated with filtered smoke.
The first peak from left represents carbon monoxide, the second peak represents
methane, the third peak represents carbon dioxide and the last peak represents
ethylene. The area under a peak is proportional to the quantity of the representing
compound.


pound.

















a00 -

200

100 *



Figure 6-19. Chromatogram of the headspace analysis over an untreated control sample. The
first peak from left represents carbon monoxide and the second peak represents
carbon dioxide. The area under a peak is proportional to the quantity of the
representing compound.


Figure 6-20. Chromatogram of the headspace analysis over a sample, purchased at a local ethnic
store. The first peak from left represents carbon monoxide, the second peak
represents methane, the third peak represents carbon dioxide and the last peak
represents ethylene. The area under a peak is proportional to the quantity of the
representing compound.























Figure 6-21. Chromatogram of the injection of 50 [l of pure filtered smoke. The first peak from
left represents carbon monoxide, the second peak represents methane, the third peak
represents carbon dioxide, the fourth peak represents ethylene and the last peak
represents ethane. The area under a peak is proportional to the quantity of the
representing compound.


1 ,A ] I
IWO -


Figure 6-22. Chromatogram of the injection of 50 pl of pure filtered smoke "B". The first peak
from left represents carbon monoxide, the second peak represents methane, the third
peak represents carbon dioxide, the fourth peak represents ethylene and the last peak
represents ethane. The area under a peak is proportional to the quantity of the
representing compound.


8i
























Figure 6-23. Chromatogram of the injection of 50 [il of pure artificial smoke. The first peak
from left represents carbon monoxide and the second peak represents carbon dioxide.
The area under a peak is proportional to the quantity of the representing compound.


iM


Figure 6-24. Chromatogram of the injection of 50 pl of air sampled from the
surrounding lab environment. The first two small peaks from left represent noise
from the injection and a very small amount of carbon monoxide. The third peak
represents carbon dioxide. The area under a peak is proportional to the quantity of the
representing compound.









CHAPTER 7
SUMMARY AND CONCLUSIONS

Smoking of meats and seafood has been a preservation method for a long period of time,

although in the recent century it became more a flavoring agent than a necessity in order to

preserve foods. From the early days, when the products were mainly cooked and dried, it became

a specialized process that induces just the right amount of flavor in sophisticated processes.

Today it is possible to smoke products at nearly 0C and in the right humidity, so that the

product does not differ in texture or appearance from a fresh product other then by its odor and

taste. However, smoke can do more than that. The next development of smoking food products is

called filtered wood smoke processing, a process that uses modem technology to clarify and

filter the natural produced smoke and high tech processing to apply this filtered smoke to the

product at the lowest possible temperature to retain a product that is nearly unchanged in texture

and odor compared to its untreated fresh counterpart. From the beginning it was believed that

filtered wood smoke has the potential to prolong the shelf life of fresh and frozen muscle foods

and to enhance their appearance and color property. Today it is known that the color retention

properties of filtered smoke relies on one of the main ingredients in filtered wood smoke, carbon

monoxide, a potential hazardous gas for humans and animals that replaces the oxygen in the

blood and muscle tissue and causes muscle relaxation and asphyxiation. However, just this effect

makes it very interesting and attractive for the meat and seafood industry, since the new complex

that is formed in this process results in a stable red color in these foods.

Filtered wood smoke is produced by burning wood chips at a high temperature without

oxygen to produce smoke that is then filtered and concentrated in compressed gas cylinders. To

compare if a mixture of the major gaseous components in filtered smoke would achieve the same









effect, artificial smoke was prepared from industrially available gases and used also in this

proj ect.

It is shown during this project that filtered and artificial smoke processing did not affect

the growth or inhibition if Salmonella spp. as they seem to be more affected by their competitive

microbial environment. No Salmonella spp. growth was reported at all in non-inoculated

samples, which raises the question whether it is a real threat in fresh tuna or not. There was some

inhibition effect of filtered and artificial smoke on the growth of aerobic spoilage bacteria during

the first 4 to 6 days of storage, however it is suggested by the author that this effect does not

enhance the shelf life of the product dramatically in this particular study. However, the combined

effect of filtered and artificial smoke processing and subsequent frozen storage at -200C leads to

a greater inhibition of aerobic spoilage bacteria then was observed in an untreated frozen

product. The main effect observed in this study for filtered and artificial smoke processing is

their ability to preserve and improve the color properties and appearance of red muscle foods as

shown in the color experiment in this project. After the visual observation of a piece of tuna that

was frozen in a vacuum bag without any treatment compared to a filtered or artificial smoke

treated product it is very clear which product looks more red and fresh. The filtered and artificial

smoke treated products have clearly an advantage over control.

During the whole study filtered smoke and artificial smoke treatments were always

compared side by side and no significant differences were detected in any of the mentioned

studies between filtered and artificial smoke treated samples. A consumer can not differentiate

whether a product was treated with filtered or artificial smoke according to the taste panel

results, based on either odor or appearance.









All these finding raise the final question what is really the difference between filtered

smoke and artificial smoke. Why would we use filtered smoke or the less expensive artificial

version? Artificial smoke contains industrial carbon monoxide, which is regulated by many

countries in the world in its use as a food ingredient or additive. But filtered smoke, although

containing the same carbon monoxide is considered a natural product, based on a long history of

food processing with wood smoke in most of these countries. Since it is not obvious just by

visual or sensorial inspection whether a product was treated with the original filtered smoke or

its artificial counterpart the rapid GC identification method was developed to detect in a simple

way whether a product was treated with filtered smoke or just with carbon monoxide. The results

show that it is possible to identify a filtered smoke treated product based on the presence of three

additional components that are not found in the artificial smoke mixture. The rapid method also

allows the quantification of the residual amount of carbon monoxide in the muscle tissue when a

standard curve for the quantification of carbon monoxide was established. It has to be

acknowledged that an individual, who wants to deliberately misguide the consumer about the

products treatment, could do so by adding all the ingredients into the artificial smoke that are

tested by the rapid identification method. But this is a question of risk for every consumer and

producer.










APPENDIX A
TASTE PANEL DEMOGRAPHICS AND COMMENTS

Comments from the Panelists for the Fresh Odor Triangle Test


Table A-1.
Name
Panelist 2
Panelist 8
Panelist 10
Panelist 13
Panelist 17
Panelist 19
Panelist 24
Panelist 25
Panelist 26
Panelist 27
Panelist 32
Panelist 37
Panelist 41
Panelist 44
Panelist 46
Panelist 48
Panelist 52
Panelist 56
Panelist 57
Panelist 59
Panelist 2
Panelist 8
Panelist 10
Panelist 12
Panelist 13
Panelist 17
Panelist 25
Panelist 32
Panelist 37
Panelist 41
Panelist 43
Panelist 44
Panelist 46
Panelist 48
Panelist 52
Panelist 59


Artificial
Artificial
Artificial
Artificial
Artificial
Artificial
Artificial
Artificial
Artificial


Smoke
Smoke
Smoke
Smoke
Smoke
Smoke
Smoke
Smoke
Smoke


Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke


Treatment
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Artificial Smoke
Artificial Smoke


Comments
this smell has a stronger smell
had a stronger aroma than the rest of them.
Smelled the same as 564\Smelled the same as 262
least fishy smell
smells like #546\smells like #262
less scent
same as the first one.\It smells odd like it was dead a week ago.
did not have as strong of a fishy smell as the other two
musky
Lighter smell
smells raw
not as strong of an odor
very little scent\little scent
This sample smells stronger than the other two.
It smelled very strong as well.\This sample smelled very strong.
smelled unfresh \, like paint\smelled unfresh, like paint
Smells like 546\seems the same as 262
It had a stronger/heavier smell
strong and smelled fatty
harsh smelling\more smelly
this smell is similar to the second one\smells like saltwater
smelled same as the 927 sample.\it really didn't smell like fish
Smelled worse (more fishy)
smells less fishy
fishy\strongest smell
sample #927 has a more potent smell, it smells saltier too.
very strong fishy smell\very strong fishy smell
smells putrid\bitter scent
strong smell\strong smell
more fishy odor
The same as the previous\Smells fresh out of the sea
This sample smells similar to 927.\This smells similar to 347.
The smell of this sample wasn't as strong as the other two.
smelled fresher, didn't smell as much like paint
The scent is less stronger
has an odor of salmon


Only panelists who identified the odd sample correct were asked to leave comments. Numbers
546 and 262 refer to the filtered smoke treated sample. Numbers 347 and 927 refer to the
artificial smoke treated sample. Samples were not previously frozen.










Comments from the Panelists for the Frozen Odor Triangle Test


Name
Panelist
Panelist


Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist


Treatment
Filtered Smoke
Filtered Smoke


Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke

Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke

Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke


Comments
smells cooked\smells cooked
This one was the same as number 466 and did not smell as strong and
fishy as number 173\This one and number 882 both smelt fishy but the
odor was not as strong and disgusting as number 173
can't really smell anything
it also smelled like chicken.\it smelled like chicken.
882 smelled like 466\It smelled like fish, and t was a bad smell.
not as strong as the others
smells less fishy
more like crab\smells more like crab
pungent smell like it had gone bad\subtle smell like canned tuna
similar to 173\scent not as strong
one of them smells less fishy than the other two.
this one smells like 466\this one smells very bitter
It has a distinc odor which can be smelled from other stink fish but it was
kind of lighter smell than the first ones.
smells not as fresh\smells not as fresh
Smells salty
Doesn't smell as strong
same as 466\different smell
smelled very plain
stronger odor
weakest scent
Same as the first one\It doesn't even smell like tuna
No fishy smell
no strong odor\no strong odor
not sure. there was a slight smell of saltyness.
it has a lighter smell.
harsh smell, rot
smells like it went bad
This one smelt extra fishy
has a smell\has a smell
smelled like there was some sort of chemical on the fish.
792 did not really have much of a smell, as opposed to the other ones that
did have a smell. It did not really smell like fish at all.
very strong\very strong
hardly has a smell\hardly smells like anything
didn't smell that much
exactly the same as 466
strongest smell
smells very fishy\it smells fishy
this one smells sweeter
smells very fishy
Does not have a strong odor\Does not have a strong odor
smells stronger\smells stronger
different smell
barely smelled at all\strong smell
weaker odor\weaker odor


Table A-2.










Continued
Treatment
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke


Comments
most 'fishy'\mild scent
Smells rotted.
fishy smell\fishy smell
it has more odor
smelled a little stronger than the other one
not as strong as 466.\it has more of the typical fish smell.
Smells not that bad like the second\I couldn't figure out its smell.


Only panelists who identified the odd sample correct were asked to leave comments. Numbers
882 and 446 refer to the filtered smoke treated sample. Numbers 792 and 173 refer to the
artificial smoke treated sample. Samples were not previously frozen.


Table A-3.


Comments from the Panelists for the Fresh Color Triangle Test


Treatment
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke

Filtered Smoke
Filtered Smoke
Filtered Smoke


Filtered Smoke
Filtered Smoke
Filtered Smoke


Filtered Smoke

Filtered Smoke
Filtered Smoke

Filtered Smoke
Filtered Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke


Comments
darker\darker
Sample 119 had thicker veins than the other two.
much darker than the other two
more red\more red
the lines in it were different.\the lines in it were different.
dark\dark
darker
Aside from seeming harder, sample 119 seems darker as well.
119 is darker than 511, but just as dark as 730.\730 is darker than 511,
but just as dark as 119.
This sample seems sliced differently than the other two samples.
This sample looks the best\There are thinner lines on this sample
Looks exactly like 119.\they are all the same color, but sample 119 and
730 are the same size and have the same amount of fat content and the
same sort of lines in them (the grain of the meat).
This has a darker color.\This has a darker color.
lighter
There is a slight indentation in this sample that looks similar to that of
sample 730, just not as long.\There is one barely noticeable line going
through it. The color is very similar to that of sample 119.
Identical to 730, darker and more grainy than 378\Identical to 119, darker
and more grainy than 378
Identical to 730.\Identical to 119.
Didn't have the defined serrations the other two samples had, also seemed
lighter.
Solid red/pink color\Similar to 730 with solid pink/red color
\
it's lighter in color
378 looked liked 511\511 looked like 378
grainier\grainier and lighter
pinker
The lines in it were different.
lighter\lighter
Sample 378 appears more tender and soft than 119.\Sample 511 appears
more tender than 119.


Table A-2.
Name
Panelist 40
Panelist 43
Panelist 47
Panelist 50
Panelist 53
Panelist 54
Panelist 24


Name
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist


Panelist
Panelist
Panelist


Panelist 47

Panelist 48
Panelist 51


Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist


Y










Table A-3.
Name
Panelist 29
Panelist 33

Panelist 34

Panelist 35

Panelist 41
Panelist 46


Panelist 54 Artificial Smoke


Continued
Sample
Artificial Smoke
Artificial Smoke

Artificial Smoke

Artificial Smoke

Artificial Smoke
Artificial Smoke

Artificial Smoke
Artificial Smoke
Artificial Smoke


Panelist
Panelist
Panelist


Only panelists who identified the odd sample correct were asked to leave comments. Numbers
119 and 730 refer to the filtered smoke treated sample. Numbers 378 and 511 refer to the
artificial smoke treated sample. Samples were not previously frozen.


Comments
it is the only pink piece (the lightest)
This sample has more uniformity than the other in its appearance.\It has a
semi-circle shape line on its top.
Sample 378 seems to have two white streaks running through it which
look like fat and, and therefore are unappetizing.
This sample is smaller than the other two and has more fat in the meat.
You can see it in the grain.
This sample has a brighter color. Distinguishable.
this sample has four visible lines going through it diagonally. The color is
also slightly darker than sample 730.
Lighter and less grainy than 730 and 119
Significantly lighter and pinker than the other samples.
Had defined serrations/grooves in it.\Had defined serrations/grooves in
sample.
Striped appearance










Table A-4. Comments from the Panelists for the Frozen Color Triangle Test


Name
Panelist
Panelist
Panelist
Panelist
Panelist


Panelist 59 Artificial Smoke


Panelist 27


Treatment
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke

Filtered Smoke

Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke

Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke
Filtered Smoke

Filtered Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke

Artificial Smoke
Artificial Smoke
Artificial Smoke

Artificial Smoke

Artificial Smoke
Artificial Smoke

Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke
Artificial Smoke


Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist
Panelist
Panelist
Panelist
Panelist

Panelist
Panelist
Panelist


Panelist 38

Panelist 44
Panelist 46


Panelist
Panelist
Panelist
Panelist
Panelist


Only panelists who identified the odd sample correct were asked to leave comments. Numbers
416 and 822 refer to the filtered smoke treated sample. Numbers 614 and 294 refer to the
artificial smoke treated sample. Samples were previously frozen.


Comments
416 has striations on it that none of the other 2 have.
lighter shade and luminous glow
it was lighter in color and had less lines
the lines in it were different
this samples\ seems layedand the grain is similar to sample 419\this
sample is more smooth with less of vertical grain
416 appears tender and soft, much lighter than 614.\822 appears tender
and light.
this one seems to be more red, a bit darker
416 is the lightest, it has little white strands in it.
It seems the same a sample 822.\it seems the same as sample 416.
416 seems to look sinewy and less like gelatin, but the difference is small
This piece has indentation in it and the shape is different than the first
two. It also has visible white lines..
a dent on the surface\a dent in the surface
3 cuts. pinkish\3 cuts and pinkish
Pretty smooth appearance
its quite coarse\quite flat with little marks
this sample had fewer white lines and looked less 'defined' than the
typical tuna steak cell structure
Lighter color, also seems to have less fatty tissue.
No striations.\No striations.
looks like the other\resembles 294
had lines\had lines
the lines in it were different\the lines in it are different
the filet seems like the grain is more vertical then horizontal
614 seems again not as soft and tender as the other samples. It also
appears a little bit darker.
same as 614\same as 294
IT has whit division lines differently than the other two.
texture is slightly different, but still similar to 614\very cleanly cut and
gelatin like
This sample does not contain any white areas or lines where the other
two samples do. It appears uniform in color and texture.
lighter
Square piece that is thinner than 614 but the same color.\Square piece
that is pink.
smooth texture
more salmon color
Striped and a bit jagged\White stripes
its more smoother than the rest
This one looked about the same compared to sample 614\This sample
looked about the same as sample 294
darker color, same as 614\Darker color, same as 294









Table A-5. Demographic Variance of the Odor Triangle Tests
Age Range Under 18 18-20 21-24 Over 24 Total
Female 2 33 3 2 40
Male 0 14 6 0 20
Total 2 47 9 2 60
Note: The demographic variance for the fresh and frozen storage odor tests are the same.

Table A-6. Demographic Variance of the Color Triangle Tests
Age Range Under 18 18-20 21-24 Over 24 Total
Female 1 31 3 1 36
Male 0 15 7 2 24
Total 1 46 10 3 60
Note: The demographic variance for the fresh and frozen storage color tests are the same.









APPENDIX B
COLOR STUDY PICTURES


w


Figure B-1. Control group images for the frozen storage study captured by the CMVS. a) Image
of samples before treatment was applied. b) Image of samples immediately after
treatment was applied and ceased (here no treatment control group). c) Image of
samples after 30 days of frozen storage (-20C) followed by 14 days of refrigerated
storage (4C).


U-


w


Figure B-2. Filtered smoke (24h-treatment) group images for the frozen storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 30 days of frozen storage (-20C) followed by 14 days of refrigerated storage
(4C).


W


w


w


Figure B-3. Filtered smoke (48h-treatment) group images for the frozen storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 30 days of frozen storage (-20C) followed by 14 days of refrigerated storage
(4C).








b W c


w


Figure B-4. Artificial smoke (24h-treatment) group images for the frozen storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 30 days of frozen storage (-20'C) followed by 14 days of refrigerated storage
(4oC).


wr


w


w


Figure B-5. Artificial smoke (48h-treatment) group images for the frozen storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 30 days of frozen storage (-20'C) followed by 14 days of refrigerated storage
(4oC).


1W


w


Figure B-6. Control group images for the fresh storage study captured by the CMVS. a) Image
of samples before treatment was applied. b) Image of samples immediately after
treatment was applied and ceased (here no treatment control group). c) Image of
samples after 14 days of refrigerated storage (4oC).


a low










wA


Figure B-7. Filtered smoke (24h-treatment) group images for the fresh storage study captured by
the CMVS. a) Image of samples before treatment was applied. b) Image of samples
immediately after treatment was applied and ceased. c) Image of samples after 14
days of refrigerated storage (4C).


w1


W


qw


Figure B-8. Filtered smoke (48h-treatment) group images for the fresh storage study captured by
the CMVS. a) Image of samples before treatment was applied. b) Image of samples
immediately after treatment was applied and ceased. c) Image of samples after 14
days of refrigerated storage (4C).


w


Figure B-9. Artificial smoke (24h-treatment) group images for the fresh storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 14 days of refrigerated storage (4C).









a -- Wb c








Figure B-10. Artificial smoke (48h-treatment) group images for the fresh storage study captured
by the CMVS. a) Image of samples before treatment was applied. b) Image of
samples immediately after treatment was applied and ceased. c) Image of samples
after 14 days of refrigerated storage (4C).









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BIOGRAPHICAL SKETCH

Stefan Crynen was born in 1976 in Monchengladbach, Germany. He attended the

"Stifitisch Humanistisches Gymnasium" (high school) and graduated with the "Abitur" in 1996.

After the 1 year mandatory military duty he attended culinary school and graduated as a

professional chef in Summer 2000. From 2000 to 2004 he studied food technology at the

University of Applied Sciences in Trier, Germany and graduated in October 2004 with the

degree "Diplom-Engineer of Food Technology". He did an internship from September 2002 to

February 2003 at the Food Science and Human Nutrition Department at the University of

Florida. He started his master's degree in 2005 under Dr. Hordur G Kristinsson.





PAGE 1

EFFECT OF FILTERED WOOD SMOKE PR OCESSING ON SPOILAGE BACTERIA, PATHOGENIC BACTERIA AND SENSORY CH ARACTERISTICS OF YELLOWFIN TUNA By STEFAN CRYNEN 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 SCIENCE UNIVERSITY OF FLORIDA 2007 1

PAGE 2

2007 Stefan Crynen 2

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To my loving parents, my brother and especially my fiance Gogce. 3

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ACKNOWLEDGMENTS I sincerely want to express my deep gratitude towards my ma jor advisor, Dr. Hordur G. Kristinsson, who helped me to complete this stud y with his advice guidan ce and support. I would also like to thank my committee members Dr. Murat Balaban and Dr. Bruce Welt for their suggestions, advice and help in th e completion of this research. I would like to thank Mr. Blane Olson. His generosity and support made this project possible. I would also like to thank my family for their love and support and especially my fiance Gogce who believed in me and supported with guidance and understandi ng thru this entire project. Finally I would like to thank Dr. Charles Si ms, Yavuz Yagiz and Lorenzo Puentes and his Team for their support with the Taste Panel expe riments, Sibel Damar and Sara Aldaous for her friendship and support and all my fellow class an d lab mates at the University of Florida. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8LIST OF ABBREVATIONS .........................................................................................................11ABSTRACT ...................................................................................................................................12CHAPTER 1 INTRODUCTION ..................................................................................................................142 LITERATURE REVIEW .......................................................................................................16Smoking of Fish and Seafood Products ..................................................................................16Filtered Smoke ........................................................................................................................17Bacteria and Other Microorganisms in Fish and Seafood ......................................................19Listeria Monocytogenes ..........................................................................................................20Clostridium Botulinum ............................................................................................................20Salmonella Spp. ......................................................................................................................21Biogenic Amines ....................................................................................................................21Parasites ..................................................................................................................................21Sensory Characteristics of Fresh and Smoked Seafood .........................................................223 OBJECTIVES .........................................................................................................................254 PRELIMINARY STUDIES ....................................................................................................26Tuna Microbiology Study .......................................................................................................26Tuna Color Study ....................................................................................................................28Identification of Filtered Smoke Treated Products .................................................................295 MATERIAL AND METHODS ..............................................................................................36Fresh and Frozen Storage .......................................................................................................36Sample Preparation and Treatments .......................................................................................37Salmonella Study ....................................................................................................................38Total Aerobic Plate Count Study ............................................................................................40Color Analysis ........................................................................................................................41Sensory Taste Panel Analysis .................................................................................................41Rapid Gas Chromatography Identification Method ...............................................................42Statistical Analysis ..................................................................................................................44 5

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6 6 RESULTS AND DISCUSSION .............................................................................................47Salmonella Results ..................................................................................................................47Fresh Storage Study .........................................................................................................48Frozen Storage Study ......................................................................................................49Total Aerobic Plate Count ......................................................................................................52Fresh Storage Study .........................................................................................................52Frozen Storage Study ......................................................................................................54Color Analysis ........................................................................................................................57Fresh Storage Study .........................................................................................................58Frozen Storage Study ......................................................................................................60Sensory Taste Panel ................................................................................................................68GC-Analysis ...........................................................................................................................69Identification of Gaseous Component s in Filtered and Artificial Smoke .......................71Analysis of Treated Samples ...........................................................................................757 SUMMARY AND CONCLUSIONS .....................................................................................80APPENDIX A TASTE PANEL DEMOGRAPHICS AND COMMENTS ....................................................83B COLOR STUDY PICTURES .................................................................................................89LIST OF REFERENCES ...............................................................................................................93BIOGRAPHICAL SKETCH .........................................................................................................97

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LIST OF TABLES Table page 5-1 Treatment combinations used in all studies of this project ............................................... 385-2 Nikon D200 Settings ......................................................................................................... 455-3 GC-Settings for the rapid identification method ............................................................... 456-1 ANOVA results for Salmonella spp. for the fresh storage study. ..................................... 506-2 ANOVA results for Salmonella spp. for the frozen storage study. .................................. 516-3 ANOVA results for aerobic plate count for the fresh storage study. ................................ 566-4 ANOVA results for aerobic plate count for the frozen storage study. .............................. 576-5 ANOVA results for the average a* -values for the fresh storage study. ............................ 626-6 ANOVA results for the average a*-v alues for the frozen storage study. ......................... 636-7 Results of the four ta ste panel triangle tests. ..................................................................... 696-8 Percentage of gaseous com ponents in the treatment gasses. ............................................ 72A-1 Comments from the Panelists for the Fresh Odor Triangle Test ...................................... 83A-2 Comments from the Panelists for the Frozen Odor Triangle Test .................................... 84A-3 Comments from the Panelists for the Fresh Color Triangle Test ..................................... 85A-4 Comments from the Panelists for the Frozen Color Triangle Test ................................... 87A-5 Demographic Variance of the Odor Triangle Tests .......................................................... 88A-6 Demographic Variance of the Color Triangle Tests ......................................................... 88 7

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LIST OF FIGURES Figure page 3-1 Tuna treated for 16 hours with 100% CO, f iltered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. .............................................................. 303-2 Tuna treated for 32 hours with 100% CO, f iltered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. .............................................................. 313-3 Tuna treated for 48 hours with 100% CO, f iltered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. .............................................................. 313-4 Tuna treated for 16 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a mixture of 18% CO with 21% CO2 balanced with nitrogen. ............................................ 323-5 Tuna treated for 32 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a mixture of 18% CO with 21% CO2 balanced with nitrogen. ............................................ 323-6 Tuna treated for 48 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a mixture of 18% CO with 21% CO2 balanced with nitrogen. ............................................ 333-7 Chromatogram of the analysis of Clearsmoke. ............................................................. 333-8 Chromatogram of the analysis of industrial carbon monoxide (~100% CO). .................. 343-9 Chromatogram of the analysis of atmospheric air. ........................................................... 343-10 Chromatogram of the analysis of nitrogen (N2). ............................................................... 343-11 Chromatogram of the analysis of carbon dioxide (CO2) .................................................. 355-1 Flow diagram of all studies conducted during this project ............................................... 466-1 Average amount of CFU/10g of tuna for Salmonella spp. for the fresh storage study. .... 506-2 Average amount of CFU/10g of tuna for Salmonella spp. for the frozen storage study. 516-3 Average amount of CFU/10g of tuna for aer obic plate count for the fresh storage study. 556-4 Average amount of CFU/10g of tuna for aer obic plate count for the fresh storage study. 566-5 Average a*-values of the color of tuna sa mples over 14 days for the frozen storage study. ........................................................................................................................................... 626-6 Average a*-values of the color of tuna sa mples over 14 days for the fresh storage study. ........................................................................................................................................... 63 8

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6-7 Average L*-values of the color of tuna sa mples over 14 days for the fresh storage study. ........................................................................................................................................... 646-8 Average L*-values of the color of tuna sa mples over 14 days for the frozen storage study. ........................................................................................................................................... 656-9 Average b*-values of the color of tuna samples over 14 days for the fresh storage study. ........................................................................................................................................... 666-10 Average b*-values of the color of tuna samples over 14 days for the frozen storage study. ........................................................................................................................................... 676-11 Tuna steaks after 30 days of frozen storage at -20C. ....................................................... 676-12 Carbon monoxide standard curve. .................................................................................... 726-13 Methane standard curve. ................................................................................................... 736-14 Carbon dioxide standard curve. ........................................................................................ 736-15 Ethylene standard curve. ................................................................................................... 746-16 Ethane standard curve. ...................................................................................................... 746-17 Chromatogram of the headspace analysis over a sample treated with artificial smoke. ... 766-18 Chromatogram of the headspace analysis over a sample treated with filtered smoke. ..... 766-19 Chromatogram of the headspace analys is over an untreated control sample. .................. 776-20 Chromatogram of the headspace analysis ove r a sample, purchased at a local ethnic store. ........................................................................................................................................... 776-21 Chromatogram of the injection of 50 l of pure filtered smoke. ...................................... 786-22 Chromatogram of the injection of 50 l of pure filtered smoke B. ............................... 786-23 Chromatogram of the injection of 50 l of pure artificial smoke. .................................... 796-24 Chromatogram of the injection of 50 l of air sampled from the surrounding lab environment. ..................................................................................................................... 79B-1 Control group images for the frozen storage study captured by the CMVS. .................... 89B-2 Filtered smoke (24h-treatment) group images for the frozen storage study captured by the CMVS. .............................................................................................................................. 89B-3 Filtered smoke (48h-treatment) group images for the frozen storage study captured by the CMVS. .............................................................................................................................. 89 9

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B-4 Artificial smoke (24h-treatment) group images for the frozen storage study captured by the CMVS. ........................................................................................................................ 90B-5 Artificial smoke (48h-treatment) group images for the frozen storage study captured by the CMVS. ........................................................................................................................ 90B-6 Control group images for the fres h storage study captured by the CMVS. ...................... 90B-7 Filtered smoke (24h-treatment) group images for the fresh storage study captured by the CMVS. .............................................................................................................................. 91B-8 Filtered smoke (48h-treatment) group images for the fresh storage study captured by the CMVS. .............................................................................................................................. 91B-9 Artificial smoke (24h-treatment) group images for the fresh storage study captured by the CMVS. .............................................................................................................................. 91B-10 Artificial smoke (48h-treatment) group images for the fresh storage study captured by the CMVS. .............................................................................................................................. 92 10

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LIST OF ABBREVATIONS ANOVA Analysis of Variance AS Artificial Smoke (A mixture of 21 % carbon monoxide, 18 % carbon dioxide, 1.1% oxygen and a balance of nitrogen) CMVS Color Machine Vision System Ctrl Control Group FID Flame Ionization Detector FS Filtered Smoke FSHN Food Science and Human Nutrition Department University of Florida GC Gas Chromatograph SAS Statistical Analysis Software 11

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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 Science EFFECT OF FILTERED WOOD SMOKE PR OCESSING ON SPOILAGE BACTERIA, PATHOGENIC BACTERIA AND SENSORY CHARACTERISTICS OF TUNA FISH By Stefan Crynen December 2007 Chair: Hordur G Kristinsson Major: Food Science and Human Nutrition Smoking is a very old technique for preser ving fish, meat and dairy products and for enhancing their flavor. Currently the preservation aspect of smoki ng fish is often ignored since other more effective preservation methods, like fr eezing and refrigeration, have been developed. Today, most smoking applications target mainly the enhanced flavor aspect of smoking, rather then the increased shelf life of food products. Filtered smoke processing is a new method that uses the preservati on effect of smoking on fish and fish products without major changes in their sensory characteri stics, like flavor or texture. The goal of this project was to study the effects of filtered smoke processing on spoilage and pathogenic bacteria, quality aspects of warm water fish species and to optimize the smoke treatment method. Unlike most preservation techniques like free zing or refrigeration, filtered smoke also enhances the appearance of red muscle products such as tuna or mahi. This work also showed that color of the warm water fish species can be enhanced through filtered smoke treatment, especially in conjunction with refrigeration and freezing. 12

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Fresh tuna steaks were treated with filtered and artificial smoke for 24 and 48 hours and then analyzed for 14 days for Salmonella spp. growth, total aerobic bacteria growth and changes in color, especially the redne ss of the samples. A similar study was conducted where the samples were frozen and stored at -20C for 30 days prior to analysis The first two studies showed that there was nearly no affect of filtered or arti ficial wood smoke processing on the growth of Salmonella spp. for either the fresh or frozen stored samples. However, there appeared to be an inhibitory effect of both the f iltered and the artificial smoke tr eatment on the growth of aerobic spoilage bacteria during the first 4 days of observation. This effect seemed to be enhanced in the samples that were stored at -20C for 30 days. However, these effects suggest no improvement in the shelf life of tuna as caused by any of th e treatments. The color analysis confirmed the effectiveness of filtered smoke processing on th e preservation of the color properties and appearance of the samples, especially after froz en storage. No significan t differences could be seen between the filtered smoke and the artifi cial smoke treatments in any of the mentioned studies. Moreover in a sensorial taste panel no significant diff erences could be found based on odor and appearance between filtered smoke and artificial smoke treated tuna. To identify whether a product was treated with filtered or artificial smoke for the purpose of quality assurance, a rapid gas chromatography identi fication method was developed to quantify the amount of residual carbon monoxide in products and at the same time verify whether a product was treated with filtered smoke or the artificial counterpart by a chromatographic fingerprint. 13

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CHAPTER 1 INTRODUCTION Smoking fish, meat and dairy products is a very old technique that has been used as a method of long term preservation for over 600 years (Cutting 1961). Until other methods like refrigeration and freezing were developed and es tablished, smoking fish and meat products was one of the most used preservation methods. The advantages of smoking food products are increased shelf life, enhanced flavor, and prevention of insect infest ation. Although traditional fish smoking can still be found in practice today, the distinct boundaries of its use have vanished (Dillon and others 1994). In industr ial countries, smoking of fish a nd meat products is used today mainly to enhance flavor and texture (Robi nson 1983). Modern forms of smoke processing provide sometimes only little protection against mi crobial spoilage and especially cold smoked products can spoil as readily as non-s moked foods (Hsu and others 1979). A new technique that uses filtered and comp ressed wood smoke to treat fish products by using a low temperature treatment process, attemp ts to use the preserving aspect of wood smoke on food products without changing their flavor or texture. Although most smoking applications today aim to change and enhance the flavor of a food product wher e it is accepted by the consumer, generally smoke flavor is not wide ly accepted. As with other flavor and taste expressions like spiciness, sweetne ss, rancidity or others, the range of likeability goes all the way from no smoke flavor over mild taste to very strong taste for smoked food products. With filtered smoke applications it is now possi ble to adjust the level of smoke flavor of a food product while at the same time increasing the antimicrobial e ffect by concentrating e ffective components. This project aims to determine the antimicr obial effects of filtere d wood smoke processing on fish products while assessing changes that occur in color, text ure, taste and overall likeability. This study will determine the eff ects of filtered wood smoke trea tment on the growth, inhibition 14

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and reduction of Salmonella spp. The effects were studied on th e natural fauna present when the fish was delivered and on inoculated. Since the filtered smoke treatment is expected to maintain and enhance color properties, especially related to the red muscle parts, it is important to determine whether a product was treated with filtered smoke or just with industrial carbon monoxi de, which is already proven to enhance the color of red muscle products. A part of this study was to develop and improve a rapid gas chromatography identification method, designated to determine whether a product was treated with filtered smoke or carbon monoxide. The method is based on a gas chromatography profile, which shows a combination of several natural gases, like methane, ethane, butane and pentanes, in different concentra tions that produce chromatographic fingerprint for smoke treated products. 15

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CHAPTER 2 LITERATURE REVIEW Smoking of Fish a nd Seafood Products Smoking fish and meat products is a very old food processing technique and has been used over a long time as a method of long term pres ervation of foods (Burgess and Bannerman 1963). Smoked foods are generally better preserved and protected against insects infestation than untreated products. Smoking fish and fish products can be divided into two distinct types, a hot smoking process and a cold smoking process. Hot smoking involves cooking products during or immediately after the smoke treatment, usually in the same chamber, at temperatures around 70 to 80C. Traditional hot smoking is actually a three step process. The product is first smoked betw een 30 and 60 minutes at 30C to dry and toughen the skin. In general the more dry the surface of the products the more smoke flavor is absorbed into the product. The s econd step is the actual smoking step where the temperature is increased to about 50C and the amount of smoke blown into the oven is raised. This is considered the smoking step. The last step is the cooking step where the product is cooked at temperatures between 70 and 90C for about 1 to 2 hours (Dillon and others 1994). For cold smoke processing, the difference to hot smoking is the temperature at which the product is smoked and finished. The purpose of cold smoking is to apply a smoky flavor to the product without cooking, so the product may be c onsumed raw, such as cold smoked salmon, or prepared as desired by the consumer. The products are smoked at temperatures below 30C with an initial heavy smoke deposit in to the chamber that is tapere d off toward the end. For some species, like herring and mackerel, the temperature is increased after the smoking step to 40C so that natural oils can come to the surface to provide the final pr oduct with a glossy appearance (Dillon and others 1994). 16

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Filtered Smoke Filtered Smoke treatment is a new technique to preserve meats and seafood by utilizing the preserving aspects of wood smoke and at the sa me time minimizing the introduction of flavor and odor giving compounds, which are filtered ou t of the smoke before it is used in the processing of the food product. The idea to use filte red smokes to preserve color and spoilage of seafood products started when modified atmosphe ric packaging became commercially available, and carbon monoxide, a main component in natural and filtered wood smoke, was used to enhance and preserve the fresh appearance of seafood (Otwell 2006). Although the use of industrial carbon monoxide to treat meat and seafood products is still debated in many countries, the use of wood smoke has been acknowledged and accepted as a preservation method by competent health author ities in every nation of the world (Olson 2006). Filtered smoke is the next step in this long hist ory of preservation that utilizes the preserving aspects of wood smoke (e.g. carbon monoxide a nd carbon dioxide) and minimizes or even eliminates unwanted changes in flav or, texture and taste to the product. Compared to the traditional hot and cold smoking, with treatment temperature from 30C (for cold smoking) up to 80C (for hot smoking), th e filtered smoke is applied to the product at 0-5C. The whole process, as described by Olson (2006), involves the generation of wood smoke by burning wood chips in a smoke chamber without air intake, to ensure the incomplete combustion of the wood, leading to smoke, then run this smoke to a series of filters to remove particles such as tar, ash and soot and finally remove most of the odor and flavor compounds with and active carbon filter. This filtered sm oke can be used immediately or stored in compressed gas cylinders for later use. The food products are then prepared for the filtered smoke treatment by filleting and steaking to increase the surface area, and then placed on racks and put into the smoking chamber. The chamber is then evacuated from the remaining air and the 17

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filtered smoke is introduced into the chamber and replaced several times during the treatment, which can take from 2 to 48 hours. The filtered wood smoked products show great adva ntages in their retail value, compared to untreated or naturally smoked products, due to the enhancement of their color, texture, odor and taste properties. The color of food products is one of the major aspects most consumers are interested. If the color of a piece of tuna is brown or grey instead of red or pink, as expected from a fresh product, the consumer will most likely reject the product. The carbon monoxide, present in filtered smoke, will bind to muscle heme proteins, which determine the color of the muscle based on their chemical state. Freezing or other long term storage and exposure to air (oxygen) will change the conformation of the heme protein, which results in brown to gray colors of the product. Carbon monoxide will bind to the heme protei n and stabilizes it so that the color change due to long term storage and freezing can be prevented and the product looks appealing red and pink (Olson 2006). The same mechanism also affects odor and tast e of the product due to oxidative rancidity of the lipids in the fish muscle. Lipid degradati on and the reactions of these degradation products lead to undesirable off-odors and flavors. It is believed that hemoglobin and myoglobin are key prooxidants that cause the oxidation and degradation of lipids that lead to off flavors and odors. By stabilizing the heme proteins with carbon mo noxide, the oxidation of lip ids and therefore the formation of off flavors and odors can be reduced. As a result, the fish product smells and tastes fresh longer (Kristinsson and others 2006b). Some research has been done on the eff ect of carbon monoxide, one of the main components of filtered smoke, on the growth of microorganisms. Several studies have been conducted with various levels of carbon monoxide on red meat and seafood to investigate how 18

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these treatments inhibit the gr owth of spoilage bacteria. Some recent studies compared the growth of total aerobic bacteria with diffe rent treatments, incl uding a control group (no treatment), a 4% CO treatment, a treatment with 100% nitrogen to test the exclusion of oxygen, a treatment with 100% CO, a treatment with filtered smoke and a treatment with 18% CO. The results showed that the greatest reduction in tota l aerobic bacteria was no ticed for the filtered smoke and the 18% CO treatment together with the 100% CO treatment (Kristinsson and others 2007). The 100% nitrogen treatment showed some bacterial count reduction, while the bacterial counts for the 4% CO treatment and the contro l group were increased. In a long term freezing study, the 18% CO and filtered smoke treatments showed an even higher bacterial count reduction (Kristinsson and others 2006b). Nevertheless, data on how filtered smoke and carbon monoxide affect the microbial flora of meat and seafood during fresh and frozen storage is limited and this area needs to be more investigated. Bacteria and Other Microorganisms in Fish and Seafood All fresh fish and fish products contain a natu ral variety of bacteria and spores (Jahncke and Herman 2001). These bacteria an d spores are partially responsib le for the spoilage of fresh fish and processed fish products (Gram and Huss 1996). Some might also be a potential hazard for human health because of their pathogenic pr operties (Jahncke and Herman 2001). The visible evidence of spoilage is usually the growth of molds and slimy bacterial colonies, but spoilage can also change the sensory characteristics of a food product, such as smell (off-odors) and taste (off-flavor) (Gram and Huss 1996). Cold-smoked fi sh products are usually ready-to-eat food products that have not received sanitizing or stabilizing heat treatment (Gram 2001b). Therefore pathogens and biological hazards are of particul ar concern for these products. Some potential hazards associated with cold-smoked fish products are: Listeria monocytogenes, Clostridium botulinum type E, Salmonella spp., biogenic amines, and parasites (Jahncke and Herman 2001). 19

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Listeria Monocytogenes Listeria monocytogenes is a widely distributed Gram-positive, food borne pathogen that naturally occurs in many raw food products. It can grow between 1-45C and between 0 and 10% NaCl. High levels of L. monocytogenes in unheated ready-to-eat food products have been associated with listerioses (McLauchlin 1997). Si nce cold-smoked fish is a ready-to-eat product it has been linked to sporadic cases of listerios es (Gram 2001b). The tradi tional way of isolating these organisms involves the use of selective media such as PALCAM agar, blood agar with nalidixic acid agar, Oxford Listeria selective agar (LSA) and enrichment and pre-enrichment broths with incubation at 30C for 48 h. Since the growth of L. monocytogenes may be hindered by other microorganisms, PALCAM agar and Oxford LSA have advantages over other media, because of their ability to reduce the presence of contaminating micro-organisms (Neamatallah and others 2003). To eliminate or control the growth of L. monocytogenes a variety of different treatments of raw and finished product have al ready been approved. Two treatments involve: (1) washing the raw fish with water that contains chlorine, and (2) treatment of the raw fish with calcium hydroxide solution (pH 12). Other treatment options include washing the raw fish with acidified sodium chloride solutions, ozone treatment, steam surf ace pasteurization and electrochemical brine tank trea tments. For the finished produ ct, freezing and addition of approved microbial growth inhibitors are options to stop or control growth of L. monocytogenes (Jahncke and others 2004). Clostridium Botulinum Clostridium botulinum is the name of a range of Gram -positive, anaerobic, spore forming bacteria that produce the botulinum neurotoxin. These bacteria and their specific neurotoxins have been divided into 7 types (A, B, C, D, E, F and G) based on their antigenic properties. The disease caused by these neurotoxin s is called botulism. Generally botulism occurs rarely today. 20

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However, the neurotoxins from Clostridium botulinum are still some of the most potent toxins known. For cold-smoked fish products, the Group II of C. botulinum and particular strains that produce the type E neurotoxin are a major concern (Gram 2001a). Salmonella Spp. Salmonella spp. can be carried by fish and shellfis h which show no signs of disease. The contamination of this organism derives from terrest rial sources and fish may serve as a vector for Salmonella spp. (Novotny and others 2004) Especially shellfish from sewage-polluted waters seems to be a major problem. Although outbreaks of Salmonella spp. have occurred, processed seafood products are usually considered to pr esent a lower risk (Heinitz and Johnson 1998). Biogenic Amines Biogenic amines are nitrogen compounds that ca n be expected in nearly all foods that contain proteins or free amino acids (Shalaby 1996). In partic ular, histamine and tyramine have been associated with some toxicological ch aracteristics and outbreaks of food poisoning. The presence of biogenic amines a bove a certain level in non-fermen ted foods indicates the presence of undesired microbial activity and could therefore be used as an indicator of microbial spoilage. While not all biogenic amines correlate with th e growth of spoilage organisms, histamine, putrescine and cadaverine levels usually increase during spoilage of fish and fish products (Santos 1996). Biogenic amines and particularly histamine have been also implicated as the causative agent in a number of scombroid food poi sonings. It is also a concern that in coldsmoked products secondary amines such as putresc ine and cadaverine can react with nitrite and form carcinogens (Flick and others 2001). Parasites Cold-smoked or cold-smoked and dried fi sh and fish products may contain human pathogenic parasites such as Anisakis spp. (a nematode or roundworm), Diphyllobothrium spp. (a 21

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cestode or tapeworm) and Nanophyetus salmincola The harvest of parasite free fish in the wild is very difficult. Some aquacultured fish, however, are considered free of parasites, since their diet and environment may be controlled. The most effective way to ensure that viable parasites are not present in cold smoked fish products is to freeze the raw fish prior to the smoking step for a prescribed time that assures destruction of all vi able parasites in that fish (Bledsoe and Oria 2001). Sensory Characteristics of Fresh and Smoked Seafood For most people, fish and seafood is associat ed with the typical fishy smell that also surrounds fishing piers and fish markets. Howeve r, only few consumers know that this fishy smell is already a sign of degradation of the fish and seafood products. Most fresh fish and seafood products have no distinct smell or odor at all when caught and processed very fresh. Fish that has been stored for a couple of days or previously been fro zen develops a slight fishy smell due to the oxidation of lipids (Olson 2006). Further degradation of fish and seafood then ultimately leads to very unpleasant fishy and spoile d smell that will tell the consumer that this product is spoiled and no longer consumable. Odor and flavor is only one of the sensory characteristics consumers rely on when making a decision to buy or prepare fish and seafood. In f act, the most important characteristic is the color of the fish or seafood, since most consum ers will make eye contact with the product the first time through a glass display, where the colo r and overall appearance is the only factor they can base their decision. A fresh re d and pink color for most seafood products rich in dark muscle is preferred over the brown and gray color of lo ng stored, untreated produ cts. The third sensory characteristic is the texture and mouth-feel of fresh and processed seafood that determines if a customer is satisfied with the product. 22

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All these sensory characterist ics change dramatically when seafood products are processed using the hot or cold smoking technique with natural wood smoke. Hot and cold smoking introduces substantial amounts of flavor compounds into the seafood, which are identified by most consumers as smoky flavor and taste. Wh ile a long time ago smoking was discovered to be a good preservation method that also introduces flavor and taste to the products, today mostly the flavor effect is desired since other better preser vation techniques are available. Hot smoking of fish and seafood results in a fully cooked product with intense smoky fla vor and taste. Some forms of hot smoking also involve an intense dr ying step which removes so much water out of the product that it becomes shelf stable at room temperature without further preservation. Cold smoking is a less intense form of smoking and allo ws the smoky flavor and taste to be introduced into the product withou t cooking it. While the hot smoking pro cess changes the color and texture of the product the cold smoking process leaves the color and texture of the seafood nearly unchanged if done at the ri ght temperature (Burt 1988). Compared to the traditional smoking proce sses, filtered smoke and carbon monoxide treatments are performed at refrigeration temp eratures and have only minimal impact on the texture of the product. Also the taste and smell are nearly unchanged due to the treatment since carbon monoxide is tasteless and most of th e taste and odor giving compounds have been removed from the filtered smoke. However, the treatments may have a long term preservation effect that exceeds the traditional smoking techniques. The taste and smell are not only unchanged, but further preserved for an extende d period, since the car bon monoxide, which is also present in filtered smoke inhibits the li pid oxidation which causes off odors and tastes (Kristinsson and others 2006b). The color of fi sh and seafood products treated with filtered 23

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smoke and carbon monoxide is e nhanced and preserved during fresh and frozen storage, which increases the consumer acceptance and likeability (Olson 2006). 24

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CHAPTER 3 OBJECTIVES The hypothesis for this study is filtered wood sm oke processing increases the shelf life and the quality of Yellowfin tuna by inhibiting pathog enic and spoilage bacter ia without altering the sensory characteristics of the produc t. The aim of this experiment was to treat Yellowfin tuna with filtered wood smoke to determine whether s poilage and pathogenic b acteria are inhibited by this treatment, and to observe any significant correlation between the inhibition of spoilage bacteria and the growth and i nhibition of pathogenic bacteria. In the first part of this experiment Yellowf in tuna steaks were tr eated with filtered wood smoke at 0C and for time periods of 24 and 48 hours. The growth or inhibition of spoilage and pathogenic bacteria was measured before and after treatment, on frozen storage, and also every second day of refrigerated storag e up to a total storage time of two weeks. The second part of this experiment aimed to determine the signifi cant changes in odor and color of the filtered smoke treated products compared to th e artificial smoke treated products. A final objective of this study was to desi gn and validate a quick gas chromatography method to identify whether a product was treate d with filtered smoke or just with carbon monoxide. The use of carbon monoxide is currently fo rbidden in Europe and strictly regulated in many other countries. 25

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CHAPTER 4 PRELIMINARY STUDIES Filtered wood smoke processing is a recently developed processing technique that is gaining widespread use throughout the globe. Only a few companies produce filtered smoked fish products so far and the market has not been fully developed yet. Since the filtered wood smoke processing is so new, not many studies ha ve been done on this particular subject. Ongoing studies in our laboratory involve the determin ation of polycyclic aromatic hydrocarbons (PAH) in filtered wood smoke and treated products, deve loping a rapid analysis to identify products treated with filtered wood smoke and general identification of th e major components of filtered wood smoke and their relevance to the process. We have also done work on the effect of filtered smoke on the quality of fish muscle, but limited to few treatment times and only a few properties of fish muscle. Tuna Microbiology Study Overall these results indicate th at filtered smoke and artificial smoke seem to improve the shelf life of tuna, but more conclusive data over a longer period is required to confirm the hypothesis. Figure 3-1 shows that colony forming units (CFU) count decreased immediately for all treatments, even for the control group, which mi ght be an indication for a reduction in CFU/g due to the stress of handling the samples. Af ter the treatment the c ontrol group, the 100% carbon monoxide and the 100% nitrogen group showed a larger increase in CFU/g, followed by the filtered smoke and the artificial filtered smoke treatments. The artificial smoke treatment showed overall the lowest bacterial count until day 6, while the filte red smoke treatment increased significantly in CFU/g after day 3. While conducting this study cr oss contamination might have occurred for one of the three monitored samples for the filtered smoke treatment, which might 26

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explain this high value. All other treatments 100% carbon monoxide, 100% nitrogen and control showed higher bacterial load until day 6 compared to the artificial smoke. Figure 3-2 shows a similar initial effect for all treatments as seen in Figure 1. The CFU count decreased for all treatment s, including the contro l group, after the treatments ended. Over the next six day the control gr oup and the 100% nitrogen group in creased the most in bacterial count. The filtered smoke treatment showed the smallest increase in bacterial load over the next six days, followed by the artificial sm oke group and the 100% carbon monoxide group. Figure 3-3 shows the CFU count for all treatm ents after 48 hours. The graph shows that there is no initial decrease in CFU count for a ny of the treatments, which could be because the treatment time of 48 hours allo wed the microorganisms to leave the lag phase al ready during the treatment and so they started growing again. St ill the control group, the 100% nitrogen group and the 100% carbon monoxide group showed initial in creases in bacterial count followed by the filtered smoke and artificial smoke treatments. After six days all treatments reached a similar bacterial load and showed no difference. A study was conducted to investigate the effect of carbon monoxide, carbon dioxide, nitrogen and filtered smoke on the overall bacterial growth on tuna steaks over one week. Tuna steaks were cut from a fresh whole tuna loin and tumbled for a minute in a sterile container to ensure equal bacterial load on the surface. Two steaks fo r each treatment were then chosen and placed into a decontaminated gas tight container. The following treatments were applied to the tuna steaks: control (no treatment), carbon monoxide (~100%), 100% nitrogen, filtered smoke, and artificial filtered smoke (18% CO, 21% CO2 and 61% nitrogen). The steaks were held at 4C for 16, 32 and 48 hours inside the decontaminated c ontainers, which were f illed with each gas, Samples were taken from each steak before and im mediately after the treatments, as well as 1, 3 27

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and 6 days after the end of the treatments. Each sample was then analyzed for the total bacterial count using total plate count Petrifilms from 3M Corp. Fi gures 3-1 to 3-3 show the average total bacterial count per gram for all treatments after 16, 32 and 48 hours of treatment. Tuna Color Study Tuna steaks were cut from one fresh tuna loin and placed into a gastight container where they were treated with carbon monoxide (~100%), 100% nitrogen, filtered smoke and artificial filtered smoke (18% CO, 21% CO2 and balanced with nitrogen) for 16, 32 and 48 hours. The control group was exposed to the surrounding air. The tuna steaks were analyzed with a color vision machine for their color value, according to the L*a*b color system. Pictures were taken before (day -1), after treatment (day 0) and on day 1, 3 and 6 after the treatment and the average color value was determined and calculated by us ing the color vision m achine and color vision software. The average L*-, a*and b*values we re recorded for each sample. Since the major interest of this study is in the maintenance and enhancement of the red color of the tuna muscle, the a* value representing the redness of the sa mple as a positive number (negative represents greenness), were compared to determine how the redness of the t una changes after the treatments. Figures 3-4 to 3-6 show the average a* values (redness) of th e untreated and treated samples over a time period of 6 days after treatment. As shown above for 16, 24 and 48 hours the cont rol (CTRL) as well as the filtered smoke (FS), the 100% carbon monoxide (100% CO) and the 18% carbon monoxide (18% CO) have increased a*-values immediate after the treatmen t. Only the sample treated with pure nitrogen (100% N2) showed an immediate decline in the a*-val ues after treatment and then continued to decline over the period of 6 day, except for the 48 hour treatment where a slight increase in a*value can be seen on day 1. Overall the nitrogen tr eatment seems to have a negative affect on the a*-values, which can be seen immediately afte r treatment. The control groups show for the 16 28

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and 48 hour treatments increases in a*-val ues and unchanged a*-values for the 24 hour treatment. From day 1 to day 6 after the treatmen t the a*-values from the control group declined rapidly for the 16, 32 and 48 hour treatment. The filtered smoke, 100% CO and 18% CO treatments showed a slight increase in a*-val ues for the 16 and 32 hour treatment and a large increase in a*-values for the 48 hour treatme nt at day 0. These three treatments show stabilization in a*-values for the 16 hour treatmen t until day 1 and then a slow decline until day 6. The 32 and 48 hour treatment show stable a*-v alues until day 3 and then a decline at day 6. Overall the 100% CO treatment showed the highes t improvement and stabilization in a*-values after the treatment and over the period of 6 days. The filtered smoke and the 18% CO treatment seemed to stabilize the a*-values nearly as good as the 100% CO treatment, especially over the extended period of 6 days. The lack of data poi nts between these measurements makes it clear that more frequent measurements are needed to describe the color changes due to filtered smoke and carbon monoxide treatments. Identification of Filtered Smoke Treated Products A study was conducted to identify gas compound s that were typical for filtered wood smoke. A gas chromatograph from Agilent (6890N) was equipped with a packed column and a nickel catalyst tube to convert carbon monoxide and carbon dioxide into methane. The nickel catalyst was needed to detect carbon monoxide and carbon dioxide with the FID (Flame ionization detector), which are otherwise not detectable. 100% carbon monoxide, filtered wood smoke and unfiltered fresh generated wood smok e were directly injected into the gas chromatograph (GC) and analyzed. The chromatograms showed a large number of peaks for the smoke samples and only one peak for the carbon monoxide sample as expected. Natural gas and refinery gas standards from Scott Specialty Gasses were then anal yzed in the GC with the same method as the smoke samples before. 29

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The previous results led to the idea to iden tify smoke treated products by analyzing gas compounds that are evaporated from smoked pr oducts. An experiment was conducted where tuna steaks were treated with 100% carbon monoxide, filtered smoke and natural wood smoke. A portion of 10 g of sample from each treatment was transferred into a 60 ml vial with a gas tight lid with septa inlet. The samples were heated in a water bath at 100C for 5 minutes. After the heat treatment a sample of 100 l was taken from the headspace of the vials with a gas tight syringe and injected into the GC. The results show that smoke treated products show two significant peaks that were not present in products that were untreate d or treated with carbon monoxide, nitrogen and carbon dioxide. Note th at carbon monoxide and carbon dioxide can not be detected directly by using the FID (Flame Ionization Detector), therefore these components will be methanized (transformed into methane) after separation on the column, which can then be detected by the FID. Figures 3-7 to 3-11 show the chromatograms collect ed from this study. Figure 3-1. Tuna treated for 16 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was exposed to the surrounding air only. The graph shows the measurement of colony forming units (CFU) at day -1 (before trea tment), day 0 (right after treatment) and then day 1, 3 and 6 after treatment. 1.00E+04 1.00E+05 1.00E+06 1.00E+07 -202468CFU per [g]Days at 4C No Treatment 16 hrs 100 % CO 16 hrs Filtered Smoke 16 hrs 100 % Nitrogen 16 hrs 18 % CO 16 hrs 30 days of frozen storage 30

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Figure 3-2. Tuna treated for 32 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was exposed to the surrounding air only. The graph shows the measurement of colony forming units (CFU) at day -1 (before trea tment), day 0 (right after treatment) and then day 1, 3 and 6 after treatment. 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 -202468CFU per [g]Days at 4C No Treatment 32 hrs 100 % CO 32 hrs Filtered Smoke 32 hrs 100 % Nitrogen 32 hrs 18 % CO 32 hrs 30 days of frozen storage Figure 3-3. Tuna treated for 48 hours with 100% CO, filtered smoke, 100% nitrogen and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group was exposed to the surrounding air only. The graph shows the measurement of colony forming units (CFU) at day -1 (before trea tment), day 0 (right after treatment) and then day 1, 3 and 6 after treatment. 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 -202468CFU per [g]Days at 4C No Treatment 32 hrs 100 % CO 32 hrs Filtered Smoke 32 hrs 100 % Nitrogen 32 hrs 18 % CO 32 hrs 30 days of frozen storage 31

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Figure 3-4. Tuna treated for 16 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group (CTRL) was exposed to the surrounding ai r only. The graph shows the measurement of average a-value at Day -1 (before treatment), Day 0 (right after treatment) and then Day 1, 3 and 6 after treatment. 0 5 10 15 20 25 day 1day 0day 1day 3day 6a valur N2 16 hr FS 16 hr 100% CO 16 hr 18 % CO 16 hr CTRL 16hr Figure 3-5. Tuna treated for 32 hours with 100% CO, filtered smoke(FS), 100% nitrogen(N2) and a mixture of 18% CO with 21% CO2 balanced with nitrogen. The control group (CTRL) was exposed to the surrounding ai r only. The graph shows the measurement of average a-value at Day -1 (before treatment), Day 0 (right after treatment) and then Day 1, 3 and 6 after treatment. 0 5 10 15 20 25 30 day 1day 0day 1day 3day 6a value N2 32 hr FS 32 hr 100 % CO 32 hr 18 % CO 32 hr CTRL 32 hr 32

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Figure 3 Figure 3 0 5 10 15 20 25 30 35 40 45a value 6. Tuna tre and a mi x (CTRL) w of averag e Day 1, 3 a 7. Chroma t monoxid e minutes a day 1 ated for 48 h x ture of 18 % w as exposed e a -value at a nd 6 after t r t ogram of t h e at around 0 a nd the last p day 0 h ours with 1 % CO with 2 1 to the surro Day -1 (bef o r eatment. 33 h e analysis o f 0 .7 minutes, p eak represe n day 1 00% CO, fi l 1 % CO2 b al a unding air o o re treatme n f Clearsmo k the second p n ts carbon d 1 day l tered smok e a nced with n o nly. The gr a n t), Day 0 (r i k e. The fi r p eak repres e d ioxide at ar o 3da y e (FS), 100 % n itrogen. T h a ph shows t h i ght after tre r st peak r ep r e nts methan e o und 3.7 mi n y 6 % nitrogen(N 2 h e control gr o h e measure m atment) and r esents carb o e at around 2 n utes N2 FS 100% CO 18% CO CTRL 48 hr 2 ) o up m ent then o n 2 .5

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34 Figure 3 Figure 3 Figure 3 8. Chroma t peak repr e 0.1% car b 9. Chroma t trace am o detectabl e 10. Chrom a amounts o t ogram of t h e sents carb o b on monoxi d t ogram of t h o unt of carb o e by the FI D a togram of t o f carbon m o h e analysis o f o n monoxid e d e in air v/v h e analysis o f o n monoxid e D t he analysis o o noxide. Ni t f industrial c e at around 0 f atmospher i e resi d ual in o f nitrogen ( t rogen and o c arbon mon o 0 .7 minutes. i c ai r The s the air. Nitr ( N2). This p o xygen are n o xide (~100 % The sample s hown peak r ogen and o x p eak also re p ot detectabl e % CO). Th i was diluted r epresents a x ygen are no p resents trac e e by the FI D i s to t e D

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35 Figure 3 11. Chrom a small am o dioxide. a togram of t o unt of carb o t he analysis o o n monoxid e o f carbon di e sample an d oxide (CO2 ) d the secon d ) The first p d peak repre s eak again s h s ents carbo n h ow a n

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CHAPTER 5 MATERIAL AND METHODS Fresh and Frozen Storage This project is mainly divided into two part s, a fresh storage and a frozen storage study. Figure 5-1 shows a scheme for th e project plan. For the frozen storage study the samples were prepared as described in Sample Preparati on and Treatments and each sample was then vacuum sealed in a Foodsaver Bag, placed in the freezer and then stored for 30 days at -20C. Samples for the frozen storage study were thaw ed out two days prior to the start of the experiments at 4C in the cold room. The frozen storage samples were not analyzed during the frozen storage, but rather after the frozen storag e in the same manner as the samples for the fresh storage study. For the fresh storage study the samples were prepared as described in Sample Preparation and Treatments and every sample was then tran sferred into a ZipLoc bag and stored in the cold room at 4C for the remainder of the experiment. Fresh and frozen storage studies were timed in a manner that fresh and frozen samples could be analyzed together at the en d of the frozen storage period. For the Salmonella Total Aerobic Plate Count and the Color-Study, the sample s were analyzed before the treatment (day 1), immediately after the treatment (day 0) and then at day 1, day 2 and every other day after that for a total of 14 days. For the frozen storage stud ies Day 1 represents the day when the thaw out process was completed, while it represents the actual first day after treatment for the fresh storage studies. For the taste panel and GCexperiments only samples representing day 1 after treatment or thaw out were chosen and analyzed. 36

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Sample Preparation and Treatments For all studies except the taste panel study samples were prepared in duplicates per treatment. For all studies fresh yellowfin tuna was ordered from Save-On Seafood (Tampa, Fl) and was processed and treated immediately upon delivery. For all stud ies the workplace was cleaned and sanitized with 70% ethanol v/v in water to minimize microbial cross contamination. The fully trimmed tuna loins were cut into 2.5 cm thick steaks, which were again trimmed and then sorted for uniformity, especially for the color and taste panel studies. All samples were then transferred into the prepared Foodsaver bags. Fr om a roll 23 by 28 cm bags were cut and sealed on three sites. The bags were then equipped with a silicon septum valve recovered from PVF gas sampling bags from LabPure Instruments. Each bag filled with one tuna steak was individually vacuum sealed and then stored on ice until treatment. For the Salmonella study the samples were inoculated with Salmonella enteriditis as described in the chapter Salmonella study. Each bag was then filled with three liters of the desi gnated treatment atmosphere except for the control which was kept vacuum sealed. The treatment gases were injected directly from the gas cylinders via a hose, equipped with a pressu re gun and a needle, thru the sept a valve into the sample bags. The amount of gas introduced to each bag was controlled with a gas flow meter from Alicat Scientific Model M-50SLPM-D which was set to Air mode since this is the closest representation of the treatment gases in terms of density. The actual volume of each treatment gas was not too important in these experiments, but rather the fact that each bag was filled with the same amount of treatment gas to en sure uniformity among the treatments. The filtered smoke was delivered in compresse d gas cylinders and ready to use for the treatments. The filtered smoke was delivered with a certified analysis stating that it contained 21% carbon monoxide, 18 % carbon di oxide, 55% nitrogen and is balanced with oxygen and other gases. Trace components from the incomple te combustion of wood chips that were not 37

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filtered out were not analyzed or determined. Acco rding to the typical treatment times used in the industry it was chosen to determine the effect of a 24 and a 48 hour treatment of filtered smoke on tuna. The second treatment gas that was used in these experiments was a pure mixture of 21 % carbon monoxide, 18 % carbon dioxide, 1.1% oxygen a nd a balance of nitrogen. This mixture was chosen since it represents the exact compos ition of the filtered smoke without the trace components that are possibly found in filtered wood smoke. This mixture was called artificial smoke and will be abbreviated as AS. The original filtered smoke will be abbreviated as FS. To compare AS to FS equal treatment times (24 and 48 hours) were chosen for the AS treatments as well. The control group for all experiments consis ts of tuna steaks which were vacuum-packed and sealed and then kept on ice for 24 hours prio r to analysis. Table 4-1 shows an overview over the treatment combinations that were applied to all studies. Table 5-1. Treatment combinations us ed in all studies of this project Label Treatment Gas Treatment Time Storage Conditions FS24F Filtered smoke (FS) 24 hours 30 days at -20C FS48F Filtered smoke (FS) 48 hours 30 days at -20C AS24F Artificial smoke (AS) 24 hours 30 days at -20C AS48F Artificial smoke (AS) 48 hours 30 days at -20C CtrlF No gas treatment 24 hours 30 days at -20C FS24 Filtered smoke (FS) 24 hours 1 day at 4C FS48 Filtered smoke (FS) 48 hours 1 day at 4C AS24 Artificial smoke (A S) 24 hours 1 day at 4C AS48 Artificial smoke (A S) 48 hours 1 day at 4C Ctrl No gas treatment 24 hours 1 day at 4C Salmonella Study To study the effect of filtered and artificial smoke on the inhibition or growth of Salmonella spp. tuna steaks were prepared as descri bed in Sample preparations and treatments with 4 steaks per treatment. Two steaks per treatment were inoculated with Salmonella enteriditis, which was obtained from the American Type Culture Collection (ATCC #13076). The culture was prepared and enriched accordin g to the instructions given by the ATCC in 38

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Nutrient Broth. For each steak 1 ml of culture wa s spread evenly over th e entire steak and the bag was shaken for 1 minute to ensure an even di stribution of the culture over the tuna steak. For the Salmonella analysis a cube of 10 g was cut from th e each inoculated tuna steak in a manner that the cube consists of three surfaces exposed an d three internal exposed s ites to ensure an even distribution of microorganisms each time the stea ks were sampled. The two other steaks were kept non-inoculated to see the effect of the treatments on th e naturally existing Salmonella spp. flora. These steaks were sampled in the same manner as the inocul ated samples. The noninoculated samples were sampled at day -1, day 0, day 1 and day 2 from that point it was decided to abandon the non-inoculated study, since no Salmonella growth could be identified. These samples were analyzed a last time at day 14 to ensure that there was actually no Salmonella growth and the decision to abandon this study wa s correct. The inoculated steaks were sampled right after the inocul ation but before the treatment (Day -1), directly after the treatment (Day 0) and at Day 1, 2 and then every ot her day until day 14. During this ti me the steaks were stored in Ziploc Bags at 4C in a cold room. Each 10 g samples for each analysis were plac ed into a sterile Nasco Whirlpack bag and 90 ml of Hardy Diagnostics Dilu-LOK II Phospha te buffer with magnesium chloride (buffer solution) added to the sample. The sample was squeezed and mixed by hand inside the bag with the buffer solution. Three Dilutions were prepared by pipetting 10 ml of the sample solution into 90 ml of sterile buffer solution. More Dilutions we re prepared if the resu lts from the previous day indicated that the first three dilutions w ould have to numerous to count microorganisms. Then 0.2 ml of each dilution were plated onto tw o preplated XLD-Agar Plates from Biomrieux Industries (0.1 ml per plate) and spread with a Lazy L spreader over the entire plate to ensure uniform growth. Always the last three dilutions were plated and counted. All plates were 39

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incubated at 35C for 24 hours. To determine the amount of Colony Forming Units (CFU) per ml of solution all yellow colonies with black cente rs were counted by hand for each plate. Which dilutions were prepared and plated was decided on the basis of th e results from the previous day. Most of the Salmonella study wa s performed under a sterile hood. Total Aerobic Plate Count Study To study the effect of filtered and artificial smoke on the inhibition or growth of all naturally present aerobic bacteria, tuna steak s were prepared as described in Sample preparations and methods with 2 steaks per treatment. During preparation and treatment all samples were handled aseptically to avoid cross contamination. All sample steaks were stored in Ziploc Bags at 4C during the en tirety of the 14 day shelf life study. For the fresh storage study the steaks were sampled before treatment (Day -1), directly after treatment (Day 0) and at Day 1, 2 and then every other day until day 14. For the frozen storage study the steaks were sampled before treatment (Day -1), directly after treatme nt (Day 0) and then after the two day defrosting process (Day 1) and at day 2 and every other day until day 14. To determine the amount of CFU per g of tuna at any given sampling time, a 10 g cube consisting of three surface-exposed and three inside-exposed sites was cut from each stea k and placed aseptically into a sterile Nasco Whirlpack bag. The bag was then filled with 90 ml of sterile premade Dilu-LOK II Butterfields buffer from Hardy Diagnostics and the sample cube was squeezed and mixed inside the bag with the buffer by hand for 1 minute. The sample so lution in the bag is then considered the first dilution. Further dilutions were prepared with the same buffer solution by transferring 9 ml of the sterile buffer solution and 1 ml of the sample solution aseptically into a sterilized culture tube. More Dilutions were prepared if the results from the previous day indica ted that the first three dilutions would have to numerous to count mi croorganisms. For each dilution and sample 2 ml 40

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were plated onto two Petrifilm Plates for Aerobic Plate Count from 3M Microbiology Products with 1 ml of solution per plate. Always the last three dilutions were plated and counted. All plates were then incubated at 34.5C for 48 hours. Each plate was counted by hand to determine the amount of CFU per gram of sample. Which dilutions were prepared and plated was determined based on the re sults from the previous day. Color Analysis The effect of filtered and artificial smoke on the color of tuna during 14 days of storage at 4C was determined by a digital Color Machin e Vision System (CMVS). The CMVS was used according to the procedure described by Balaba n and Luzuriaga (2001). The CMVS can report the average L*-(lightness), a*-(redness) and b*-(y ellowness) values for each sample. For this experiment the main focus was on the changes in th e average a*values since the redness of tuna is one of the main quality criterion for the industry and especially the consumer. Pictures were taken before treatment (Day -1), direct after treatm ent (Day 0) and at day 1 (after thaw out period for the frozen storage study), day 2 and then every second day until day 14. The samples were placed in a light box and top lighting with two fl uorescent lights each to simulate illumination by noonday summer sun (D65 illumination). The door remained closed while images were capturedto assure uniformity of light inside and to minimize the effect of outside light. Images were captured using a camera (Nikon D200 Digital Camera, Nikon Corp., Japan) located inside the chamber mounted to face the bottom of the light box. The Nikon D200 Settings used are described in Table 5-2. A red reference tile was la id into each picture to compensate for changes in light and camera settings in the co mputer analysis of the pictures. Sensory Taste Panel Analysis To determine if there were any human detect able differences between a filtered and an artificial smoked tuna steak in appearance and smell, a sensory taste panel was conducted with 41

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60 random untrained panelists. The taste panel an alysis was conducted on two different days, with the first day focusing on the odor differences among the samples and on the second day the color differences were analyzed. Only the 48 hour treatments with filtered smoke and artificial smoke for the frozen and the fresh storage study were compared, since it was known that these treatments would give the most odor and color di fferences. Four triangle tests were conducted to determine if there were any detectable differe nces in odor or color between 48 hour treated filtered and artificial smoked tuna after frozen (30 days at -20C) and fresh (1 day at 4C) storage. The design, test and an alysis of this study were conducted in the FSHN taste panel facility with the use of the Compusense so ftware program. The detailed design for each triangle test can be found in Appendix A Taste Panel Design Sheets Each panelist who identified the odd sample as the different samp le was asked to write down any comments he might have to describe the difference. Tables B-1, B-2, B-3 and B-4 show a summery of all comments given by the panelists. Rapid Gas Chromatography Identification Method The main components of filtered and artifici al smoke are carbon monoxide, carbon dioxide and nitrogen, as well as a small amount of oxygen. However, filtered smoke contains more then just these components. A GC method which wa s mainly developed to quantify the amount of carbon monoxide in red muscle food might also have the pote ntial to determine whether a product was treated with real filte red wood smoke or just a combina tion of 4 industrial gases that we call artificial smoke. The first step was to determine which components can be identified in filtered smoke that are not present in artificial smoke with the simple GC method developed for carbon monoxide quantificati on. An Agilent Technologies 6890N Network GC System, equipped with a Flame Ionization Detector (F ID), a Supelco 80/100 Porapak Q Column (1.82 m long) and a hydrogen aided Nickel Catalyst (to convert carbon m onoxide and carbon dioxide into 42

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methane and make it detectable for the FID) was used for this study. The settings for the rapid identification procedure are s hown in Table 5-3. PVF Gas sampling bags from LabPure Laboratory Instruments were used to collect the pure gasses and I-Che m Economy 100 series 60 ml glass vials with a Teflon-fluor ocarbon-resin/silicone septa lid were used for gas dilutions and sampling. The glass vials were flushed with nitrogen prior to sampling to minimize any contamination from the surrounding air. To iden tify the gas components in filtered smoke its chromatogram was compared with the chromatogram of the Refinery Gas Test Sample from Agilent Technologies. After the all peaks from the filtered smoke chromatogram had been identified all these gases were ordered in hi gh purities to create standard curves (for quantification) for each of them. Ethylene ( 99.5% purity) and ethane (99.0% purity) were obtained from Scott Specialty Gasses. Meth ane (Ultra High Purity Grade), carbon monoxide (CP-Grade) and carbon dioxide (CPGrade) were obtained from Airgas. Standard curves for each of these gasses were produced by diluting e ach gas several times and injecting different amounts of each dilution into the GC. The areas under the corresponding peaks were then plotted against the actual quantity of gas injected. Af ter a linear regression an alysis an equation was obtained for each gas that allows th eir quantification by peak area. Two different samples of filtered smoke, the artificial smoke and a filtered smoke, which was obtained from another source (filtered smoke B), were analyzed with the GC for their content of each of th e individual gasses. The final step was to identify whether a piece of tuna was treated with fi ltered or artificial wood smoke and to quantify the am ount of carbon monoxide per gram of muscle tissue. For this analysis 10 g of sample from each treatment a nd each storage study were minced and transferred quickly into the 60 ml glass vial s and the vials were sealed. The samples were then heated at 43

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100C for five minutes and then cooled down to room temperature for another five minutes. A quantity of 100 l of the headspace atmosphere from each vial was then injected into the GC and analyzed for the previously mentioned gas components. Since the artificial smoke does not contain any methane, ethylene or ethane, it would be unlikely for the tuna to absorb these gases and release them into the headspace of the vial as it would do with a sample treated with filtered smoke. Therefore the presence of these gases in the headspace over a sample would indicate a treatment with filtered smoke. Statistical Analysis For the Salmonella total aerobic plate count and color-studies the data for the fresh and the frozen storage was analyzed separately. Analysis of Variance (ANOVA) was used to determine significant differences between all treatments at all sampling times. When significant differences among the sample means were detected Tukeys St udentized Range (HSD) test was used to do a pair wise comparison between these sample mean s. The level of significance for all of the ANOVA and Tukeys test were set to 5 % or 0.05. Simple linear regression analysis was used to determine the standard curve equations for the GC analysis of carbon monoxide, carbon di oxide, methane, ethyl ene and ethane. The Statistical Analysis Software (SAS) and Microsoft Excel were used for the analysis of the data. 44

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Table 5-2. Nikon D200 Settings Setting Specification Device Nikon D200 Lens VR 18-200 mm F 3.5-5.6 G Focal length 36 mm Sensitivity ISO 100 Optimize image Custom High ISO NR Off Exposure mode Manual Metering mode Multi-pattern Shutter speed and ap erture 1/3s-F/11 Exposure compensation (in camera) 0 EV Focus mode AF-S Long exposure NR Off Exposure compensation (by capture NX) 0 EV Sharpening Auto Tone compensation Auto Color mode Model Saturation Normal Hue adjustment 0 White balance Direct sunlight Table 5-3. GC-Settings for th e rapid identification method Parameters Settings Injection Temperature 100C Carrier Gas and Flow rate Helium at 26.9 ml/min (splitless) Nickel Catalyst Temperature 375C Oven Temperature 30C isotherm Runtime 5 minutes Detector Temperature 250C Column 80/100 Porapak Q packed column (1.82 m) 45

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Figure 5-1. Flow diagram of all st udies conducted duri ng this project 46

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CHAPTER 6 RESULTS AND DISCUSSION Salmonella Results The first part of this project was to investigat e what effect a filtered smoke and artificial smoke treatment will have on the growth of Salmonella enteriditis on Yellowfin tuna after fresh and frozen storage. It should be noted at this point that it was planned to follow the Salmonella growth over a period of 14 days for in oculated and non-inoculated samples. Salmonella spp. is the leading cause of several food poisoning related diseases on humans (Scott 1996) and Salmonella enteriditis is one of the most aggressive species (Butt and others 2004). Salmonella enteriditis was therefore obtained from the American Type Culture Collection (ATCC). The noninoculated samples showed absolutely no Salmonella growth during the firs t 4 days of the study for all samples and all treatments. It was ther efore decided to abandon the analysis of the noninoculated samples and keep them under the same conditions as the inoculated samples to analyze them a last time at day 14. When no growth of Salmonella was detected on the noninoculated samples at day 14, it was decided not to include any co llected data on the noninoculated samples since there was obvi ously nothing to report. Although no Salmonella spp. were found on the non-inoculated samples, seve ral studies still repor t incidences with Salmonella spp. in raw and cold smoked seafood, where the chance of a Salmonella incident is usually higher in imported seafood (H einitz and Johnson 1998; He initz and others 2000). The focus was laid on the an alysis of the growth of Salmonella spp. on the inoculated samples after filtered smoke and artificial smoke treatments for the fresh and frozen studies, since Salmonella species were detected in several smoke d fish samples from different countries (Heinitz and Johnson 1998; Fell and others 2000). Figures 6-1 and 6-2 show the average amount of Colony Forming Units (CFU) for each treatment at any measured time point for the fresh and 47

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frozen studies, respectively. For both the fres h and the frozen study, Analysis of Variance (ANOVA) tests were conducted fo r every observed day to detect any significant differences among the average CFU count for all treatments. Tables 6-1 and 6-2 show the results of the ANOVA tests as well as the results of the Tuke y pair wise comparisons which were conducted when a significant difference wa s detected by an ANOVA test. Fresh Storage Study As Table 6-1 for the fresh study shows, a significant difference among the sample means could only be detected at day 4 of the study. Howeve r, the data for Tukeys test shows that there is no significant difference among all treatments except for the 48 hour artificial smoke treatment, which is significantly different from all other treatments except the 24 hour filtered smoke treatment. It is very likely that this difference is based on a random effect and an experimental error than an eff ect of the 48 hour artificial smoke treatment. It should be noted here that to conserve time a nd resources for all experiments duplicate samples were prepared and analyzed. All statistical analysis is based on the analysis of du plicate samples. Nevertheless it can be clearly seen that none of the applied treatments seems to have a major effect on the growth or inhibition of growth of Salmonella spp. It is of course well observed in Figure 6-1 that from the day of inoculation during the comp lete time of observation, the growth of Salmonella spp. was inhibited and the count of Salmonella CFU decreased. A possible explanation for this effect might be the competition of the Salmonella bacteria with the na tural existing microbial flora present on the tuna samples (Revolledo and others 2003; Liao 2007). Therefore it is suggested that not so much any specific treatmen t, artificial or filtered smoke, inhibited the growth of the Salmonella spp. but rather the growth of naturaly existing microorganisms present on the tuna samples. Another observation supporting these findings is the fact that no Salmonella growth was detected in the noninoculated samples. Heinitz a nd others (2000) reported a 10% 48

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chance of Salmonella spp on raw imported seafood and onl y a 2.8% chance on raw domestic seafood based on samples taken by USDA field laboratories over a period of 9 years. Frozen Storage Study The results for the frozen storage study were quite similar to the fresh storage study, as shown in Figure 6-2. There were no significant differences among the treatment means for all observed days as shown in Table 6-2, which s hows the p-values of the ANOVA tests that were conducted to detect any significant differences among the sample means for each observed day. It seems that in Figure 6-2 compared to the fresh storage study there was some growth of Salmonella spp. observed between the inoc ulation and the end of the tr eatments, but this can be explained by a minor experime ntal flaw that was eliminat ed immediately after the first measurement. When the steaks were inoculated with Salmonella enteriditis the culture was kept in a buffer solution and 1 ml of this buffer solu tion was spread over the entire surface of the sample steak as evenly as possible. A sample for analysis was then taken immediately, as described in Materials and Methods, before th e sample was vacuum packed for the treatment. It was later discovered that the vacuum packing actually contribu tes to the distribution of the culture on the sample through the force of atmospheric pressure on the outside of the packaging material. For the fresh storage study all samples were vacuum packed after the inoculation and then reopened and sampled before the treatment to improve the even distribution of the culture. Since in fact all samples were vacuum packed before any treatment was applied to the samples there should have been no further imp acts on the outcome of this analysis. It can therefore be concluded that filtered smoke and artifi cial smoke processing have no immediate effect on the growth or inhibition of Salmonella spp. whether the sample were stored frozen or kept at 4C. 49

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Although it was not observed in this experi ment, the inhibition or growth of Salmonella spp. can be highly influenced by the natural mi crobial flora present on the product (Liao 2007). Table 6-1. ANOVA results for Salmonella spp. for the fresh storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value 0.79 0.05 0.09 0.14 0.01 0.51 0.46 0.30 0.73 0.47 Ctrl a a a a a a a a a a FS 24 a a a a ab a a a a a FS 48 a a a a a a a a a a AS 24 a a a a a a a a a a AS 48 a a a a b a a a a a P-Values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other Figure 6-1. Average amount of CFU/10g of tuna for Salmonella spp. for the fresh storage study. The graph shows the average CFU/10g of tuna at day -1 (before any treatment was applied), day 0 (right after the treatment) and day 1 though day 14. The samples were kept at 4C during the entire time of observa tion. All samples were inoculated with Salmonella enteriditis before the day -1 measurement. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 1.00E+02 1.00E+03 1.00E+04 1.00E+05 20246810121416CFUDays Ctrl FS 24 FS 48 AS 24 AS 48 50

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Figure 6-2. Average amount of CFU/10g of tuna for Salmonella spp. for the frozen storage study. The graph shows the average CFU/10g of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were frozen for 30 days at -20C between day 0 and day 1.The samples were then kept at 4C during the rema ining time of observation. All samples were inoculated with Salmonella enteriditis before the day -1 measurement. The samples were treated with filtered smoke for 24 (FS24) and 48 ( FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The contro l samples (Ctrl) remained untreated. 1.00E+02 1.00E+03 1.00E+04 1.00E+05 20246810121416CFUDays Ctrl FS 24 FS 48 AS 24 AS 4830 days frozen storage Table 6-2. ANOVA results for Salmonella spp. for the frozen storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value 0.05 0.78 0.20 0.75 0.72 0.16 0.28 0.41 0.75 0.81 Ctrl a a a a a a a a a a FS 24 a a a a a a a a a a FS 48 a a a a a a a a a a AS 24 a a a a a a a a a a AS 48 a a a a a a a a a a P-values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other 51

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Total Aerobic Plate Count In the early days of natural wood smoke pr ocessing the main purpose was to preserve meats and seafood for a prolonged period. Although ot her factors like the he at treatment and the dehydration of the product (Burt 1988), which accompanied the smoke treatment, are affecting the microbial flora present on the product, it wa s suggested that certain components, present in natural wood smoke might inhibit the growth of spoilage bacter ia and enhance their shelf life (Kristinsson and others 2006b). This study therefor e focuses on the effect of filtered wood smoke on the growth or inhibition of aerobic bacteria naturally present on Yellowfin tuna. Earlier studies already suggested that filtered wood smoke processing inhibits the growth of aerobic bacteria during the first 6 days of storage (Danya li 2004). In this study the effect of filtered wood smoke and artificial wood smoke processing for 24 and 48 hours on fresh and frozen tuna was analyzed over a period of 14 days of storage at 4C. The fro zen tuna was treated prior to subjecting it to 30 days of frozen storage. Figur es 6-3 and 6-4 show the results of the average CFU count for all treatments at all observed time points for the fresh and frozen storage studies, respectively. For each single time point, ANOVA te sts were conducted to detect any significant differences among the sample means of the trea ted and untreated products. When significant differences were detected Tuke ys pair wise comparison tests were conducted to determine exactly which treatments are significantly different at this time point. Table 6-3 and 6-4 show the p-values of the ANOVA tests for each time point as well as the results of the pair wise comparison for the fresh and frozen studies respectively. Fresh Storage Study Results of the fresh storage study show that no significant differences could be detected for most of the observed days, except day 0 (immedia tely after treatment) an d day 2. The p-value of the ANOVA tests for day 1 shows that no significant difference could be detected at a 52

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significance level of 0.05, but there was a significant difference at a level of 0.09 and higher. As described before, all experiments were conduc ted in duplicates. Therefore one outlier can influence the outcome. Still it has to be observed that at day 1 the control group showed a much higher CFU count than all other groups. These fi ndings are similar to the results described by Kristinsson and others (2006b) It can be said that all treatment s seem to suppress the growth of aerobic bacteria during the firs t 4 days of observation. Total aerobic bacterial count then suddenly stopped increasing at day 4 and reached an equal level for all treatments, including the control group. From day 4 to day 14 there were no significant differences detectable between the control group and any of the treatments. The redu ced CFU count at day 4 represents the end of the lag phase of a typical bacterial growth curve (Creager and others 1990), followed by the log or exponential phase (Tortora a nd others 1992) from day 4 to da y 6 and the stationary phase from day 8 to day 14. The end of the stati onary phase can not be seen here since no measurements were taken after day 14. Although no significant differences could be detected after day 4 it seems like there is a small second la g phase for the sample treated with filtered and artificial smoke at day 8 compared to the cont rol group, however the lack of significance and of data surrounding this event makes these findings inconclusive. Overall it can be said that the filtered and artificial smoke treated samples appear to be better protected against aerobic bacteria growth during the first three days of storage at 4C after the treatme nt. However no significant improvement on shelf life can be reported afte r the exponential growth of the organisms was initiated. There is also no evidence in this study suggesting any signifi cant difference between the filtered and artificial smoke treatments or a prolonged 48 hour treatment compared to the 24 hour treatment for both treatment gasses. 53

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Frozen Storage Study The results for the frozen storage study show ed that there are no significant differences among the sample means for day -1 (before treatme nt), day 0 (after treatment) and day 1, day 10, day 12 and ay 14. At day 1, this is the first sa mpling day after the 30 day frozen storage period, all samples seem to have the same CFU count regardless of their treatment. This can be explained by the fact that so shortly after the thaw out process only few viable cells could be sampled and survived the stress of freezing a nd thawing (Speck and Ray 1977; Bhaduri and Cottrell 2004). However, already at day 2 it ca n be observed that CFU count for the control group is significantly higher then the CFU count for all other treatm ents. It should be noted that, although not statistically significan t, the CFU count for the control group seems already to be higher directly after the treatment and before the samples were placed into frozen storage at day 0. Furthermore should be noted that at day 0 it seems that the 48 hour filtered smoke and artificial smoke treatments inhibit the growth of aerobic bacteria more effectively then the 24 hour treatments, respectively. From day 4 to day 6 the same behavior is observed as compared to the fresh storage study, where day 4 marks the end of the lag phase of the bacterial growth and the beginning of the log or exponential phase. From day 6 to day 14 a more or less stable stationary phase is observed. The slight increase in CFU count for all samples during the last 8 days could suggest that there ar e still microorganisms recovering from the 30 day frozen storage (Speck and Ray 1977). Nevertheless the cont rol group, although not longer statistically significant, has higher CFU counts than the treatm ent groups at all times from day 6 to 14. The statistical analysis shows that for most of the time points no significant difference could be detected between filtered and artificial smoke processed samples, whether they were treated for 24 or 48 hours. These findings again suggest that a 48 hour treatment does not improve the overall results compared to a 24 hour treatment. Also, the artificial smoke treatment seems to 54

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affect the growth of aerobic bacteria in a similar way as filtered smoke treatment contrasted to the results from other studies which suggest an advantage of the filtered smoke treatment over the artificial smoke treat ment (Danyali 2004; Kristinsson and others 2007). Overall, both the filtered and the artificial smoke treatment, show an effect on the growth of aerobic bacteria on tuna, espe cially before the exponential growth phase is initiated. Furthermore the combined effect of freezing and filtered or artificial smoke seems to prolong the shelf life of tuna fish after thawing by inhibiti ng aerobic bacterial growth during the first 6 days after thawing. Similar findings ha ve been reported by Rawles a nd others (1996) and Demir and others (2004) Figure 6-3. Average amount of CFU/10g of tuna for aerobic plat e count for the fresh storage study. The graph shows the average CFU/10g of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were kept at 4C during the entire time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 20246810121416CFUDays Ctrl FS 24 FS 48 AS 24 AS 48 55

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Figure 6-4. Average amount of CFU/10g of tuna for aerobic plat e count for the fresh storage study. The graph shows the average CFU/10g of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were frozen for 30 days at -20C between day 0 and day 1.The samples were then kept at 4C during the rema ining time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 20246810121416CFUDays Ctrl FS 24 FS 48 AS 24 AS 48 30 days frozen storageTable 6-3. ANOVA results for aerobic plat e count for the fresh storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value 0.85 >0.01 0.08 >0.01 0.86 0.47 0.52 0.52 0.39 0.11 Ctrl a a a a a a a a a a FS 24 a b a b a a a a a a FS 48 a b a c a a a a a a AS 24 a b a bc a a a a a a AS 48 a b a c a a a a a a P-values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other 56

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Table 6-4. ANOVA results for aerobic plat e count for the frozen storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value 0.72 0.28 0.49 >0.01 0.02 0.01 >0.01 0.29 0.06 0.12 Ctrl a a a a a a a a a a FS 24 a a a b b b b a a a FS 48 a a a ab b b c a a a AS 24 a a a b b b c a a a AS 48 a a a b b b c a a a P-values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other Color Analysis The appearance and color of fresh and frozen seafood is one of the major characteristics consumers use to judge the quality of a product. Most of the time the consumer is not able to taste or smell the actual product si nce it is either vacuum packed a nd frozen or it is laid out on a tray behind the counter where the consumer can only visually examine the product. Therefore a fresh color and appearance are of major importance. For fresh Yellowfin tuna a bright red to dark red color is desired for consumer acceptance. Carbon monoxide processing and modified atmospheric packaging have been known and used for a while to preserve and enhance the color properties and especially the redness of fish and seafood pr oducts (Mancini and Hunt 2005; Sorheim and others 2006). For this experiment a Color Machine Vision System was used to analyze the color and color chan ges of the samples during this study. Since the degree of redness (a*-value) is one of the most im portant quality indicators in red muscle seafood, such as tuna, the average a*-value of each sample was determined for each treatment at any time point. Figures 65 and 6-6 show the average a*-values for each tr eatment measured over a period of 14 days for the fresh and frozen study, respectively. ANOVA tests were conducted to detect any significant differences among the treatment means for each obs erved day of the fresh and frozen storage studies. Table 6-5 and 6-6 show the results of the ANOVA tests as well as the results of the Tukeys pair wise comparisons, which were cond ucted to determine which sample means where 57

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different from each other. Reading the table column by column, same letters assigned to sample means show there are no significant differences among these means. The L*-values (lightness) and b*-values (yellowness) were also analyzed and are shown in Figures 6-7 to 6-10. Kristinsson and others (2006a) reported that in several studies the redness of the fish muscle was highly influenced by carbon monoxide but not the lightne ss (L*-values) and the ye llowness (b*-values). Fresh Storage Study Table 6-5 shows that the ANO VA tests detected significant differences among the sample means for all observed days of the fresh storage st udy. It can also be seen that for all observed days, except the first day before any treatment were applied to the samples, the control group means are always significantly different from th e other treatment means, according to Tukeys pairwise comparison. Similar to the frozen stor age study there are signif icant differences among the samples before any treatment were applied, which again can be explained by the way sample steaks were visually sorted and assigned to the treatments, as shown in Figure 6-5. In comparison to the frozen storage study however we were starting clearly at the same a*-value level. It also seems that there is a difference between the 24 a nd 48 hour treatments at this point that can not be explained by the treatments since no treatments were applied at this time. At day 0, directly after the treatments all samples showed an impr ovement in a*-values, even the control group, which could be explained that all samples were transferred directly into the open Ziploc bags after the treatment (the control group too) wher e the are immediately e xposed to the oxygen in the air, compared to the frozen study where the samples were kept in the vacuum bags for the frozen storage. Similar to the frozen storage study, the 48 hour treatments for filtered and artificial smoke showed a grea ter improvement in redness than the 24 hour treatment or the control group. At day 1 after the treatment, the filtered smoke treated samples showed a great improvement in the redness, similar to the frozen storage study, while the artificial smoke treated 58

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samples show no improvement in redness from this point until day 6. Actually, the redness in the artificial smoke treated sample decreased slight ly from day 4 to day 6. The a*-values of the filtered smoke treated samples follow nearly the same pattern as recorded for the frozen storage study. The control group also follows a similar pattern in the development of the a*-values as compared to the frozen study, where there can be seen a decline in redness until day 6 and then a slight increase until day 14. The artificial smoke treated samples seem to follow now a similar pattern where a decline in redness can be from day 0 until day 6 and then a slight increase until day 14. Increased overall variation among the samp le means, indicated by increased standard deviation values, suggests, similar to the frozen storage study, that more factors than just the treatments start to influence the color developmen t of the samples starting from day 6. So far, no reasonable explanation could be found why the ar tificial smoke treatments does not show the same effect as the filtered smoke treatment in the fresh storage study. Still, the improvement in redness among the artificial smoke treated samples exceeds the redness of the control group by far and results in fresh and red looking tuna steaks, as shown in Figure 6-6. The results from the analysis of L*-values (b rightness) and b*-values (yellowness) for the fresh storage study were quite similar to the findi ngs of the frozen storage study (Figures 6-7 and 6-9), where the L*and b*values follow the patte rn of the a*-values and show large increases in brightness and yellowness for all treatments during the first 6 days of observation while the control group stays behind. These findings again c onfirm that there are changes in the L*and b*values affected by the filtered smoke and artificial smoke treatment, contrary to the reports of several other studies (Danyali 2004 ; Garner 2004; Balaban and others 2005), where little effects on L*and b*values were reported. 59

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It should be noted at this poi nt that overall no differences were observed whether a sample was treated for 24 or 48 hours, excep t when analyzed right after th e treatment. It is therefore suggested that a 24 hour treatment of either filtered or artificial smoke should be sufficient to improve the color properties of tuna for fresh or frozen storage. Frozen Storage Study As Table 6-6 indicates, for every observed day, except the last day of the frozen storage study there are significant differences among the treatment means. Furthermore Tukeys test results show that for every observed day (excep t day 14) the control gr oup is significantly different from all other treatments. Surprisingly the control group means are even significantly different from all other treatment means before a ny treatment was applied to any of the samples. This could be explained by a minor flaw in the way the sample steaks were sorted and assigned to the treatments visually prio r to the beginning of this experi ment. However, Figure 6-6 shows clearly that in the overall de velopment of redness (a*-values ) of the samples the initial measurements are very close to each other with re latively small standard deviations resulting in a highly detectable difference among the samples fo r the ANOVA test. It is also clearly visible that the value of the standard deviations increased over time, wh en other factors like microbial spoilage, oxidation and degradation influence the appearance of each sample individually. Therefore, at day 14 no significant differences can be detected among the treatments and the control samples. Immediately afte r treatment Figure 6-6 indicates that the 48 hour treatment with filtered and artificial smoke re sults in higher a*-values then the 24 hour treatment, which is confirmed significant by Tukeys test (Table 6-6). This can be expl ained by the fact that the 48 hour treated samples spent 24 more hours in trea tment prior to the analysis and the carbon monoxide had therefore more time to penetrate into the muscle and form the carboxy-myoglobin complex, which is responsible for the redness of the sample. It is also clearly visible that there 60

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are no significant differences between the filt ered and artificial smoke treatments. Day 1 represents the first day of analys is after the 30 day frozen storag e period, and it is clearly visible that the a*-values, and therefore the redness, of all treatments greatly improved while the control group lacks significantly behind. A small increase can be seen in the control group, possibly to the effect of oxygen exposure of the sample, when the bags where opened for analysis after the thaw-out process. The a*-values for all treatments seem to be stab le for a period of about 4 days of storage at 4C, from which point they slowly decline. At the end of the 14 day study the differences among the samples are so big, indica ted by increased standard deviations, that no significant differences can be detected among an y treatment and the control group. The control groups shows a decrease in their a*-v alue for the first 4 days of the analysis and then a very slow increase, which could be a result of increased spoilage and oxidation, but they never reach the redness as resulted by the treatments. Figure 6-11 shows visual comparison of untreated tuna steaks and filtered smoke treated tu na steaks after 30 days of fro zen storage right after thawing. The differences in color and overall appearance are clearly visible. The control group has a dark grayish appearance whereas the filtered smoke treated samples look nice red and fresh. The L*values (Figure 6-8) show a similar pattern as de scribed for the a*-values above, an effect that was also reported by Balaban and others (2006). The lightness of the 24 hour treated samples (filtered and artificial smoke) increased dramatica lly after the frozen storage, while the 24 hour treatments increased only half as much in lightness and the control groups stoud unchanged. Also the b*-values (Figure 6-10) show a increase in yellowness for the 48 hour treated samples compared to the 24 hour treated samples and the control group. These findings are quite interesting since Kristinsson and others (2006a) reported that no significant changes have been found in several studies among the L*and b*values for carbon m onoxide treated fish. 61

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Table 6-5. ANOVA results for the average a*-values for the fr esh storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 Ctrl a a a a a a a a a a FS 24 b c c c d c bc b b b FS 48 a b b b b b b bc b b AS 24 b d d c d c c c b b AS 48 a b b n c b b bc b b P-values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other Figure 6-5. Average a*-values of the color of tu na samples over 14 days for the frozen storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were kept at 4C during the entire time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 20246810121416a* ValuesDays Ctrl FS 24 FS 48 AS 24 AS 48 62

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Figure 6-6. Average a*-values of the color of tuna samples over 14 days for the fresh storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were frozen for 30 days at -20C between day 0 and day 1.The samples were then kept at 4C during the rema ining time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 2024681012141a* ValuesDays 6 Ctrl FS 24 FS 48 AS 24 AS 48 30 days frozen storageTable 6-6. ANOVA results for the average a* -values for the frozen storage study. Days -1 0 1 2 4 6 8 10 12 14 p-Value >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 0.05 0.12 Ctrl a a a a a a a a a a FS 24 b b b b b b b ab ab a FS 48 b b b b b b b bc ab a AS 24 b c b b b b c bc b a AS 48 b c b b b b bc c ab a P-values smaller then 0.05 indicate a signifi cant difference among the average CFU counts for the specific treatments. Treatment means with the same letter are not significantly different from each other 63

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Figure 6-7. Average L*-values of the color of tuna samples over 14 days for the fresh storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were kept at 4C during the entire time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 25.00 30.00 35.00 40.00 45.00 50.00 2024681012141L* valuesDays 6 Ctrl FS 24 FS 48 AS 24 AS 48 64

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Figure 6-8. Average L*-values of the color of tu na samples over 14 days for the frozen storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were frozen for 30 days at -20C between day 0 and day 1.The samples were then kept at 4C during the rema ining time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 25.00 30.00 35.00 40.00 45.00 50.00 2024681012141L* valuesDays 6 Ctrl FS 24 FS 48 AS 24 AS 48 30 days frozen storage 65

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Figure 6-9. Average b*-values of the color of tuna samples over 14 days for the fresh storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were kept at 4C during the entire time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 0.00 5.00 10.00 15.00 20.00 25.00 2024681012141b* valuesDays 6 Ctrl FS 24 FS 48 AS 24 AS 48 66

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Figure 6-10. Average b*-values of the color of tuna samples over 14 days for the frozen storage study. The graph shows the average a*values of tuna at day -1 (before any treatment was applied), day 0 (right af ter the treatment) and day 1 though day 14. The samples were frozen for 30 days at -20C between day 0 and day 1.The samples were then kept at 4C during the rema ining time of observation. The samples were treated with filtered smoke for 24 (FS24) and 48 (FS48) hours and with artificial smoke for 24 (AS24) and 48 (AS48) hours. The control samples (Ctrl) remained untreated. 0.00 5.00 10.00 15.00 20.00 25.00 2024681012141b* valuesDays 6 Ctrl FS 24 FS 48 AS 24 AS 48 30 days frozen storage Figure 6-11. Tuna steaks after 30 days of frozen storage at -2 0C. A) Shows the untreated control sample. B) Shows the filtered smoke treated sample. C) Shows the artificial smoke treated sample 67

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Sensory Taste Panel The sensory characteristics of a product such as appearance, smell and taste are very important factors for consumers to make their de cision to buy a product. Since the use of filtered and artificial smoke had similar results in thei r effect on color and appearance, as well as microbial growth of the samples, the task was no w to verify if there are any human detectable differences in smell and appearance among filtere d and artificial smoke treated samples. Four triangle tests were conducted and 60 random untrained panelists per te st were asked if they could identify the odd sample by odor (first and second triangle tests) and by appearance (thirds and fourth triangle tests). The desi gn, execution and analysis of thes e triangle tests were conducted with the Compusense Software. Table 6-7 shows th e results of all four tr iangle tests, where the actual number of correct answers is compared to the number of correct answers necessary to establish a level of significance at 5%. This means that a lower number of correct answers indicate that there are no significant differences detectable in a human sensory panel between the treatments. It can be seen that for all four te sts no significant differences have been detected between filtered and artificial smoke treated sa mples in odor and appearance for the fresh and frozen storage studies. All samples were prepar ed in the same manner and presented to the panelists immediately after trea tment or after thawing. All sa mples were treated for 48 hours since a longer treatment sugge sted a greater effect on colo r and odor from preliminary experiments. To gain additional information, each panelist who identified the odd sample correctly was asked to note any comments he or she might have about the differences among the samples that led to their decision of choosi ng the odd sample. Tables A-5 to A-8 in Appendix A show all comments given by the panelists for all four tests. For the color analysis, most of the panelists described the filtered smoke sample to appear darker and more red then the artificial smoke 68

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sample for the fresh storage study, while nearly equal number of panelists describe either the artificial smoked or the filtered smoked sample as lighter in color for the frozen storage study. This confirms in part the findings of the co lor study, where for the fr ozen storage study no significant differences were found at day 1 between the filtered and artificial smoke treated samples. The samples for the color studies and th e taste panel analysis were obtained separately from each other at different time points and also treated separately and at different times. Comments regarding the odor triangle tests show that an equal amount of people noticed a stronger smell in either the filtered smoke or the artificial smoke treated samples for both the fresh and frozen storage studies. It is therefore inconclusive wh ether a particular smell or odor was responsible for these panelists to decide whic h the odd sample was. It should be noted at this point that only the panelists who identified the odd sample co rrectly were asked to comment their decision and that there is a possibility that some of th ese panelists found the odd sample by chance and not based on a real difference. Th e Compusense analysis software includes the possibility of a right answer by guessing in thei r statistical analysis. Also the demographical variety was limited to students from the University of Florida between the ages of 18 to 24 years. Table 6-7. Results of the four taste panel triangle tests. Tests 1 2 3 4 Total number of panelists 60 60 60 60 Number of correct answers needed 27 27 27 27 Actual Number of correct answers 23 26 19 19 Significance (p-Value) 0.244 0.068 0.654 0.654 Number 1 represents the odor te st with frozen samples, number 2 the odor test with fresh samples, number 3 the color test with frozen sa mples and number 4 the color test with fresh samples. GC-Analysis Carbon monoxide is known to preserve and enhance the color prope rties of red muscle foods and is used in several applications t oday. Small quantities of carbon monoxide (> 0.5%) are used today in modified atmospheric packaging to preserve the redness of fresh meats, such as 69

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ground beef (Sorheim and others 1999; Kusmider and others 2002; Hunt and others 2004) and pork sausages (Sorheim and others 2001; Martinez and others 2005; Sorheim and others 2006). A short term treatment with high er percentages of carbon monoxide (up to 100%) is used in the seafood industry to retain the colo r of certain seafoods prior to frozen storage. However, carbon monoxide is not a brand new comp onent in the process of preserving meats and seafood. Natural wood smoke, produced by the incomplete combusti on of wood chips, always contains a certain amount of carbon monoxide, which is applied to the product during the smoking process. During cold smoking, a process where the product is kept at a low temperature (usually below 15C) while the smoke is applied, the pr oduct is not cooked and therefor e retains its color and texture properties on the inside. The outside of the product usually turns into a sli ght brown to grey color because of solid particles of the smoke that condense on the product during smoking. Filtered wood smoke is a relatively new invention where th ese particles and most of the odor and flavor components are filtered out and therefore don t condense onto the product. According to the analysis of the manufacturer the main components of the filtered smoke us ed in this experiment are carbon monoxide (21%), carbon dioxide (1 8%), oxygen (1.1%) and a balance of nitrogen. However, filtered smoke contains many more components that have not yet been identified. Since the production of filtered wood smoke can be a complicated and costly process, a cheaper alternative is a treatment where the menti oned gas components are mixed together from commercially available gasses and used in the same way as the filtered smoke. For this reason we called this mixture artificial smoke (AS). As the previous studies showed it had a similar effect on the color properties, sens ory characteristics and microbial gr owth as the original filtered smoke. To differentiate whether a product was treat ed with the original filtered smoke or the artificial smoke mixture, a rapid gas chromat ography method was developed that focuses mainly 70

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on the identification and quantification of other gaseous components which are present in filtered smoke but not in artificial smoke. Identification of Gaseous Components in Filtered and Artificial Smoke The fist step in the development was to identify the gaseous components in filtered smoke and artificial smoke and devel op a standard curve for each of these components with the GCMethod mentioned in the chapter Materials and Methods. Filtered smoke was injected into the GC and 5 peaks could be verified with retention times less than five minutes. Two of these peaks (retention times around 0.49 minutes and 1.52 minut es) were known already from previous experiments as carbon monoxide and carbon dioxide respectively. The other three peaks were not identified yet. It was also known from pr evious experiments that only low molecular hydrocarbons will be eluted from the GC column in less than five minutes. It was therefore decided to use refinery gas standard from Ag ilent technologies, whic h is a mixture of low molecular weight hydrocarbons (like methane, ethyl ene, ethane, propane and so on), to identify the three unknown components found in filtered smoke. It should be noted at this poi nt that there are most likely many more gaseous components contained in filtered smoke, but with retention times much longer than 30 minutes in this particular GC setup and ther efore do not contribute to a rapid identification method. With a comparison of the filtered smoke and the gas standard chromatograms the three unknown components could be identified as methan e, ethylene and ethane respectively in the order of their elution. The second step was now to obtain these five gases found in filtered smoke in the highest purities available and produce standard curves for each of them to quantify them in the filtered smoke, artificial smoke and the treated samples. Fi gures 6-12 to 6-16 show these standard curves and display the resulting equations that were us ed to quantify the amount of each gas, based on 71

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and injection volume of 100 l a nd their respective peak area. Now the five components found in filtered smoke with this method could be identifi ed and quantified. As a comparison the artificial smoke and a filtered smoke (FS-B), which wa s obtained from a different company, were analyzed with this method. Table 6-8 shows the pe rcentage of each of these five gasses found in filtered smoke (FS), filtered smoke B (FS-B) and artificial smoke (AS). Since it is was clear that the artificial smoke lacks any me thane, ethylene and ethane, the next step was to experiment whether any of these five gasses can also be found absorbed in the actual products. Table 6-8. Percentage of gaseous components in the treatment gasses. Treatment Gas Filtered smoke A Filtered smoke B Artificial Smoke Carbon Monoxide 22.53 11.00 22.20 Methane 9.84 2.85 0.00 Carbon Dioxide 14.32 12.55 19.43 Ethylene 0.94 0.14 0.00 Ethane 0.17 0.20 0.00 Figure 6-12. Carbon monoxide standard curve. This graph shows the average peak area for the injection of different quan tities of carbon monoxide. Th e displayed equation results from the linear regression of the displayed da ta can be used to calculate the amount of carbon monoxide in a 100l samp le based on the peak area y = 0.0002002x + 0.0003965 R = 0.9999182 0.000 0.200 0.400 0.600 0.800 1.000 1.200 0100020003000400050006000l Carbon Monoxide/100 lPeak Area [pA] 72

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Figure 6-13. Methane standard curve. This graph shows the average peak area for the injection of different quantities of methane. The displayed equation results from the linear regression of the displayed data can be used to calculate the am ount of methane in a 100l sample based on the peak area y = 0.0003414x 0.0006153 R = 0.9999588 0.000 0.200 0.400 0.600 0.800 1.000 1.200 0500100015002000250030003500l Methane/100 lPeak Area [pA] Figure 6-14. Carbon dioxide standard curve. This graph shows the average peak area for the injection of different quan tities of carbon dioxide. The displayed equation results from the linear regression of the displayed da ta can be used to calculate the amount of carbon dioxide in a 100l sample based on the peak area y = 0.0001941x + 0.0003750 R = 0.9999415 0.000 0.200 0.400 0.600 0.800 1.000 1.200 0100020003000400050006000l Carbon Dioxide/100 lPeak Area [pA] 73

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Figure 6-15. Ethylene standard curve. This graph shows the average peak area for the injection of different quantities of ethylene. The displayed equation results from the linear regression of the displayed data can be used to calculate the amount of ethylene in a 100l sample based on the peak area y = 0.0000994x + 0.0030171 R = 0.9999063 0.000 0.200 0.400 0.600 0.800 1.000 1.200 020004000600080001000012000l Ethylene/100 lPeak Area [pA] Figure 6-16. Ethane standard curv e. This graph shows the averag e peak area for the injection of different quantities of ethane. The disp layed equation results from the linear regression of the displayed data can be used to calculate the amount of ethane in a 100l sample based on the peak area y = 0.0000995x + 0.0020152 R = 0.9997862 0.000 0.200 0.400 0.600 0.800 1.000 1.200 020004000600080001000012000l Ethane/100 lPeak Area [pA] 74

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Analysis of Treated Samples The last step in the development of the ra pid GC identification method was to find a way to actually determine whether a product was treated with filtered smoke or just a mixture of carbon monoxide and carbon dioxide, called artificia l smoke. Previous expe riments showed that when a certain amount of treated muscle tissue is heated at 100C for about 5 minutes in a sealed vial, hydrocarbons that were absorbed into the tissue during the treatment will get released into the headspace of the vial where the can be samp led with a syringe and th en analyzed with the GC. Samples were taken from each treatment at day 2 after treatment and analyzed with the procedure described in Material and Methods. Figures 6-17 to 6-24 di splay the chromatograms obtained by this analysis that represent the filtered smoke and artificial smoke treated samples as well as well as the control samples. Figure 6-21 shows the chromatogram of pure filtered smoke, directly analyzed with the GC. By comparing th ese figures it is obvious that in the headspace from artificial smoke treated samples the peaks for methane, ethylene and ethane are missing, as well as in the control samples. The filtered smoke treated samples show the peaks for carbon monoxide, methane, carbon dioxide and ethylene in th e order they were eluted from the column respectively. Only the ethane peak is missing from the filtered smoke treated samples as well. A possible explanation for the missing ethane peak in the filtered smoke treated sample could be that ethane is not as well absorbed as other hydrocarbons or not as easily released by the sample. Nevertheless all chromatograms from the artifi cial smoke treated samples showed the same results as well as did the filtered smoke trea ted among each other and the control samples. Finally filtered smoke treated frozen tuna was obtained from a local et hnic grocery store and tested the same way. The chromatogram of this sa mple is shown in figure 6-20. Note that for all treatments several samples were obtained and an alyzed with the GC method. For every sample two chromatograms were obtained and analy zed to ensure the method works accurately. 75

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76 Figure 6 Figure 6 17. Chrom a smoke. T represent s represent i 18. Chrom a The first p methane, ethylene. compoun d a togram of t T he first pea k s carbon dio i ng compou n a togram of t p eak from l e the third pe a The area u n d t he headspa c k from left r e xide. The a r n d. t he headspa c e ft represent s a k represent n der a peak i s c e analysis o e presents c a r ea under a p c e analysis o s carbon m o s carbon di o s proportio n o ver a sampl e a rbon mono x p eak is prop o o ver a sampl e o noxide, the o xide and th e n al to the qu a e treated wi t x ide and the o rtional to t h e treated wi t second pea k e last peak r e a ntity of the t h artificial second pea k h e quantity o t h filtered s m k represents e presents representin g k o f the m oke. g

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77 Figure 6 Figure 6 19. Chrom a first peak carbon di o represent i 20. Chrom a store. T h represent s represent s represent i a togram of t from left re p o xide. The a i ng compou n a togram of t h e first peak f s methane, t h s ethylene. T i ng compou n t he headspa c p resents car b a rea under a n d. t he headspa c f rom left r e p h e third pea k T he area un d n d. c e analysis o b on monoxi peak is pro p c e analysis o p resents car b k represents d er a peak is o ver an untr e de and the s p ortional to t o ver a sampl e b on monoxi d carbon dio x proportiona e ated contro l econd peak t he quantity e purchase d d e, the seco n x ide and the l to the qua n l sample. T h represents of the d at a local e t n d peak last peak n tity of the h e t hnic

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78 Figure 6 Figure 6 21. Chrom a left repre s represent s represent s represent i 22. Chrom a from left r peak repr e represent s represent i a togram of t s ents carbo n s carbon dio s ethane. Th i ng compou n a togram of t r epresents c e sents carb o s ethane. Th i ng compou n t he injection n monoxide, xide, the fo u e area unde r n d. t he injection arbon mono o n dioxide, t h e area unde r n d. of 50 l of p the second p u rth peak re p r a peak is p r of 50 l of p xide, the se c h e fourth pe a r a peak is p r p ure filtere d p eak represe p resents eth y r oportional t p ure filtere d c ond peak r e a k represen t r oportional t d smoke. T h nts methan e y lene and th e t o the quanti t d smoke B e presents m e t s ethylene a n t o the quanti t h e first peak e the third p e e last peak t y of the The first p e thane, the t h n d the last p t y of the from e ak p eak h ird p eak

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79 Figure 6 23. Chrom a from left r The area u Figure 62 surround i from the i represent s represent i a togram of t r epresents c u nder a pea k 2 4. Chrom a i ng lab envi r i njection an d s carbon dio i ng compou n t he injection arbon mono k is proporti o a togram of t h r onment. T h d a very sm a xide. The a r n d. of 50 l of p xide and th e o nal to the q h e injection o h e first two s a ll amount o f r ea under a p p ure artifici a e second pe a q uantity of t h o f 50 l of a s mall peaks f f carbon mo n p eak is prop o a l smoke. T a k represent s h e represent i a ir sampled f f rom left re p n oxide. The o rtional to t h T he first pea k s carbon dio x i ng compou n f rom the p resent nois e third peak h e quantity o k x ide. n d. e o f the

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CHAPTER 7 SUMMARY AND CONCLUSIONS Smoking of meats and seafood has been a pres ervation method for a long period of time, although in the recent century it became more a flavoring agent than a necessity in order to preserve foods. From the early days, when the pr oducts were mainly cooked and dried, it became a specialized process that induces just the right amount of flavor in sophisticated processes. Today it is possible to smoke products at nearly 0C and in the right humidity, so that the product does not differ in texture or appearance from a fresh pr oduct other then by its odor and taste. However, smoke can do more than that. Th e next development of smoking food products is called filtered wood smoke processing, a proce ss that uses modern technology to clarify and filter the naturaly produced smoke and high tech processing to apply this filtered smoke to the product at the lowest possible temp erature to retain a product that is nearly unchanged in texture and odor compared to its untreated fresh counter part. From the beginning it was believed that filtered wood smoke has the potential to prolong the shelf life of fresh and frozen muscle foods and to enhance their appearance and color prope rty. Today it is known that the color retention properties of filtered smoke relies on one of the main ingredients in filtered wood smoke, carbon monoxide, a potentialy hazardous gas for humans and animals that replaces the oxygen in the blood and muscle tissue and causes muscle relaxation and asphyxiation. However, just this effect makes it very interesting and attractive for the meat and seafood industry, since the new complex that is formed in this process result s in a stable red co lor in these foods. Filtered wood smoke is produced by burning wood chips at a high temperature without oxygen to produce smoke that is then filtered and concentrated in compressed gas cylinders. To compare if a mixture of the major gaseous compon ents in filtered smoke would achieve the same 80

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effect, artificial smoke was prepared from industr ially available gases an d used also in this project. It is shown during this projec t that filtered and artificial sm oke processing did not affect the growth or inhibition if Salmonella spp. as they seem to be more affected by their competitive microbial environment. No Salmonella spp. growth was reported at all in non-inoculated samples, which raises the question whether it is a r eal threat in fresh tuna or not. There was some inhibition effect of filtered and artificial smoke on the growth of aerobic spoilage bacteria during the first 4 to 6 days of storage, however it is suggested by the author that this effect does not enhance the shelf life of the produc t dramatically in this particular study. However, the combined effect of filtered and artificial smoke processing and subsequent frozen storage at -20C leads to a greater inhibition of aerobic spoilage bacteria then was obs erved in an untreated frozen product. The main effect observed in this study for filtered and artificial smoke processing is their ability to preserve and impr ove the color properties and appearance of red muscle foods as shown in the color experiment in this project. Af ter the visual observation of a piece of tuna that was frozen in a vacuum bag without any treatmen t compared to a filtered or artificial smoke treated product it is very clear which product look s more red and fresh. The filtered and artificial smoke treated products have clearl y an advantage over control. During the whole study filtered smoke and ar tificial smoke treatments were always compared side by side and no significant differen ces were detected in any of the mentioned studies between filtered and artificial smoke treated samples. A consumer can not differentiate whether a product was treated with filtered or artificial smoke according to the taste panel results, based on either odor or appearance. 81

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All these finding raise the final question what is really the difference between filtered smoke and artificial smoke. Why would we use f iltered smoke or the less expensive artificial version? Artificial smoke contains industrial carbon monoxide which is regulated by many countries in the world in its use as a food i ngredient or additive. But filtered smoke, although containing the same carbon monoxide is considered a natural product, based on a long history of food processing with wood smoke in most of th ese countries. Since it is not obvious just by visual or sensorial inspection whether a product wa s treated with the original filtered smoke or its artificial counterpart the rapid GC identification method wa s developed to detect in a simple way whether a product was treated with filtered smoke or just with carbon monoxide. The results show that it is possible to identify a filtered smoke treated product based on the presence of three additional components that are not found in the ar tificial smoke mixture. The rapid method also allows the quantification of the residual amount of carbon monoxide in the muscle tissue when a standard curve for the quantif ication of carbon monoxide was es tablished. It has to be acknowledged that an in dividual, who wants to deliberately misguide the consumer about the products treatment, could do so by adding all the ingredients into the ar tificial smoke that are tested by the rapid identification method. But this is a question of risk for every consumer and producer. 82

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APPENDIX A TASTE PANEL DEMOGRAPHICS AND COMMENTS Table A-1. Comments from the Panelists for the Fresh Odor Triangle Test N ame Treatment Comments Panelist 2 Filtered Smoke this smell has a stronger smell Panelist 8 Filtered Smoke had a stronger aroma than the rest of them. Panelist 10 Filtered Smoke Smelled the same as 564\Smelled the same as 262 Panelist 13 Filtered Smoke least fishy smell Panelist 17 Filtered Smoke smells like #546\smells like #262 Panelist 19 Filtered Smoke less scent Panelist 24 Filtered Smoke same as the first one.\It smells odd like it was dead a week ago. Panelist 25 Filtered Smoke did not have as strong of a fishy smell as the other two Panelist 26 Filtered Smoke musky Panelist 27 Filtered Smoke Lighter smell Panelist 32 Filtered Smoke smells raw Panelist 37 Filtered Smoke not as strong of an odor Panelist 41 Filtered Smoke very little scent\little scent Panelist 44 Filtered Smoke This sample smells stronger than the other two. Panelist 46 Filtered Smoke It smelled very strong as well.\This sample smelled very strong. Panelist 48 Filtered Smoke smelled unfresh \, like paint\smelled unfresh, like paint Panelist 52 Filtered Smoke Smells like 546\seems the same as 262 Panelist 56 Filtered Smoke It had a stronger/heavier smell Panelist 57 Filtered Smoke strong and smelled fatty Panelist 59 Filtered Smoke ha rsh smelling\more smelly Panelist 2 Artificial Smoke this smell is similar to the second one\smells like saltwater Panelist 8 Artificial Smoke smelled same as the 927 sample.\it really didn't smell like fish Panelist 10 Artificial Smoke Smelled worse (more fishy) Panelist 12 Artificial Smoke smells less fishy Panelist 13 Artificial Smoke fishy\strongest smell Panelist 17 Artificial Smoke sample #927 has a more potent smell, it smells saltier too. Panelist 25 Artificial Smoke very strong fishy smell\very strong fishy smell Panelist 32 Artificial Smoke smells putrid\bitter scent Panelist 37 Artificial Smoke strong smell\strong smell Panelist 41 Artificial Smoke more fishy odor Panelist 43 Artificial Smoke The same as the previous\Smells fresh out of the sea Panelist 44 Artificial Smoke This sample smells similar to 927. \This smells similar to 347. Panelist 46 Artificial Smoke The smell of this sample wasn't as strong as the other two. Panelist 48 Artificial Smoke smelled fresher, didnt smell as much like paint Panelist 52 Artificial Smoke The scent is less stronger Panelist 59 Artificial Smoke has an odor of salmon Only panelists who identified the odd sample correct were asked to leave comments. Numbers 546 and 262 refer to the filtered smoke treated sample. Numbers 347 and 927 refer to the artificial smoke treated sample. Sa mples were not previously frozen. 83

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Table A-2. Comments from the Panelists for the Frozen Odor Triangle Test N ame Treatment Comments Panelist 2 Filtered Smoke smells cooked\sm ells cooked Panelist 3 Filtered Smoke This one was the same as number 466 and did not smell as strong and fishy as number 173\This one and number 882 both smelt fishy but the odor was not as strong and disgusting as number 173 Panelist 5 Filtered Smoke can't really smell anything Panelist 8 Filtered Smoke it also sme lled like chicken.\it smelled like chicken. Panelist 9 Filtered Smoke 882 smelled like 466\It smelled like fish, and t was a bad smell. Panelist 11 Filtered Smoke not as strong as the others Panelist 12 Filtered Smoke smells less fishy Panelist 13 Filtered Smoke more like crab\smells more like crab Panelist 14 Filtered Smoke p uungent smell like it had gone bad\ subtle smell like canned tuna Panelist 15 Filtered Smoke similar to 173\scent not as strong Panelist 18 Filtered Smoke one of them smells less fishy than the other two. Panelist 20 Filtered Smoke this one smells like 466\this one smells very bitter Panelist 24 Filtered Smoke It has a distinc odor which can be smelled from other stink fish but it was kind of lighter smell than the first ones. Panelist 26 Filtered Smoke smells not as fresh\smells not as fresh Panelist 29 Filtered Smoke Smells salty Panelist 30 Filtered Smoke Doesnt smell as strong Panelist 33 Filtered Smoke same as 466\different smell Panelist 35 Filtered Smoke smelled very plain Panelist 36 Filtered Smoke stronger odor Panelist 40 Filtered Smoke weakest scent Panelist 43 Filtered Smoke Same as the first one\It doesnt even smell like tuna Panelist 47 Filtered Smoke N o fishy smell Panelist 50 Filtered Smoke no strong odor\no strong odor Panelist 53 Filtered Smoke not sure. th ere was a slight smell of saltyness. Panelist 54 Filtered Smoke it has a lighter smell. Panelist 59 Filtered Smoke harsh smell, rot Panelist 2 Artificial Smoke smells like it went bad Panelist 3 Artificial Smoke This one smelt extra fishy Panelist 5 Artificial Smoke has a smell\has a smell Panelist 8 Artificial Smoke smelled like there was some sort of chemical on the fish. Panelist 9 Artificial Smoke 792 did not really have much of a smell, as opposed to the other ones that did have a smell. It did not really smell like fish at all. Panelist 11 Artificial Smoke very strong\very strong Panelist 12 Artificial Smoke hardly has a smell\hardly smells like anything Panelist 13 Artificial Smoke didn't smell that much Panelist 14 Artificial Smoke exactly the same as 466 Panelist 15 Artificial Smoke strongest smell Panelist 18 Artificial Smoke smells very fishy\it smells fishy Panelist 20 Artificial Smoke this one smells sweeter Panelist 26 Artificial Smoke smells very fishy Panelist 29 Artificial Smoke Does not have a strong odor\Does not have a strong odor Panelist 30 Artificial Smoke smells stronger\smells stronger Panelist 33 Artificial Smoke different smell Panelist 35 Artificial Smoke barely smelled at all\strong smell Panelist 36 Artificial Smoke weaker odor\weaker odor 84

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Table A-2. Continued N ame Treatment Comments Panelist 40 Artificial Smoke most 'fishy'\mild scent Panelist 43 Artificial Smoke Smells rotted. Panelist 47 Artificial Smoke fishy smell\fishy smell Panelist 50 Artificial Smoke it has more odor Panelist 53 Artificial Smoke smelled a little stronger than the other one Panelist 54 Artificial Smoke not as strong as 466.\it has more of the typical fish smell. Panelist 24 Artificial Smoke Smells not that bad like the second\I couldn't figure out its smell. Only panelists who identified the odd sample correct were asked to leave comments. Numbers 882 and 446 refer to the filtered smoke treated sample. Numbers 792 and 173 refer to the artificial smoke treated sample. Sa mples were not previously frozen. Table A-3. Comments from the Panelists for the Fresh Color Triangle Test N ame Treatment Comments Panelist 6 Filtered Smoke darker\darker Panelist 7 Filtered Smoke Sample 119 ha d thicker veins than the other two. Panelist 8 Filtered Smoke much darker than the other two Panelist 16 Filtered Smoke more red\more red Panelist 23 Filtered Smoke the lines in it were different.\the lines in it were different. Panelist 24 Filtered Smoke dark\dark Panelist 25 Filtered Smoke darker Panelist 27 Filtered Smoke Aside from seeming harder, sample 119 seems darker as well. Panelist 29 Filtered Smoke 119 is darker than 511, but just as dark as 730.\730 is darker than 511, but just as dark as 119. Panelist 33 Filtered Smoke This sample seems sliced differently than the other two samples. Panelist 34 Filtered Smoke This sample looks the best\There are thinner lines on this sample Panelist 35 Filtered Smoke Looks exactly like 119.\they are all the same color, but sample 119 and 730 are the same size and have the sa me amount of fat content and the same sort of lines in them (the grain of the meat). Panelist 41 Filtered Smoke This has a darker color.\T his has a darker color. Panelist 44 Filtered Smoke lighter Panelist 46 Filtered Smoke There is a slight indentation in this sample that looks similar to that of sample 730, just not as long.\There is one barely noticeable line going through it. The color is very similar to that of sample 119. Panelist 47 Filtered Smoke Identical to 730, darker and more grainy than 378\Identical to 119, darker and more grainy than 378 Panelist 48 Filtered Smoke Identical to 730.\Identical to 119. Panelist 51 Filtered Smoke Didn't have the define d serrations the other two samples had, also seemed lighter. Panelist 54 Filtered Smoke Solid red/pink color\Similar to 730 with solid pink/red color Panelist 58 Filtered Smoke \ Panelist 6 Artificial Smoke it's lighter in color Panelist 7 Artificial Smoke 378 l ooked liked 511\511 looked like 378 Panelist 8 Artificial Smoke grainier\grainier and lighter Panelist 16 Artificial Smoke p inker Panelist 23 Artificial Smoke The lines in it were different. Panelist 25 Artificial Smoke lighter\lighter Panelist 27 Artificial Smoke Sample 378 appears more tender and soft than 119.\Sample 511 appears more tender than 119. 85

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Table A-3. Continued N ame Sample Comments Panelist 29 Artificial Smoke it is the only pink piece (the lightest) Panelist 33 Artificial Smoke This sample has more uniformity than the other in its appearance.\It has a semi-circle shape line on its top. Panelist 34 Artificial Smoke Sample 378 seems to have two white streaks running through it which look like fat and, and therefore are unappetizing. Panelist 35 Artificial Smoke This sample is smalle r than the other two and has more fat in the meat. You can see it in the grain. Panelist 41 Artificial Smoke This sample has a brighter color. Distinguishable. Panelist 46 Artificial Smoke this sample has four visible lines going through it diagonally. The color is also slightly darker than sample 730. Panelist 47 Artificial Smoke Lighter and less grainy than 730 and 119 Panelist 48 Artificial Smoke Significantly lighter and pinker than the other samples. Panelist 51 Artificial Smoke Had defined serrations/grooves in it.\Had defined serrations/grooves in sample. Panelist 54 Artificial Smoke Striped appearance Only panelists who identified the odd sample correct were asked to leave comments. Numbers 119 and 730 refer to the filtered smoke treated sample. Numbers 378 and 511 refer to the artificial smoke treated sample. Sa mples were not previously frozen. 86

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Table A-4. Comments from the Panelists for the Frozen Color Triangle Test N ame Treatment Comments Panelist 4 Filtered Smoke 416 has striations on it that none of the other 2 have. Panelist 17 Filtered Smoke light er shade and luminous glow Panelist 22 Filtered Smoke it was lighter in color and had less lines Panelist 23 Filtered Smoke the lines in it were different Panelist 26 Filtered Smoke this samples\ seems layedand the gr ain is similar to sample 419\this sample is more smooth with less of vertical grain Panelist 27 Filtered Smoke 416 a ppears tender and soft, much lighter than 614.\822 appears tender and light. Panelist 28 Filtered Smoke this one seems to be more red, a bit darker Panelist 29 Filtered Smoke 416 is the li ghtest, it has little white strands in it. Panelist 33 Filtered Smoke It seems the same a sample 822.\i t seems the same as sample 416. Panelist 36 Filtered Smoke 416 seems to look sinewy and less like gelatin, but the difference is small Panelist 46 Filtered Smoke This piece has indentation in it and th e shape is different than the first two. It also has visible white lines.. Panelist 49 Filtered Smoke a dent on the surface\a dent in the surface Panelist 50 Filtered Smoke 3 cuts. pinkish\3 cuts and pinkish Panelist 54 Filtered Smoke Pretty smooth appearance Panelist 56 Filtered Smoke its quite coarse\quite flat with little marks Panelist 58 Filtered Smoke this sample had fe wer white lines and looked less 'defined' than the typical tuna steak cell structure Panelist 59 Filtered Smoke Lighter color, also seems to have less fatty tissue. Panelist 4 Artificial Smoke N o striations.\No striations. Panelist 17 Artificial Smoke looks like the other\resembles 294 Panelist 22 Artificial Smoke had lines\had lines Panelist 23 Artificial Smoke the lines in it were different\the lines in it are different Panelist 26 Artificial Smoke the filet seems lik e the grain is more vertical then horizontal Panelist 27 Artificial Smoke 614 seems again not as soft and tender as the other samples. It also appears a little bit darker. Panelist 29 Artificial Smoke same as 614\same as 294 Panelist 33 Artificial Smoke IT has whit division lines differently than the other two. Panelist 36 Artificial Smoke text ure is slightly different, but still similar to 614\very cleanly cut and gelatin like Panelist 38 Artificial Smoke This sample does not contain any white areas or lines where the other two samples do. It appears uniform in color and texture. Panelist 44 Artificial Smoke lighter Panelist 46 Artificial Smoke Square piece that is thinner than 614 but the same color.\Square piece that is pink. Panelist 49 Artificial Smoke smooth texture Panelist 50 Artificial Smoke more salmon color Panelist 54 Artificial Smoke Striped and a bit jagged\White stripes Panelist 56 Artificial Smoke its more smoother than the rest Panelist 58 Artificial Smoke This one looked about the same compared to sample 614\This sample looked about the same as sample 294 Panelist 59 Artificial Smoke darker color, same as 614\Darker color, same as 294 Only panelists who identified the odd sample correct were asked to leave comments. Numbers 416 and 822 refer to the filtered smoke treated sample. Numbers 614 and 294 refer to the artificial smoke treated sample. Samples were previously frozen. 87

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Table A-5. Demographic Variance of the Odor Triangle Tests Age Range Under 18 18-20 21-24 Over 24 Total Female 2 33 3 2 40 Male 0 14 6 0 20 Total 2 47 9 2 60 Note: The demographic variance for the fresh and frozen storage odor tests are the same. Table A-6. Demographic Variance of the Color Triangle Tests Age Range Under 18 18-20 21-24 Over 24 Total Female 1 31 3 1 36 Male 0 15 7 2 24 Total 1 46 10 3 60 Note: The demographic variance for the fresh an d frozen storage color tests are the same. 88

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APPENDIX B COLOR STUDY PICTURES Figure B-1. Control group images for the frozen storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased (here no treatment control group). c) Image of samples after 30 days of frozen storage (-2 0C) followed by 14 days of refrigerated storage (4C). Figure B-2. Filtered smoke (24h-treatment) group im ages for the frozen storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 30 days of frozen storage (-20C) fo llowed by 14 days of refrigerated storage (4C). Figure B-3. Filtered smoke (48h-treatment) group images for the frozen storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 30 days of frozen storage (-20C) fo llowed by 14 days of refrigerated storage (4C). 89

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Figure B-4. Artificial smoke (24h-tr eatment) group images for the frozen storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 30 days of frozen storage (-20C) fo llowed by 14 days of refrigerated storage (4C). Figure B-5. Artificial smoke (48htreatment) group images for th e frozen storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 30 days of frozen storage (-20C) fo llowed by 14 days of refrigerated storage (4C). Figure B-6. Control group images for the fresh storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased (here no treatment control group). c) Image of samples after 14 days of re frigerated storage (4C). 90

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Figure B-7. Filtered smoke (24h-treatment) group images for the fresh storage study captured by the CMVS. a) Image of samples before tr eatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 14 days of refrigerated storage (4C). Figure B-8. Filtered smoke (48h-treatment) group images for the fresh storage study captured by the CMVS. a) Image of samples before tr eatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 14 days of refrigerated storage (4C). Figure B-9. Artificial smoke (24htreatment) group images for th e fresh storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 14 days of refrig erated storage (4C). 91

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Figure B-10. Artificial smoke (48h-treatment) gr oup images for the fresh storage study captured by the CMVS. a) Image of samples before treatment was applied. b) Image of samples immediately after treatment was applied and ceased. c) Image of samples after 14 days of refrig erated storage (4C). 92

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LIST OF REFERENCES Balaban MO, Kristinsson HG, Olson B. 2006. Color Enhancement and Potential Fraud in Using CO. In: Otwell WS, Kristinsson HG, Balaba n MO, editors. Modified atmospheric processing and packaging of fish : filtered smokes, carbon monoxide, and reduced oxygen packaging. 1st ed. Ames, Iowa: Blackwell Pub. p 127-40. Balaban MO, Kristinsson HG, Otwell WS. 2005. Evaluation of color parameters in a machine vision analysis of carbon monoxide -treated fish Part I: Fr esh tuna. Journal of Aquatic Food Product Technology 14(2):5-24. Balaban MO, Luzuriaga DA. 2001. Measuring Colo r in Foods. Machine vision system provides program for analysis. Resource, Engineer ing,and Technology for a Sustainable World 8(8):10-1. Bhaduri S, Cottrell B. 2004. Survival of cold-s tressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage. Appl Environ Microbiol 70(12):7103-9. Bledsoe GE, Oria MP. 2001. Potential hazards in cold-smoked fish: parasites. J Food Sci 66(7):S1100-3. Burgess GHO, Bannerman AM. 1963. Fish Smoking, A Torry Kiln Operators' Handbook. In: Station TR, editor: Ministry of Ag riculture, Fisheries and Food, UK. Burt JR. 1988. Fish smoking and drying : the eff ect of smoking and dr ying on the nutritional properties of fish. New York, NY: Elsevier Applied Science Publishers. xii, 166 p. Butt AA, Aldridge KE, Sanders CV. 2004. Infections related to the ingesti on of seafood Part I: viral and bacterial infecti ons. The Lancet Infectious Diseases 4(4):201-12. Creager JG, Black JG, Davison VE. 1990. Microbio logy principles and a pplications. Englewood Cliffs, N.J.: Prentice Hall. Cutting CL. 1961. Historical aspects of fish. In: Borgstrom G, editor. Fish as food. New York,: Academic Press. p v. Danyali N. 2004. The Effect of Carbon of Monoxide and Filtered Smoke on Quality and Safety of Yellowfin Tuna. Gainesvill e: University of Florida. Demir N, Kristinsson HG, Balaban MO, Otwell WS. 2004. Quality changes in mahi mahi (Coryphaena hippurus) f illets treated by different carbon monoxide concentrations and filtered smoke as assessed by color m achine vision and lipid oxydation. IFT Annual Meeting. New Orleans, LA: In stitute of Food Technologist. Dillon R, Patel TR, Martin AM. 1994. Microbiol ogical control for fish smoking operations. In: Martin AM, editor. Fisheries processing : biotechnological applications. 1st ed. London ; New York: Chapman & Hall. p xiv, 494. 93

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Fell G, Hamouda O, Lindner R, Rehmet S, Lieseg ang A, Prager R, Gericke B, Petersen L. 2000. An outbreak of Salmonella blockley infecti ons following smoked eel consumption in Germany. Epidemiology a nd Infection 125(1):9-12. Flick GJ, Oria MP, Douglas L. 2001. Potential h azards in cold-smoked fish: biogenic amines. J Food Sci 66(7):S1088-99. Garner K. 2004. The Effect of Carbon Monoxide on Muscle Quality of Spanish Mackerel. Gainesville: University of Florida. Gram L. 2001a. Potential hazards in cold-smoked fish: Clostridium botulinum type E. J Food Sci 66(7):S1082-7. Gram L. 2001b. Potential hazards in cold-smoke d fish: Listeria monocytogenes. J Food Sci 66(7):S1072-81. Gram L, Huss HH. 1996. Microbiolo gical spoilage of fish and fish products. J Food Microbiol 33(1):121-37. Heinitz ML, Johnson JM. 1998. The incidence of Li steria spp., Salmonella spp., and Clostridium botulinum in smoked fish and sh ellfish. J Food Prot 61(3):318-23. Heinitz ML, Ruble RD, Wagner DE, Tatini SR. 2 000. Incidence of Salmonella in fish and seafood. J Food Prot 63(5):579-92. Hsu LA, Fisher RW, Daun H. 1979. Meat Preservation by Smoke Curing in DevelopingCountries. Food Tech 33(5):86-&. Hunt MC, Mancini RA, Hachmeister KA, Kropf DH, Merriman M, DelDuca G, Milliken G. 2004. Carbon monoxide in modified atmosphere packaging affects color, shelf life, and microorganisms of beef steaks and gr ound beef. J Food Sci 69(1):FCT45-FCT52. Jahncke ML, Collette R, Hicks D, Wiedmann M, Scott VN, Gall K. 2004. Treatment Options to Eliminate or Control Growth of Lister ia monocytogenes on Raw Material and on Finished Product for the Smoked Fish Industry. Food Prot Trends 24(8):612-9. Jahncke ML, Herman D. 2001. Control of Food Safety Hazards During Cold-Smoked Fish Processing. J Food Sci 66(7):S1104-12. Kristinsson HG, Balaban MO, Otwell WS. 2006a. The Influence Of Carbon Monoxide And Filtered Wood Smoke On Fish Muscle Colo r. In: Otwell WS, Kristinsson HG, Balaban MO, editors. Modified atmospheric processing and packaging of fish : filtered smokes, carbon monoxide, and reduced oxygen packaging. 1st ed. Ames, Iowa: Blackwell Pub. p 29-52. 94

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Kristinsson HG, Balaban MO, Otwell WS. 2006b. Microbial and Quality Consequences of Aquatic Foods Treated with Carbon Monoxide or Filtered Wood Smoke. In: Otwell WS, Kristinsson HG, Balaban MO, editors. Modified atmospheric processing and packaging of fish : filtered smokes, carbon monoxide, and reduced oxygen packaging. 1st ed. Ames, Iowa: Blackwell Pub. p 65-86. Kristinsson HG, Crynen S, Yagiz Y. 2007. Effect of a filtered wood smoke treatment compared to various gas treatments on aerobic bacteria in yellowfin tuna steaks. LWT Food Science and Technology In Pr ess, Corrected Proof. Kusmider EA, Sebranek JG, Lonergan SM, Honeyman MS. 2002. Effects of carbon monoxide packaging on color and lipid stability of irradiated ground beef. J Food Sci 67(9):3463-8. Liao CH. 2007. Inhibition of foodborne pathogens by native microflora recovered from fresh peeled baby carrot and propagated in cultures. J Food Sci 72(4):M134-M9. Mancini RA, Hunt MC. 2005. Current research in meat color. Meat Science 71(1):100-21. Martinez L, Djenane D, Cilla I, Beltran JA, Roncales P. 2005. Eff ect of different concentrations of carbon dioxide and low concentration of carbon monoxide on the shelf-life of fresh pork sausages packaged in modified atmosphere. Meat Science 71(3):563-70. McLauchlin J. 1997. The pathogenicity of Listeria monocytogenes: A public health perspective. Rev Med Microbiol 8(1):1-14. Neamatallah AAN, Dewar SJ, Austin B. 2003. An improved selective isolation medium for the recovery of Listeria monocytogenes from sm oked fish. Lett Appl Microbiol 36(4):230-3. Novotny L, Dvorska L, Lorencova A, Beran V, Pavlik I. 2004. Fish: a potential source of bacterial pathogens for human bei ngs. Vet Med Czech 49(9):343-58. Olson BE. 2006. Commercial Aspects of Filte red Wood Smoke Technology Compared to Carbon Monoxide Gassing of Seafood Products. In: Otwell WS, Kristinsson HG, Balaban MO, editors. Modified atmospheric processing and packaging of fish : filtered smokes, carbon monoxide, and reduced oxygen packaging. 1st ed. Ames, Iowa: Blackwell Pub. p 3-13. Otwell WS. 2006. Use of Filtered Smokes and Carbon Monoxide in Fish Processing. In: Otwell WS, Kristinsson HG, Balaban MO, editors. Modified atmospheric processing and packaging of fish : filtered smokes, carbon monoxide, and reduced oxygen packaging. 1st ed. Ames, Iowa: Blackwell Pub. p 3-13. Rawles DD, Flick GJ, Martin RE. 1996. Biogenic amines in fish and shellfish. Advances in Food and Nutrition Research. p 329-65. Revolledo L, Ferreira CSA, Ferreira AJP. 2003. Comparison of experimental competitiveexclusion cultures for contro lling Salmonella colonization in broiler chicks. Brazilian Journal of Microbiology 34(4):354-8. 95

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Robinson RK. 1983. The vanishing ha rvest : a study of food and its conservation. Oxford ; New York: Oxford University Press. 273 p. Santos MHS. 1996. Biogenic amines: Their impor tance in foods. J Food Microbiol 29(2-3):21331. Scott E. 1996. Foodborne disease and other hygiene issues in the home. Journal of Applied Bacteriology 80(1):5-9. Shalaby AR. 1996. Significance of biogenic amines to food safety and human health. Food Res Int 29(7):675-90. Sorheim O, Langsrud O, Cornforth DP, Johannesse n TC, Slinde E, Berg P, Nesbakken T. 2006. Carbon monoxide as a colorant in cooked or fermented sausages. J Food Sci 71(9):C549C55. Sorheim O, Nissen H, Aune T, Nesbakken T. 2001. Use of carbon monoxide in retail meat packaging. J Dairy Sci 84(Supplement 1):58. Sorheim O, Nissen H, Nesbakken T. 1999. The storage life of beef and pork packaged in an atmosphere with low carbon monoxide and hi gh carbon dioxide. Meat Science 52(2):15764. Speck ML, Ray B. 1977. Effects of Freezing and St orage on Microorganisms in Frozen Foods Review. J Food Prot 40(5):333-6. Tortora GJ, Funke BR, Case CL. 1992. Microb iology an introduction. Redwood City, Calif.: Benjamin/Cummings. 96

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97 BIOGRAPHICAL SKETCH Stefan Crynen was born in 1976 in Mnc hengladbach, Germany. He attended the Stifitisch Humanistisches Gymnasium (high school) and graduated with the Abitur in 1996. After the 1 year mandatory military duty he attended culinary school and graduated as a professional chef in Summer 2000. From 2000 to 2004 he studied food technology at the University of Applied Sciences in Trier, Germany and graduated in October 2004 with the degree Diplom-Engineer of Food Technology. He did an internship from September 2002 to February 2003 at the Food Science and Human Nu trition Department at the University of Florida. He started his ma sters degree in 2005 under Dr. Hordur G Kristinsson.


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