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Effect of High Pressure and Irradiation Treatment on Quality Changes in Fish Muscle

Permanent Link: http://ufdc.ufl.edu/UFE0022466/00001

Material Information

Title: Effect of High Pressure and Irradiation Treatment on Quality Changes in Fish Muscle
Physical Description: 1 online resource (176 p.)
Language: english
Creator: Yagiz, Yavuz
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: astaxanthin, atlantic, fatty, high, irradiation, lipid, quality
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Seafood is a major economic contributor in many countries, including the US. Since fish is a highly perishable commodity, proper processing and storage are very important factors to maintain or extend its shelf-life. There is an increasing interest in the application of high pressure processing (HPP) technology and irradiation to obtain fresh-like quality, and safe seafood products. These novel technologies provide long shelf-life and minimum quality loss since they do not have many of the undesirable changes that are associated with thermal processing. Limited information exists on these two technologies with fish, so it is therefore of great interest and importance to investigate these technologies further. The first part of the project was to investigate the effect of different high pressures (150, 300, 450, and 600 MPa for 15 min) on the quality changes (color, lipid oxidation, texture and total aerobic plate count) of a) fresh water species (rainbow trout) b) salt water species (mahi mahi) and c) brackish water species (Atlantic salmon). The second part was to investigate the effect of different irradiation doses on the quality changes for fresh and frozen, and dark and light muscle of Atlantic salmon. Similar experiment design was performed for irradiation at different doses for fresh (0, 1, 1.5 2, 3 kGy ) and frozen Atlantic salmon fillets (0, 1, 2, 3, 5 kGy) including analysis of color, astaxanthin, lipid oxidation, fatty acids content, total aerobic count and sensory. HPP significantly reduced microbial load, with HPP treated samples at 450 and 600 MPa having the most reduction in microbial growth. It was demonstrated that 300 MPa for rainbow trout and 450 MPa for mahi mahi are the optimum HPP conditions for controlling microbial load, lipid oxidation and color changes. For Atlantic salmon, HPP and cooking significantly reduced microbial growth. The 150 MPa had a lesser effect on the color compared to cooking and 300 MPa. Cooking and 150 MPa led to similar oxidation development as untreated sample. Cooking significantly reduced the amount of total PUFA n-6 and PUFA n-3, including EPA and DHA fatty acids, however, HPP did not change the level of those fatty acids. Fresh and frozen salmon fillets were treated with various doses of irradiation and were stored at 4 degrees C for 6 days. There was a significant decrease in the microbial growth of both fresh and frozen irradiation treated fillets at 1 kGy and higher doses. Correlation was obtained between average amount of astaxanthin and average a*-value (redness) during different irradiation treatments in fresh and frozen light and dark muscle of Atlantic salmon for each storage period up to 6 days at 4 degrees C. It was found that redness of salmon light muscle was more related to astaxanthin content, while that of salmon dark muscle was related more to the lipid content and the degree of lipid oxidation. Irradiation treatments for fresh and frozen light and dark muscle did not change lipid oxidation and the level of fatty acids compared to untreated samples. Sensory score of 2 kGy and higher doses was significantly lower in color, odor and overall acceptability compared to untreated samples, and the sensory score decreased with an increase in irradiation level. The 1 kGy treated fresh and frozen samples was a successful treatment, with no significant effect on color, sensory as well as providing inactivation of microbial growth during 6 days storage at 4 degres C compared to untreated samples.
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 Yavuz Yagiz.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Marshall, Maurice R.
Local: Co-adviser: Kristinsson, Hordur G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022466:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022466/00001

Material Information

Title: Effect of High Pressure and Irradiation Treatment on Quality Changes in Fish Muscle
Physical Description: 1 online resource (176 p.)
Language: english
Creator: Yagiz, Yavuz
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: astaxanthin, atlantic, fatty, high, irradiation, lipid, quality
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Seafood is a major economic contributor in many countries, including the US. Since fish is a highly perishable commodity, proper processing and storage are very important factors to maintain or extend its shelf-life. There is an increasing interest in the application of high pressure processing (HPP) technology and irradiation to obtain fresh-like quality, and safe seafood products. These novel technologies provide long shelf-life and minimum quality loss since they do not have many of the undesirable changes that are associated with thermal processing. Limited information exists on these two technologies with fish, so it is therefore of great interest and importance to investigate these technologies further. The first part of the project was to investigate the effect of different high pressures (150, 300, 450, and 600 MPa for 15 min) on the quality changes (color, lipid oxidation, texture and total aerobic plate count) of a) fresh water species (rainbow trout) b) salt water species (mahi mahi) and c) brackish water species (Atlantic salmon). The second part was to investigate the effect of different irradiation doses on the quality changes for fresh and frozen, and dark and light muscle of Atlantic salmon. Similar experiment design was performed for irradiation at different doses for fresh (0, 1, 1.5 2, 3 kGy ) and frozen Atlantic salmon fillets (0, 1, 2, 3, 5 kGy) including analysis of color, astaxanthin, lipid oxidation, fatty acids content, total aerobic count and sensory. HPP significantly reduced microbial load, with HPP treated samples at 450 and 600 MPa having the most reduction in microbial growth. It was demonstrated that 300 MPa for rainbow trout and 450 MPa for mahi mahi are the optimum HPP conditions for controlling microbial load, lipid oxidation and color changes. For Atlantic salmon, HPP and cooking significantly reduced microbial growth. The 150 MPa had a lesser effect on the color compared to cooking and 300 MPa. Cooking and 150 MPa led to similar oxidation development as untreated sample. Cooking significantly reduced the amount of total PUFA n-6 and PUFA n-3, including EPA and DHA fatty acids, however, HPP did not change the level of those fatty acids. Fresh and frozen salmon fillets were treated with various doses of irradiation and were stored at 4 degrees C for 6 days. There was a significant decrease in the microbial growth of both fresh and frozen irradiation treated fillets at 1 kGy and higher doses. Correlation was obtained between average amount of astaxanthin and average a*-value (redness) during different irradiation treatments in fresh and frozen light and dark muscle of Atlantic salmon for each storage period up to 6 days at 4 degrees C. It was found that redness of salmon light muscle was more related to astaxanthin content, while that of salmon dark muscle was related more to the lipid content and the degree of lipid oxidation. Irradiation treatments for fresh and frozen light and dark muscle did not change lipid oxidation and the level of fatty acids compared to untreated samples. Sensory score of 2 kGy and higher doses was significantly lower in color, odor and overall acceptability compared to untreated samples, and the sensory score decreased with an increase in irradiation level. The 1 kGy treated fresh and frozen samples was a successful treatment, with no significant effect on color, sensory as well as providing inactivation of microbial growth during 6 days storage at 4 degres C compared to untreated samples.
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 Yavuz Yagiz.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Marshall, Maurice R.
Local: Co-adviser: Kristinsson, Hordur G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022466:00001


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1 EFFECT OF HIGH PRESSURE AND IRRADIATION TREATMENT ON QUALITY CHANGES IN FISH MUSCLE By YAVUZ YAGIZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Yavuz Yagiz

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3 To my parents, and family, who supported and encouraged me; and to my wife and best friend, Dilber Dagdelen. Without her support a nd devotion I could not have done this.

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4 ACKNOWLEDGMENTS I want to ex press my infinite gratitude to my chair, Dr. Maurice R. Marshall; and my cochair, Dr. Hordur G. Kristinsson for the guida nce, direction, insight and unconditional support that made all this work possible. I sin cerely appreciate, my supervising committee, Dr. Murat O. Balaban and Dr. Bruce A. Welt, for the valuable time devoted to this project, their help, suggestions, and words of encouragement along this research. I would also like to th ank Florida Accelerator Services and Technology (F.A.S.T) and Food Tec hnology Service Inc. (F.T.S.I) facilities for their help on the irradiation study. I thank Dr. Siva Raghavan for his unconditional friendship. I am thankful to all my friends at the University of Florida, especial my friends at Dr. Kristinssons and Dr. Balabans lab for their friendship and supports. Special thanks go to FETL employees, especially Amanda Hogle for technical assistance. Finally, I would like thank my entire family members including Dilek and Ismail. My deepest gratitude and love go to my wife, D ilber, who was always there to support me. She believed in me throughout this journey.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................11 ABSTRACT...................................................................................................................................13 CHAP TER 1 INTRODUCTION..................................................................................................................15 Research Objectives............................................................................................................ ....16 Project Significance........................................................................................................... .....17 2 LITERATURE REVIEW.......................................................................................................18 Quality Attributes of Fish.......................................................................................................18 Lipid Oxidation...............................................................................................................18 Microbial Changes........................................................................................................... 20 Color Changes.................................................................................................................21 Texture Changes..............................................................................................................22 Carotenoids in Foods....................................................................................................... 22 Applications of High Pressure Processing (HPP) on Seafoods .............................................. 24 Applications of Irradiation on Seafoods................................................................................. 26 3 EFFECT OF HIGH PRESSURE TREATMENT ON THE QUALITY OF RAINBOW TROUT ( Oncorhynchus mykiss ) AND MAHI MAHI ( Coryphaena hippurus).....................32 Introduction................................................................................................................... ..........32 Materials and Methods...........................................................................................................34 High Pressure Processing................................................................................................ 34 Microbial Analysis.......................................................................................................... 35 Lipid Oxidation Analysis................................................................................................35 Color Analysis.................................................................................................................36 Texture Profile Analysis ................................................................................................. 36 Statistical Analysis.......................................................................................................... 37 Results and Discussion......................................................................................................... ..37 Microbial Analysis.......................................................................................................... 37 Lipid Oxidation Analysis................................................................................................38 Color Analysis.................................................................................................................40 Texture Profile Analysis.................................................................................................. 41 Conclusion..............................................................................................................................43

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6 4 EFFECT OF HIGH PRESSURE PROCESSING AND HEAT TREATMENT ON THE QUALIT Y OF ATLANTIC SALMON.................................................................................. 51 Introduction................................................................................................................... ..........51 Materials and Methods...........................................................................................................53 High Pressure Processing................................................................................................ 53 Cooking Treatment..........................................................................................................54 Microbial Analysis.......................................................................................................... 54 Color Analysis.................................................................................................................54 Texture Profile Analysis.................................................................................................. 55 Lipid Oxidation Analysis................................................................................................55 Lipid Extraction...............................................................................................................56 Preparation of Fatty Acid Methyl Esters......................................................................... 57 Gas Chromatography (GC) Analysis............................................................................... 57 Statistical Analysis.......................................................................................................... 58 Results.....................................................................................................................................58 Microbial Analysis.......................................................................................................... 58 Color Analysis.................................................................................................................59 Texture Profile Analysis.................................................................................................. 59 Lipid Oxidation Analysis................................................................................................60 Fatty Acids Analysis........................................................................................................ 60 Discussion...............................................................................................................................62 Conclusion..............................................................................................................................65 5 EFFECT OF IRRADIATION TREATM ENT ON THE QUALITY OF FRESH ATLANTIC SALMON MUSCLE .........................................................................................75 Introduction................................................................................................................... ..........75 Materials and Methods...........................................................................................................77 Irradiation Treatment....................................................................................................... 77 Dosimeter Calibration..................................................................................................... 78 Microbial Analysis.......................................................................................................... 79 Color Analysis.................................................................................................................79 HPLC Procedure for Quantification of Astaxanthin.......................................................80 Lipid Oxidation Analysis................................................................................................81 Measurement of thiobarbituric acid reactive substances ......................................... 81 Measurement of lipid hydroperoxides...................................................................... 81 Lipid Extraction for Fatty Acids Methyl Esters.............................................................. 82 Preparation of Fatty Acid Methyl Esters......................................................................... 82 Gas Chromatography (GC) Analysis............................................................................... 83 Sensory Evaluation..........................................................................................................83 Statistical Analysis.......................................................................................................... 84 Results.....................................................................................................................................85 Microbial Analysis.......................................................................................................... 85 Color Analysis.................................................................................................................85 Astaxanthin Analysis....................................................................................................... 86 Correlation between Amount of Astaxanthin and a* Value ............................................ 87

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7 Lipid Oxidation Analysis................................................................................................87 Fatty Acids Analysis........................................................................................................ 89 Sensory Evaluation..........................................................................................................90 Discussion...............................................................................................................................91 Conclusion..............................................................................................................................95 6 EFFECT OF IRRADIATION TREATM ENT ON THE QUALITY OF FROZ EN ATLANTIC SALMON MUSCLE.......................................................................................115 Introduction................................................................................................................... ........115 Materials and Methods.........................................................................................................117 Irradiation Treatment..................................................................................................... 117 Dosimeter Calibration................................................................................................... 118 Microbial Analysis........................................................................................................119 Color Analysis...............................................................................................................119 HPLC Procedure for Quantification of Astaxanthin.....................................................120 Lipid Oxidation Analysis..............................................................................................121 Measurement of thiobarbituric acid reactive substances ....................................... 121 Measurement of lipid hydroperoxides.................................................................... 121 Lipid Extraction for Fatty Acids Methyl Esters............................................................ 122 Preparation of Fatty Acid Methyl Esters....................................................................... 122 Gas Chromatography (GC) Analysis............................................................................. 123 Sensory Evaluation........................................................................................................123 Statistical Analysis........................................................................................................ 124 Results...................................................................................................................................125 Microbial Analysis........................................................................................................125 Color Analysis...............................................................................................................125 Astaxanthin Analysis..................................................................................................... 126 Correlation between Amount of Astaxanthin and a* Value .......................................... 127 Lipid Oxidation Analysis..............................................................................................128 Fatty Acids Analysis...................................................................................................... 129 Sensory Evaluation........................................................................................................130 Discussion.............................................................................................................................131 Conclusion............................................................................................................................136 7 SUMMARY AND CONCLUSION..................................................................................... 157 APPENDIX COMPARISON OF MINOLTA AND MAC HINE VISION SYSTEM IN MEASURING COLOR OF IRRADIATED ATLANTIC SALMON.................................. 159 Introduction................................................................................................................... ........159 Materials and Methods.........................................................................................................160 Irradiation Treatment..................................................................................................... 160 Color Analysis...............................................................................................................161 Result and Discussion.......................................................................................................... .161

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8 LIST OF REFERENCES.............................................................................................................166 BIOGRAPHICAL SKETCH.......................................................................................................176

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9 LIST OF TABLES Table page 3-1 Changes in a* value (redness), L* value (lightness) and b* value (yellow ness) for rainbow trout muscle after HPP treatment......................................................................... 443-2 Changes in a* value (redness), L* value (lightness) and b* value (yellowness) for mahi mahi muscle after HPP treatment............................................................................. 453-3 Texture profile analysis (TPA) in term s of hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness for (a) Rainbow trout and (b) Mahi mahi........... 464-1 Changes in a* value (redness), L* value (lightness) and b* value (yellowness) for Atlantic salmon dark and light muscle after HPP treatment.............................................. 664-2 Texture profile analysis (TPA) in term s of hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness fo r Atlantic salmon mu scle after HPP................674-3 Fatty acid compositions of Atlantic sa lmon dark muscle after HPP treatment or cooking followed by storage for day 0.............................................................................. 684-4 Fatty acid compositions of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 2.............................................................................. 694-5 Fatty acid compositions of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 4.............................................................................. 704-6 Fatty acid compositions of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 6.............................................................................. 715-1 Changes in L* value (lightness), a* value (redness), b* value (yellowness) and E of fresh Atlantic salmon light muscle after irradiation.......................................................... 965-2 Changes in L* value (lightness), a* va lue (redness), b* value (yellowness) and E of fresh Atlantic salmon dark muscle after irradiation treatment........................................... 975-3 Fatty acid compositions of fresh Atlan tic salmon light muscle after irradiation treatment (day 0).............................................................................................................. ..985-4 Fatty acid compositions of fresh Atlan tic salmon light muscle after irradiation treatment and storage for 2 days at 4C............................................................................. 995-5 Fatty acid compositions of fresh Atlan tic salmon light muscle after irradiation treatment and storage for 4 days at 4C........................................................................... 1005-6 Fatty acid compositions of fresh Atlan tic salmon light muscle after irradiation treatment and storage for 6 days at 4C........................................................................... 101

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10 5-7 Fatty acid compositions of fresh Atlan tic salm on dark muscle after irradiation treatment (day 0).............................................................................................................. 1025-8 Fatty acid compositions of fresh Atlan tic salmon dark muscle after irradiation treatment and storage for 2 days at 4C........................................................................... 1035-9 Fatty acid compositions of fresh Atlan tic salmon dark muscle after irradiation treatment and storage for 4 days at 4C........................................................................... 1045-10 Fatty acid compositions of fresh Atlan tic salmon dark muscle after irradiation treatment and storage for 6 days at 4C........................................................................... 1056-1 Changes in L* value (lightness), a* value (redness), b* value (yellowness) and E for frozen Atlantic salmon light mu scle after irradi ation treatment................................1376-2 Changes in L* value (lightness), a* value (redness), b* value (yellowness) and E for frozen Atlantic salmon dark mu scle after irradi ation treatment.................................1386-3 Fatty acid composition of frozen Atlant ic salmon light muscle after irradiation treatment, thawing at 4C and following storage for day 0 at 4C.................................. 1396-4 Fatty acid compositions of frozen Atlan tic salmon light muscle after irradiation treatment, thawing at 4C and following storage for day 2 at 4C.................................. 1406-5 Fatty acid compositions of frozen Atla ntic salmon light muscle after irradiation treatment, thawing at 4C and following storage for day 4 at 4C................................. 1416-6 Fatty acid compositions of frozen Atlan tic salmon light muscle after irradiation treatment, thawing at 4C and following storage for day 6 at 4C.................................. 1426-7 Fatty acid compositions of frozen Atlan tic salmon dark muscle after irradiation treatment, thawing at 4C and following storage for day 0 at 4C.................................. 1436-8 Fatty acid compositions of frozen Atlan tic salmon dark muscle after irradiation treatment, thawing at 4C and following storage for day 2 at 4C.................................. 1446-9 Fatty acid compositions of frozen Atla ntic salmon dark muscle after irradiation treatment, thawing at 4C and following storage for day 4 at 4C.................................. 1456-10 Fatty acid compositions of frozen Atlantic salmon dark muscle after irradiation treatment, thawing at 4C and following storage for day 6 at 4C.................................. 146A-1 Average L*a*b* values from Machine Vision and Minolta system at different treatment doses of Atlantic salmon, a nd that of a standard red plate.............................. 164A-2 Color representations of the Minolta and Machine visi on reading results, and actual pictures of differently treated salm on fillets and standard red plate................................ 165

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11 LIST OF FIGURES Figure page 2-1 Lipid oxidation in a po lyunsaturated fatty acid ................................................................. 302-2 Chemical structure of various carotenoids.........................................................................313-1 The 600 MPa process showing temp erature and pressure profiles.................................... 473-2 Total aerobic plate count.................................................................................................. ..483-3 Changes in lipid oxidation (sec ondary oxidation products) as TBARS............................ 494-1 Total aerobic plate count for Atlant ic salmon after 15 min of high pressure processing (150 and 300 MPa) followed by storage at 4 C for 6 days............................ 724-2 Images for Atlantic salmon dark and light muscle at different pressures after high pressure processing treatment............................................................................................ 734-3 Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances (TBARS) for da rk muscle of Atlantic salmon................................... 744-4 Temperature profile of Atlantic salmon during heating and cooling................................. 745-1 Total aerobic plate count of fresh A tlantic salmon after i rradiation treatment followed by storage at 4 C for 6 days............................................................................. 1065-2 Calibration curve for absorbed dose from alanine dosimeter versus specific absorbance from PMMA dosimeter.................................................................................1075-3 Images of fresh Atlantic salmon light and dark muscle after bei ng treated at different irradiation doses and subsequent storage for 6 days at 4 C............................................1085-4 Amount of astaxanthin in fresh light and dark muslce.................................................... 1095-5 Correlation between the amount of astaxant hin and average a*-value during different dose of irradiation treatment in fr esh light muscle during storage at.............................. 1105-6 Correlation between the amount of astaxant hin and average a*-value during different dose of irradiation treatment in fr esh dark muscle during storage at............................... 1115-7 Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances (TBARS).......................................................................................... 1125-8 Changes in lipid oxidation as measured by the formation of peroxide value (PV) for .. 1135-9 Effect of different irradi ation doses on sensory evaluati on of color, odor and overall acceptability of fresh Atlantic salmon fillets................................................................... 114

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12 6-1 Total aerobic plate count for frozen A tlantic salmon after irradiation treatm ent, thawing at 4C and following storage at 4C for 6 days................................................. 1476-2 Astaxanthin analysis HPLC chromatogram of Atlantic salmon at 445 nm UV-visible range.................................................................................................................................1486-3 Images for frozen Atlantic salmon light and dark muscle at different doses after irradiation treatment and subsequent storage for 6 days at 4 C..................................... 1496-4 Amount of astaxanthin in frozen light and dark muscle.................................................. 1506-5 Correlation between amount of astaxanthin and average a*-value for different irradiation treatments for frozen light muscle stored at 4C for 6 days........................... 1516-6 Correlation between amount of astaxanthin and average a*-value for different irradiation treatments for frozen dark muscle stored at 4C for 6 days...........................1526-7 Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances .......................................................................................................... 1536-8 Changes in lipid oxidation as measured by the formation of peroxide value .................1546-9 Effect of different irradi ation doses on sensory evaluati on of color, odor and overall acceptability for frozen Atlantic salmon fillets ............................................................... 1556-10 Temperature profile of Atlantic salmon during freezing at -20 C..................................156

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF HIGH PRESSURE AND IRRADIATION TREATMENT ON QUALITY CHANGES IN FISH MUSCLE By Yavuz Yagiz August 2008 Chair: Maurice R. Marshall Cochair: Hordur G. Kristinsson Major: Food Science and Human Nutrition Seafood is a major economic contributor in ma ny countries, including the US. Since fish is a highly perishable commodity, proper processing and storage are very important factors to maintain or extend its shelf-life. There is an increa sing interest in the application of high pressure processing (HPP) technology and irradiation to obtain fresh-like quality, and safe seafood products. These novel technologies provide long shelf-life and mi nimum quality loss since they do not have many of the undesirable changes th at are associated with thermal processing. Limited information exists on these two technologies with fish, so it is ther efore of great interest and importance to investigate these technologies fu rther. The first part of the project was to investigate the effect of different high pr essures (150, 300, 450, and 600 MPa for 15 min) on the quality changes (color, lipid oxida tion, texture and total aerobic plate count) of a) fresh water species (rainbow trout) b) salt water species (m ahi mahi) and c) brackish water species (Atlantic salmon). The second part was to investigate the eff ect of different irradiation doses on the quality changes for fresh and frozen, and dark and light muscle of Atlantic salmon. Similar experiment design was performed for irradiation at different doses for fresh (0, 1, 1.5 2, 3 kGy ) and frozen Atlantic salmon fillets (0, 1, 2, 3, 5 kGy) including analysis of color, astaxanthin, lipid oxidation,

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14 fatty acids content, total aerobic count and se nsory. HPP significantly re duced microbial load, with HPP treated samples at 450 and 600 MPa having the most reduction in microbial growth. It was demonstrated that 300 MPa for rainbow tr out and 450 MPa for mahi mahi are the optimum HPP conditions for controlling microbial load, lipid oxidation and color ch anges. For Atlantic salmon, HPP and cooking significantly reduced microbial growth. The 150 MPa had a lesser effect on the color compared to cooking a nd 300 MPa. Cooking and 150 MPa led to similar oxidation development as untreated sample. Cooking significantly reduced the amount of total PUFA n-6 and PUFA n-3, including EPA and D HA fatty acids, however, HPP did not change the level of those fatty acids. Fresh and frozen salmon fillets were treated w ith various doses of irradiation and were stored at 4C for 6 days. There was a significant decrease in th e microbial growth of both fresh and frozen irradiation treated fillets at 1 kGy and higher doses Correlation was obtained between average amount of astaxanthin and average a* -value (redness) during different irradiation treatments in fresh and frozen light and dark muscle of Atlantic salmon for each storage period up to 6 days at 4 C. It was found that redness of salmon light muscle was more related to astaxanthin content, while that of salmon dark muscle was related more to the lipid content and the degree of lipid oxidation. Irra diation treatments for fresh and frozen light and dark muscle did not change lipid oxidation a nd the level of fatty acids compared to untreated samples. Sensory score of 2 kGy and higher doses was si gnificantly lower in co lor, odor and overall acceptability compared to untreated samples, and th e sensory score decreased with an increase in irradiation level. The 1 kGy treated fresh and fr ozen samples was a successful treatment, with no significant effect on color, sensor y as well as providing inactivati on of microbial growth during 6 days storage at 4 C compared to untreated samples.

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15 CHAPTER 1 INTRODUCTION Recognition of the health benefits associated with the consumption of omega-3 fatty acids from seafoods is one of the most promising developments in nutrition research in the past 20 years (Nestel, 1990). The consumption of seaf ood in the U.S. has increased 16.5 pounds per person in 2006 (NOAA, 2007). Seafoods are highly peri shable with a 14 day shelf-life for a fresh or thawed product. Usually beyond 7 days of cold storage the product is considered being of a lower grade and frequently sold at reduced co st or discarded. Moreove r, seafoods are more susceptible to post-mortem texture deterioration than meats from land animals (Ashie and others 1996). Processing techniques that can extend th e shelf-life of seafood past 14 days can dramatically change the sensor y attributes and characteristics of the product beyond the fresh quality demanded by consumers. Heat processing can lead to inactivatio n of microbial growth and lipolytic enzymes. However, it may damage vitamins, flavor compounds and polyunsaturated fatty. High pressure processing (HPP) has been appl ied to foods as a preservation method with its major advantage being the maintenance of the fresh quality attributes of foods. High pressure processing is a promising seafood preserva tion method. This novel technology reportedly provides long shelf-life and minimum quality loss since it does not have many of the undesirable changes that are associated with thermal processing. There is limited information on the influence of high pressure on th e oxidative stability a nd quality changes of fish muscle. The major purpose of HPP is to enhance the safe ty of mostly raw seafoods by inactivating microorganisms and parasites without changing sens orial quality attributes (Cheftel and Culioli, 1997).

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16 The irradiation is a cold process treatment due to only a few degrees temperature rise in foods from the radiation energy absorbed, even at a sterilization dose. Therefore, radiation treatment causes minimal changes in appearance and provides good nutrient retention. Radiation does not leave any chemical resi due and thus can substitute fo r chemical fumigation; thereby reducing the need for chemical substances and allowing for the treatment of products with a wide range of sizes and shapes (Nawar 1995). In general, there are two main advantages of using low dose ionizing radiation (< 3 kGy) as a processing method. Firstly, it will reduce or eliminate the microorganisms responsible for spoilage and subs equently extend the fres h-storage shelf-life. Secondly, the low dose irradiation al so has the ability to reduce or eliminate specific pathogenic bacteria commonly associated with seafood (Grodner and Andrews, 1991). Research Objectives This res earch is intended to provide a better understanding of th e effect of high pressure and irradiation processing on fish muscle and quality, and to use this information obtained to rationalize more effective strategies for pr eserving the premium quality of fresh fish. Specific objectives are; 1. To investigate the effect of high pressure treatment on the quality of rainbow trout (fresh water species) and mahi mahi (salt water species) based on microbial activity, lipid oxidation, color, and texture during 6 days storage. 2. To investigate the effect of high pressure treatment and heat treatment on the quality of Atlantic salmon (brackish water species) based on microbial activity, lipid oxidation, fatty acids profile, color and texture during 6 days storage.

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17 3. To investigate the effect of different irra diation conditions on quality of fresh Atlantic salmon dark and light muscles based on microbi al activity, color, astaxanthin analysis, lipid oxidation, fatty acids composition and sensory during 6 days storage. 4. To investigate the effect of different irradi ation conditions on quality of frozen Atlantic salmon dark and light muscles based on microbi al activity, color, astaxanthin analysis, lipid oxidation, fatty acids composition and sensory during 6 days storage. Project Significance High pressure and irradiation processing to pr eserve foods are em ergi ng technologies with excellent potential to improve the market ability of current and underutilized seafood commodities. However, there are limited studies on the effect of HPP and irradiation on seafood. Thus, developing effective preservation strate gies for important health components will ultimately increase the wholesomeness of fish, and improve and extend quality.

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18 CHAPTER 2 LITERATURE REVIEW Quality Attributes of Fish Seafoods such as fish and shell fish are hi ghly susceptible to en zym atic, chemical and microbial spoilage(Ashie and ot hers 1996). Hence, preservative me thods are usually designed to sustain freshness and quality, beginning from the point of harvest through storage, processing, and distribution, until the point of consumption. The effectiveness of any method or set of storage conditions to control fish and shellf ish spoilage depends largely on the spoilage mechanisms involved. In fresh seafood, the prim ary mechanisms which adversely affect the quality include a) bacterial spoilage resulting in off-odors and formation of toxic compounds and b) oxidation of unsaturated lip ids and other lipid-soluble pigm ents resulting in undesirable flavors and colors (intVeld, 1996). Thus, an understanding of the postmortem biochemical changes in fish and shellfish spoi lage and their relationship involve d in spoilage is essential in order to formulate and rationalize effective strategi es for controlling these deteriorations (Ashie and others 1996). Lipid Oxidation The total amount of fat in fish is highly va riable. It ranges from 0.2 to 23.7%. Fish can be classified based on their fat conten t into four main categories such as lean (<2% fat), low (2-4% fat), medium (4-8% fat) and high (8% fat>) (Ackma n, 1994). In lean fish species such as cod and haddock, the lipids are present primarily as memb rane phospholipids. Fatty fish which are good sources of omega-3 fatty acids would include all types of salmon and tuna, sardines, herring, mackerel, black bass, and bluefish. The polyunsat urated nature of fish lipids makes them susceptible to a variety of chemical and enzyma tic degradations. In fatty fish species, lipid oxidation represents a serious problem. These fish often contain more free lipids and more dark

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19 muscle, which can promote oxidation more rapi dly than light muscle. The lipids of the subcutaneous layer and skin are particularly susc eptible to oxidation beca use of a closer contact with lipoxygenase and atmospheric air (Sikorski and Kolakowska, 1990). In addition to the high concentration of highly unsaturat ed fatty acids, fish muscle al so contains a variety of prooxidants. These include transition metals such as copper and iron, the latter of these being also found in heme proteins such as myoglobin and hemoglobin (Hultin, 1992) where it is complexed to a heme group. Both hemoglobin and myoglobin are active catalysts of lipid oxidation in postmortem fish muscle (Richards and others 2002; Kristinsson, 2002; Hultin, 1992). Total iron, bound or unbound, varies considerably with muscle type, being higher in dark muscle compared to light muscle, which also contains more of the highly oxidizable membrane phospholipids. Membrane phospholipids themselves are highly sus ceptible to oxidation due to their relatively high degree of polyunsaturation a nd large surface area. Being bound to proteins, the activity of iron as a catalyst of oxidation is greatly influenced by the conforma tional state of the protein. On denaturation, heme proteins become very activ e pro-oxidants as the ir on containing heme group becomes exposed (Kristinsson, 2002). Other important pro-oxidants in muscle are enzymes such as lipoxygenases whose activity is affected by de naturing conditions. In addition to a variety of pro-oxidants, fish muscle also has a number of compounds capable of inhibiting and delaying lipid oxidation. These molecules or compounds which are involved in delaying lipid oxidation are termed antioxidants. These include lipid so luble inhibitors such as tocopherol and water soluble compounds such as as corbate, metal chelators, and enzymes (Hultin, 1992). It is generally accepted that lipid oxidati on in muscle foods proceeds by the classic mechanism of free radical attack on unsaturated substrates (Fi gure 2-1), generating further free radicals that accelerate the rate of oxidation with heme and non-heme iron being the major

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20 catalyst (Khayat and Schwall, 1983). Recent evid ence, however from both mammalian and fish systems indicates the presence of enzymatic lipi d-oxidizing systems that might influence meat quality. Those systems are thought to be closely re lated to membrane deterioration caused in part by microsomal lipid oxidation; a system that is r easonably stable during stor age and is of interest from a fish quality point of view because lipi d oxidation can proceed significantly even at temperatures of 0C and below (Kanner and others 1987). Significant lipid degradation in fi sh muscle during refrigerated and frozen storage is due to enzymatic hydrolysis and leads to both a decrease in phospholipids a nd an increase in free fatty acids (Hardy and others 1979; Dyer and Fraser, 1959; Lovern a nd others 1959). Free fatty acids are regarded as more susceptible to autoxi dation and usually cause subsequent oxidative rancidity of fish (Gould and Pete rs, 1965). Moreover, textural changes in fish muscle have been reported to be caused by an increase in free fatty acid content (Fazal and Srikar, 1989; Woyewoda and others 1986). Restriction of the accumulation of free fatty acids could, therefore, be effective in preserving the quality of fish during storage. Microbial Changes Spoilage refers to any cha nge in the condition of food in which the latter becom es less palatable, or even toxic, and these changes ma y be accompanied by alterations in taste, smell, appearance or texture (Singl eton and Sainsbury, 2001). A se nsorial quality or storage characteristic of fish depends on the total number of bacteria on fish. These bacterial populations may play an important role in th e spoilage of fish upon harvest. Fish and shellfish have four major component s: proteins, lipids, carbohydrates, and water. The relative proportions of all th ese components give fish and sh ellfish their ch aracteristic

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21 structure, flavor, texture and color. Other compounds such as vitamins and other minor components are also important in the spo ilage process (Ashie and others 1996). During post-mortem storage, bacteria in the gills, gut, and skin begin to metabolize the surrounding low-molecular-weigh t compounds, producing the volat ile compounds associated with spoilage. Under refrigerated storage c onditions, this is a surface phenomenon. However, under temperature abuse conditions or via cuts in the fish skin, such as during improper handling, the microorganisms can invade the relativ ely sterile muscle tiss ue through the lateral line pores as well as the gut, resulting in more rapid spoi lage(Ashie and others 1996). Color Changes Consum ers often associate th e color of fish and seafood products with their quality (Sawyer and others 1988). Hence, the problem of discoloration is one of the more serious concerns for the seafood industry. Discoloration can be formed in different ways. For example, brownish discolorations in fish are mostly th e result of oxidation of residual blood pigments and/or lipids. In fresh fis h, heme proteins have their iron in the reduced state (Fe2+) and have either a red color (when heme is bound to oxygen) due to the formation of oxymyoglobin or a purple color (when no oxygen is bound) due to the formation of deoxymyoglobin. However on storage, freezing and processing, th e heme iron becomes oxidized (Fe3+) to form an undesirable brown color. Another color change that occurs with seafoods is greening in certain species like tuna and mullet. The greening phenomenon, which is related to fish myoglobin, has been linked to oxidation of an exposed cysteine residue on myoglobin by trimethylamine oxide (TMAO). Yellow discoloration of certain species can be caused by the relocation of carotenoid pigments from chromatophore or carotenoprotein complexes in the skin to the subcutaneous fat layers. Oxidation of carotenoid pigments in some species (e.g. salmon and trout) can also results in fading of the pink or red color of fish flesh or sk in when stored on ice or at chilling temperatures.

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22 A lipoxygenase-like enzyme, in addition to a myel operoxide found in fish leukocytes have been thought to be involved in this process (Haard, 1992). Texture Changes In general, tenderness is the one of the m ost important quality determinants and probably the most important sensory characteristic of fish and other muscle tissues The duration of rigor mortis of terrestrial animals is higher than that of fish. Fish and shellf ish muscles also have a smaller amount of connective tissue than mamma ls, and in addition, the cross-links formed by their collagens are not as exte nsive thus giving them a more tender texture (Suyama and Konosu, 1987). The interaction of lipids with proteins and other muscle constituents have numerous implications with regard to nut rient content and characteristics of muscle foods. Free fatty acid levels, which increase during fish storage, also lead to protein denaturation, and unfavorably affect texture and water-holding capacity or drip loss (Anderson and Ravesi, 1969; Love and Elerian, 1964). Carotenoids in Foods Carotenoid s can be found in variety of food s ources of importance in nutrition and health including fruits, vegetables, algae, chicken, e ggs, and fish. Carotenoid s consist of over 600 fatsoluble pigments which are synthesized de novo in higher plants, mosses algae, bacteria and fungi (Goodwin, 1980). Carotenoids are usually yellowred isoprenoid polyene pigments extensively distributed in natu re. Carotenoid structure, deri ved by lycopene, consists of 40 carbon atoms which contain two terminal ring systems joined by conjugated double bonds or polyene system. Carotenes have only carbon and hydrogen and the xantho phylls are oxygenated derivatives. This oxygen can be present as OH groups, oxy-groups or both OH and oxy-groups, called zeaxanthin, canthaxanthin or astaxanthin, re spectively (Figure 2-2). The presence of two hydroxyl groups and conjugated double bonds give pow erful antioxidant capacity to carotenoids.

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23 There has been great interest in carotenoids as they could have a crucial role in the maintenance and promotion of good health and in the preven tion of chronic diseases (Mares-Perlman and others 2002). In addition, carotenoids have been connected with a reduced incide nce and severity of cancer as well as cardiovascular and degenerative diseases due to their antioxidant capabilities (Young and Lowe, 2001). However, carotenoids, c ould be unstable because of long conjugated double bond system, and are also greatly sensitive to light, oxygen, heat, acid and alkali medium or combination of these cond itions (Foss and others 1987). Astaxanthin is one of the main caratonoi d pigments found in seafood including salmon, trout, shrimp, lobster and fish egg (Torrissen, 1 989; Haard, 1992). The intensity of red hue in the muscle of shrimp, salmon, rockfish, and snapper is directly linked to the grading or pricing of those species (Sacton, 1986). Seaf ood muscles, mostly consist of polyunsaturated fatty acids (PUFA), especially highly unsaturated fatty aci ds (HUFA) of the omega-3 and omega-6 family, are susceptible to quick oxidative deterioration. Oxidation of oils generally depends on their degree of unsaturation, as represented by double-bond index or met hylene bridge index (Senanayake and Shahidi, 2002). It has been stated that astaxanthin has an antioxidant activity, 10 times greater than other carotenoids such as zeaxanthin, lutein, canthaxantin, and -carotene; and 100 times more than -tocopherol. Hence, astantanthin ha s been dubbed a super vitamin E (Miki, 1991). Astaxanthin acts as an antioxidant by quenching singlet ox ygen and free radicals. This powerful antioxidant ability of astaxanthi n comes from unique molecular structure of 11 conjugated double bonds and two hydroxyl groups.

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24 Astaxanthin, is much more sensitive to light and oxygen in its free stat e in ether than when it is bound to protein. It could be easily degraded under those conditions (Storebakken and others 1987). Applications of High Pressure Processing (HPP) on Seafoods One of the effective processes for reducing the hydrolytic degradat ion of fish muscle lipids is heat processing, which would lead to inactiv ation of the lipolytic enzymes. However, muscle components such as vitamin derivatives, and polyunsaturated fatty acids are sufficiently labile that autoxidation coul d be accelerated by cooking. Rece ntly, treatment of food and foodstuffs with high pressure pr ocessing (HPP) has been tentativ ely applied to food processing and preservation (Hoover and others 1989). Although the first attempts to apply high pressure technology to food processing dates back to the late 19th century, the great potential of HPP technology for the food industry was first recognized in the 1980s, and HPP technology has since become one of the most popular subjects of st udy in food engineering and technology. Several researchers have discussed the engineering aspect s of HPP applications in the context of food processing, safety and quality, and the effect of HPP on ice-water transitions (Knorr, 1993; Balny and Masson, 1993; Messens and others 1997; Kalichevsky and others 1995; Sanz and others 1997; Thakur and Nelson, 1998). One of the distinct advantages claimed for pressurization in food processing and preservation is that heat -labile compounds undergo limited degradation compared with heat processing (Hay ashi and others 1989). High pressure processing however usually leads to denaturation of pr otein (Bridgman, 1914; Kurth, 1986). Although this characteristic feature of high pressure has be en effectively applied to food preservation by preventing microbial activities (T imson and Short, 1965; Hoover and others 1989), there is little information on seafoods.

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25 The response of food products to HPP processi ng is complex and is affected by processing parameters, and product characteristics, such as applied pressure, dur ation of compression, temperature, product pH and water activity. The literature about th e effect of high pressure on lipids is indeed sparse as is its affect on fish lipids. Ohshima and others (1993) used pressure levels between 200 to 610 MPa for 15 to 30 min a nd suggested that isolat ed extracted marine lipids were more stable against autoxidation than lipids present in intact muscle. Angsupanich and Ledward (1998) observed little change s in thiobarbituric acid (T BA) values, an index of lipid oxidation, on cod muscle pressurized at 200 MPa for 20 min. However, in the same experiment, TBA values increased when samples were treated at 400 MPa or higher for 20 min and continued to increase during the 7 days of storage. Recently Sequ eira-Munoz and others (2005) suggested that HPP treatmen t of fish fillets prior to fro zen storage may have a curtailing effect on the oxidation of lipids in carp fillets. High pressure may significantly affect the catalytic role proand antioxidants play in fish muscle. High pressure is well known to denatu re proteins, and has been reported to denature hemoglobin and myoglobin where the heme environment was substantially perturbed (Morishima and Hara, 1983), which could translate to their increa sed ability to oxidize lipids. Alternatively, high pressure may inactivate fi sh lipoxygenases. The integrity of cellular membranes is likely affected by the application of high pressure. This ma y not only increase the susceptibility of the membrane lipids to unde rgo oxidation, but may also lead to the decompartmentalization of soluble proand an tioxidant compounds in cellular and sub-cellular structures, which could have an unforeseen effect on the pressu rized muscle oxidative stability and thus quality. As very limited information exists on the effect of high pressure on the

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26 oxidative stability and integrity of fish lipids, it is of much importance to obtain information on this as high pressure is rapidly becoming a promising seafood processing method. Applications of Irradiation on Seafoods Irrad iation processing of food products is a widely studied field of research and is currently being practiced on several commercial food products, worldw ide (Hayashi, 2007; Hileman, 2007) Microbial safety is a potential problem associated with seafood products. Recently, irradiation treatment has been re cognized as a promising method to enhance shelf-life and safety of products introduced to consumers. The purpose of food irradiation is to enhance the shelf-life and inactivate or decrease the mi crobial safety level. Mainly, the effect of irradiation in food systems is to split the water molecules into molecular oxygen and hydrogen, as well as hydrogen and hydroxyl radicals. In addition, irradiatio n damages DNA, RNA, proteins and cellular structures of microorganisms result ing in cell injury and death (D iehl, 1995). The irradiation is a cold process treatment due to only a few degrees temperature rise in foods from the radiation energy absorbed, even at a sterilization dose. Therefore, radiation treatment causes minimal changes in appearance and prov ides good nutrient re tention. It does not leave any chemical residue, and thus, can substitute for chemical fumigation; thereby reducing the need for chemical substances and allowing for the treatment of pr oducts with a wide range of sizes and shapes (Nawar, 1995). In general, there are two main advantages of using low dose ionizing radiation (< 3 kGy) as a processing method. Firstly, it will reduce or eliminate the microorganisms responsible for spoilage and subsequently exte nd the fresh-storage shelf-life. Secondly, the low dose irradiation also has the ability to reduce or eliminate specific pathogenic bact eria commonly associated with seafood (Grodner and Andrews, 1991).

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27 The effect of irradiation on the nutritional content of lipids is minimal at low and medium doses. It is also important to note that those doses will not cause the formation of aromatic or heterocyclic rings, or the conde nsation of aromatic rings, which are carcinogenic and are known to form at high cooking temperatures. However, irra diation of lipids at high dose in the presence of oxygen can result in the formation of liquid hydroperoxides, which is not dangerous but have undesirable odors and flavors (rancidity). The unsatur ated fatty acids are more prone to develop rancidity. Lipid oxidation can be significantly reduced by freezing, and/ or by oxygen removable before irradiation trea tments (Miller, 2005). The possible reaction below could be occu rring due to the ir radiation on lipids: R-CH2-O-CO-(CH2)nCH3 RCH2-O-CO-(CH2)nCH3 + e (ionization) RCH2-O-CO-(CH2)nCH3(excitation) The initial reaction of cation radicals is deprotonation, followed by dimerization or disproportionation. Excited triglycerides due to pr otonation or deprotonati on during irradiation could undergo a wide variety of reactions re sulting in different products including esters, ketones, hydrocarbons and diglycerid es (Diehl, 1995; Nawar, 1982). Fish lipids are more susceptible than red meats due to the unsaturated nature of fish lipids (Khayat and Schwall, 1983). Irradia tion of foods also produces ozone due to presence of oxygen, which leads to lipid oxidation and heme protei n oxidation. In order to solve this problem, samples should be kept in vacuum packages. In addition, it is assumed th at irradiation in the presence of oxygen triggers autoox idation as the pathways are the same as in the light-induced or metal-catalyzed autoxidation. Venugopal (1981) reported that higher lipid oxidation was observed in the presence of air. Kumta and others (1971) stated that myoglobins present in the muscle of a number of finfish species may also enhance radia tion-induced lipid oxidation. They

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28 reported no significant difference between thiobarb ituric acid (TBA) values for non-irradiated and irradiated fish fillets at 0.05 kGy. Przybylski and others (1989) repo rted that irradiation of catf ish fillets at 0.5 kGy had no significant effect on lipid oxidation for both air and CO2/air atmospheric packages. They also concluded that the optimum irradiation dose was about 2 kGy. Ghadi and Venugopal (1991) investigated the influence of ga mma irradiation up to 5 kGy on li pid oxidation in skin and flesh fractions of Indian mackerel, white pomfret, and seer during ice storage. They found increase in TBA values in both control and irradiated fish, especially in mackerel and seer meat. Rao and Bandyopadhyay (1982) observed that i rradiation did not influence hydr olysis of triglycerides in Indian mackerel; there was a progressive decrease in the initial content of triglycerides with an increase in free fatty acids during refrigerated storage of both unirradiated and irradiated fish. Adam and others (1982) found no significant difference in th e proportion of polyunsaturated fatty acids from vacuum packed herring at 50 kGy. AlKahtani and others (1996) investigated the influence of irradiation on lipid composition of tilapia and Spanish mackerel at 1.5 to 10 kGy and reported that some fatty acids decreased. However, Armstrong and othe rs (1994) did not find any changes in fatty acids composition of two Aust ralian marine fish species at doses up to 6.0 kGy. The authors also stated that variations in fatty acid composition between individual samples were greater than any radiation-induced changes. One of the major differences between s eafood from other muscle foods is the predominance of polyunsaturated fatty acids (PUFA) in the lipid composition of fish. PUFA has a higher amount of unsaturated fatty acids compared to saturate or mono unsaturated fatty acids, and PUFA is generally more susceptible to oxida tion than saturated fatty acids. Therefore, PUFA might be more irradiation sensitive than other lipid componen ts; however, Diehl (1995) stated

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29 that evidence from meat studies showed that th e protein components of m eat may protect lipids from oxidative damage. Adam and others (1982) conducted research on the effects of radiation on the concentration of PUFA in herring by ir radiating herring fillets at 50 kGy, which is a higher dose than recommended for molluscan shellf ish. The authors concluded that there was no effect on the concentration of PUFA. Sant'Ana and Mancini (2000) studied th e effects of radiation on the composition of fatty acids in fish fillets. The authors researched two monounsaturated fatty acids and seven PUFA including three different omega-3 fatty acids before and after irradiation treatment at dos es up to 3 kGy. The concentrati on of total monounsaturated fatty acids changed very little while approximately 13% reduction was seen in total PUFA at the highest dose, largely attributable to a loss of the long chain PUFA, including DHA (docosahexaenoic acid). However, the overall change for essential fatty acids including linoleic and linolenic acids was negligible. In addition, th e concentrations of PUFA in various seafoods was not significantly affected by irradiation (Morehouse and Ku, 1992). Although seafood irradiation processing has been studied for over 45 years and is also available in European and Asian markets, F DA has not approved it in the United States. Currently, there are two active petitions for mollu scan shellfish and crustaceans pending in the FDA Office of Food Additive Safety (previ ously the Office of Premarket Approval). In conclusion, irradiation is an effective tool that can improve the safety and quality of seafood when used properly. It is a unique col d process to reduce the risk of bacterial contaminants, improve shelf-life and maintain qu ality of seafood. Many studi es have proven that the effect of irradiation on the nutritional content of lipids is mi nimal at low and medium doses, which will not cause the formation of aromatic or heterocyclic rings, or the condensation of aromatic rings, which are carcinogenic and known to form at high cooking temperatures. PUFA

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30 is generally more susceptible to oxidation than saturated fatty acids, thus PUFA might be more radiation sensitive than other lipid components, on the other hand, some studies showed no significant change in fatty aci d profiles upon irradiation at low and medium dose. Seafood irradiation is underutilized in the current seafood market in the United States due to FDA regulations.H. R-H R.ROOH RO. + OH. 2ROOH ROO. + RO. + H2O R. + O2ROO. ROO. + RH ROOH + R. RO. + RH ROH + R. R. + R. R-R ROO. + R. ROOR ROO. + ROO. ROOR + O2 Figure 2-1. Lipid oxidation in a polyunsaturated fatty acid (R H) involves a) initiation b) propagation and c) termination.

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31 Figure 2-2. Chemical structure of va rious carotenoids (a) Lycopene (b) -carotene (c) Zeaxanthin (d) Cantaxanthin (e) As taxanthin Source: (Urich, 1994).

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32 CHAPTER 3 EFFECT OF HIGH PRESSURE TREATMENT ON THE QUALITY OF RAINBOW TROUT ( Oncorhynchus mykiss) AND M AHI MAHI ( Coryphaena hippurus) Introduction Although the first attem pts to apply high pre ssure processing (HPP) technology to foods dates back to the late 19th century, the grea t potential of HPP technol ogy for the food industry was first recognized in the 1980s. Treatment of food with high hydrostati c pressures has been applied to food processing and preservation (Hoover and others 1989), and HPP technology has since become one of the most popular subjects of study in food engineering and technology. One of the distinct advantages clai med for pressurization in food pro cessing and preservation is that it can destroy microorganisms while heat-labile compounds undergo limited degradation compared with heat processing (Hayashi and others 1989). HPP of foods can redu ce the microbial load without adding any chemical substances, while fl avor, vitamins, color and other properties of foods are unchanged, or changed only to a small extent (Grant and ot hers 2000; Hendrickx and others 1998; Ohshima and others 1993). Seafoods are highly perishable and usually spo il faster than other muscle foods. Moreover, they are more vulnerable to post-mortem textur e deterioration than other meats (Ashie and Simpson, 1996; Simpson, 1998) and th eir shelf-life is limited by th e growth of different strains of spoilage bacteria (Lambert a nd others 1991). The effects of pre ssure on the structural, textural and color changes of seafoods are more obvious and variable compared to many other foods, being beneficial in some cases and detrimental in others. The major purpose of HPP is to enhance the safety of mostly raw seafoods by inactivating micro-organisms and parasites without changing sensorial quality attribut es (Cheftel and Culioli, 1997). Lipid oxidation of seafood is well known and many researchers have demonstrated that various proteins from seafood are involved as catal ysts. However, very little information exists

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33 on HPP of seafood, and more specifi cally, its effect on lipid oxida tion. Dark muscle has not only a higher amount of unsaturated lipi ds than light muscle but also a higher amount of pro-oxidants such as iron and heme proteins (Hultin, 1994). The lipids in dark muscle are more susceptible to lipid oxidation than light muscle as dark musc le has more unsaturated membrane lipids. Since fish has a high concentration of polyunsaturated lipids, oxidative cha nges induced by pressure could be very significant and st udies in this area are very limited. The oxidative changes during processing and subsequent storage directly a ffect the quality of s eafood products. Previous studies have reported an increase in the levels of both peroxide value (PV) and tiobarbituric acid reactive substances (TBARS) for fish muscle trea ted with high pressure. This could be because certain metal ions or heme proteins may cause an increase in autoxidation of lipids. Purified lipids on the other hand have been found to be stab le against high pressure (Ohshima and others 1993). Color and appearance of muscle have a dir ect influence on value a nd acceptance of most fish species and meat products. Total myoglobin c ontent has a great impact on muscle color. The relative proportions of oxy-myoglobin/hemoglobin (bright red), myoglobin/hemoglobin (dark red), and met-myoglobin/hemoglobin (grey-brown) produce various colors in meat. Denaturation of these proteins shifts them initially to the oxidized met-myoglobin/hemoglobin form and then to irreversibly denatured forms, significantly affecting muscle color. High pressure could denature heme proteins in muscle, and thus, have an impact on color. HPP can also have an impact on other changes in the meat such as muscle proteins, which could change their functional properties resulti ng in textural changes. Although there is increasing inte rest in the application of high pressure processing technology to fish-based products, limited research has been performed regarding the use of HPP

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34 in the development of high quality fresh s eafood products. The purpose of this study was to investigate the effect of differe nt pressures on the quali ty changes (color, lipid oxidation, texture and total plate count) for rainbow trout (a fres h water species) and mahi mahi (a salt water species) during cold storage. Materials and Methods Fresh whole m ahi mahi ( Coryphaena hippurus) were purchased from a local seafood supplier (Save-on-Seafood, St Peters burg, FL). Fresh rainbow trout ( Oncorhynchus mykiss ) was purchased from a local market. Fish samples were filleted aseptically an d vacuum packaged. All equipment were sterilized by using bleach and followed by ethanol before and during the entire experiments to minimize contamination. High Pressure Processing The high-pressure equipm ent consisted of a St ansted laboratory scale unit (Stansted Fluid Power, Stansted, Essex, UK) with a pressuri zation chamber of 114 mm diameter and 243 mm height, providing a usable volume of approximately 2.5 L. A minimum of three fillets were used for each pressure level and all fi llets were randomly chosen. Skinne d fillets from fresh fish were vacuum packaged and treated at different pressu res (from 150 to 600 MPa) for 15 min initially at room temperature. Temperature and pressure pr ofiles of HPP at 600 MPa for 15 min is shown in Figure 3-1 in order to give general idea about the processing condi tions including maximum temperature achieved. Processing temperature wa s 17.4C at the beginning of treatment. The temperature increased to a maximum of 33.9 C at 600 MPa for 15 min pressurization. The temperature then gradually decreased and when pressure was completely released the final temperature was 5.4C. After treatment samples we re stored for 6 days at 4C. Samples were removed aseptically from vacuum bag and placed into oxygen permeable bags for storage. Red muscle was analyzed every two days for lipid oxidation by measuring thiobarbituric reactive

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35 substances (TBARS). Total aerobic count and colo r analysis were also performed every two days on the whole muscle. Images of the fillets were captured using a color machine vision system (CMSV) equipped with a video camera and aver age color parameters (L*, a* and b* values) were determined (Luzuriaga and others 1997). Texture profile analysis was also performed (Angsupanich and Ledward, 1998; Bourne, 1978). Microbial Analysis Total aerobic m icrobial growth before and after HPP treatment was determined using PetrifilmTM (3M Laboratories, St. Paul MN) acco rding to the AOAC Official Method 990.12 (AOAC., 1995). The 3M PetrifilmTM aerobic plate is a ready ma de medium that contains standard nutrients, a cold water soluble gel ling agent and a tetrazolium indicator dye, which facilitates colony enumeration. Analysis was done on 10 g fish muscle mixed with 90 ml sterile pre-filled dilution vials of 0.3 mM monopotassium phosphate buffe r solution at pH 7.2 (Hardy Diagnostic, Santa Maria CA). Th e solution was then mixed in a stomacher for 1 min and the pH adjusted to 6.6 7.2 with 1N Na OH and then serially diluted (10-1 10-7). For inoculation, 3M Petrifilm TM was placed on a sterile flat surface and 1.0 ml of the sample was placed at the center of the film and spread by a sterile plastic spreader to an area of ~20 cm2. Duplicate inoculations were conducted for each dilution and no more th an 10 plates were stacked at 35.5C for an incubation time of 48 hours. Lipid Oxidation Analysis Developm ent of lipid oxidation in fresh-water and salt-water fish species was measured by analyzing secondary products of oxidation in the dark muscle according to the thiobarbituric acid reactive substance (TBARS) met hod (Raghavan and Hultin, 2005). Dark muscle tissue (5 g) was blended with 15 ml of TCA extracting solution (7.5% trichloroacetic acid in water, 0.1% propyl gallate and 0.1% EDTA) using a Waring co mmercial blender (War ing Products Division,

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36 Dynamics Corp. of America, CT) for 30 s in a plastic beaker, the suspension filtered using Whatman #1 filter paper and then 2 ml of suspension was mixed with 2 ml of TBA (thiobarbituric acid) in a scre wed cap tube. The tube was vortexed for 10 s, and placed into boiling water for 40 min. Finally, the tube was placed in ice for 5 min, and absorbance of samples was measured at 530 nm. A standard pl ot was prepared using tetraethoxypropane (TEP). As each mole of TEP would yield one mole of malonaldehyde, the results were expressed as micromoles of malonaldehyde (MDA) per kg tiss ue. All analyses were done in triplicate. Color Analysis Color was measured throughout storage by th e Color Machine Visi on System (CMVS) consisting of a light box and a CCD color cam era connected to a computer with a firewire connection. A software program developed was used to capture images, and to obtain color results based on L* (lightness), a* (redness), and b* (yellowness) values. Fish fillets were placed in the light box and the digital camera captured a picture of the fillets for each analysis time point. CMSV was calibrated by a standard us ing red plate ( L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). Average L*, a*, b* va lues of whole fillet surface were calculated using a color analysis program (Luzuriaga and others 1997; Yor uk and others 2004). The reason for choosing the color machine vision system is discussed in Appendix. Texture Profile Analysis (TPA) Pressure treated rainbow trout a nd m ahi mahi fillets were cut into rectangular shapes with dimensions 2x2x1 cm and 2x2x1.5 cm, respectively. R ectangular cuts were taken from either side of the midsection of fillets. All samples were dried with filter paper after treatment and stored at 4C prior to TPA, which was performed using an Instron Univer sal Testing Instrument, model 4411 (Canton, MA) at room temper ature. Samples we re stored at 4oC for not more than 1 h prior to TPA analysis. Eight replicates for ea ch treatment were comp ressed twice to 70% of

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37 their original height at 100 mm/min speed and 100 Newton comp ression load using a cylindricalshaped probe (38mm in diameter). Texture an alysis parameters (hardness, adhesiveness, chewiness, springness, cohesiveness and gumminess) were calculated using the Blue Hill Software (Norwood, MA). Statistical Analysis Color data (L*, a*, b* values) was reported as m ean and standard deviation of all pixels for the whole su rface of the fillets. Texture, microbi al analysis, and lipid oxidation data were analyzed by analysis of variance (ANOVA) and the mean separations were performed by LS Means Tukey HSD (P<0.05) using the JMP 5 so ftware (SAS Institute Inc., Cary, NC). Results and Discussion Microbial Analysis Total m icrobial count is an im portant criterion for quality evaluation in fresh and frozen seafood products. According to the Internationa l Commission of Microbi ological Standards for Foods (ICMSF, 1978), the maximum acceptable microbial limit in fresh and frozen fish is 107 CFU/g. Microbial evaluation of pressure treated rainbow trout sa mples is presented in Figure 32a. It was evident that higher pressures (450 and 600 MPa) reduce the aerobic microbiological counts by 4-6 log cycles. Processi ng rainbow trout at 450 to 600 MP a for 15 min did not lead to any bacterial growth during 6 days of subsequent storage. Extended storage studies are needed to determine the length of shelf-lif e after high pressure treatment Although there was no significant (P>0.05) difference in microbia l load between 150 MPa and untreated samples at day 1, 150 MPa samples had significantly (P<0.05) lower microbiological counts than untreated samples at day 3 and day 6. The effectiveness of 300 MPa in reducing microorganism was in between that of 150 MPa and 450 MPa.

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38 Figure 3-2b represents the effect of different pressures on micr obial levels in mahi mahi muscle during a six day storage period after HPP. Si milar results were obtained for mahi mahi as rainbow trout, as the control had a typical rise in bacteria l numbers through storage. The 150 MPa process was not very different from cont rol during storage, except for day 3 where a reduced load was seen compared to contro l. The 450 and 600 MPa treatments were very effective and no bacterial growth was detected on those samples even after 6 days storage. Overall, high pressure processing can eliminate microbial loads and extend shelf-life. In another study, it was seen that pressures above 300 MPa resulted in no bacteria l growth on Atlantic salmon muscle (Yagiz and others 2005). These results were in agreement with the findings of mahi mahi and rainbow trout. The effect of hi gh pressure processing on mahi mahi and rainbow trout fillets showed similar tr ends on the microbial evaluations during 6 days storage. Other studies have demonstrated that HPP reduces mi crobial load on various foods such as mango puree (Guerrero-Beltran and ot hers 2006), green beans (Krebbers and others 2002), pork (Shigehisa and others 1991), octopus (Hurtado and others 2001) and al bacore tuna (RamirezSuarez and Morrissey, 2006). Lipid Oxidation Analysis Lipid oxidation was studied us ing the TBARS m ethod to mon itor levels of secondary oxidation products formed. TBARS measures a va riety of secondary products, but mainly malondialdehyde (MDA). This method has been f ound to agree well with rancidity development in seafood based systems when compared to sensory analysis (Richards and Hultin, 2002; Richards and others 2002; Ri chards and Hultin, 2000). Lipid oxi dation in rainbow trout dark muscle increased as pressure increased beyond the 300 MPa treatment (Figure 3-3a). Lipid oxidation further significantly (p<0 .05) increased at the two higher pressure treatments (450 and 600 MPa) as rainbow trout was stored at refri gerated conditions for 6 days. Lipid oxidation

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39 increased from 6 mol MDA/kg fish to almost 70 mol MDA/kg fish after 3 days storage and from 13 mol MDA/kg fish to almost 100 mol MDA/kg after 6 days storage for rainbow trout as pressure increased. Polyunsaturated fats are susceptible to lipid oxi dation; however, further analysis of the fatty acids after treatment is re quired to determine the effect on polyunsaturated fatty acids in rainbow trout. Mahi mahi control and mahi mahi treatment at 150 MPa showed similar trends in oxidation, with the highest oxidati on occurring after 3 days storag e and then decreasing after 6 days storage (Figure 3-3b). The 150 MPa treatm ents did however have significantly (p<0.05) higher oxidation values than the control tr eatment. The higher pr essures (300, 450, and 600 MPa) showed similar trends with a continued increase in lipid oxidation to day 6 storage. The highest lipid oxidation occurred in fish treated at 300 MPa. The result at pressure level of 150 MPa for 15 min for rainbow trout showed no significant (p>0.05) difference from untreated samples on lipid oxidation. This result is in agreement with research by Chevalier and others (2001) who reported that 14 0 MPa pressure and below for 15 and 30 min had little effect on lipid oxidation of turbot muscle while 200 MPa for 30 min had significant effect on lipid oxida tion. Angsupanich and Ledward (1998) found that the initial TBARS value of pressurized cod muscle changed little compared with fresh samples; however, they stated that 400 MPa or highe r pressure levels resulted in increasing TBARS values during 7 days storage. Their findings were in accordance with both mahi mahi and rainbow trout muscle, which showed higher lipid oxi dation, as TBARS, for 450 MP a and 600 MPa compared with untreated samples during 6 days storage. The acceleration of oxidati on could be caused by denaturation of heme protein by high pressure releasing heme and metal ions, which promote auto-oxidation of lipids (Ohshima and ot hers 1993; Tanaka and others 1991).

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40 Color Analysis Table 3-1 shows the changes in L*, a* and b* values in pressure treated and untreated sam ples during 6 days storage. L* values increa sed as pressure increas ed and did not change after 3 days storage. However, L* values increased after 6 days storage compared to the other two days. After HPP, there was a decrease in a* values, or redness. It wa s reported that loss of red color occurs in parallel wi th TBARS development during cold storage of yellowfin tuna (Lee and others 2003) and washed cod muscle (W etterskog and Undeland, 2004). Changes in color (L* a*, b* values) for mahi mahi muscle after HPP treatment followed by storage for 6 days at 4C are shown in Table 3-2. L* value for mahi mahi increased slightly as a function of increased pressure; however, there was little difference be tween L* values during storage. The control showed the lowest L* value of all treatments. The a* value, which represents redness when positive, decreased as the pressure treatment increased. The a* value also tended to decrease as storage time increased, demonstrating a loss in re dness of the muscle with pressure and storage time. The b* value, which represents yellowness when positive, increased over the control for all pressures tested. The muscle developed a cooked appearance as the pressu re treatment increased. This would also cause an increase in b* value as the muscle coagulates forming a whitishyellowish appearance. This is also reflected in the increase in L* values for the high pressure processed samples. The color results for rainbow trout and mahi mahi agreed with Yagiz and others (2005), who reported that redness of A tlantic salmon fillets d ecreased with increasing pressure while L* value significantly in creased with increasing pressure. A cooked appearance is very common in high pressure processing of muscle systems and demonstrates that high pressure can denature both sarcoplasmic and myofibrillar proteins in muscle (Angsupanich and Ledward, 1998). Since some of these proteins are also catalysts (prooxidants) for lipid oxidation, futu re studies should focus on eval uating the specific pro-oxidant

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41 components and how they influence lipid oxidatio n with high pressure processing. Additionally, it is interesting that after 150 MPa treatment, rainbow trout (Figure 3-4a) demonstrated a completely cooked appearance but mahi mahi (Fi gure 3-4b) did not. Mahi mahi is a warm water species while rainbow trout is a cold water species. It is likely that the proteins in mahi mahi are more stable towards denaturation, and thus, tolerant to higher pressures than proteins in rainbow trout (Hastings and others 1985). Chaijan and others (2006) re ported that oxidation and denaturation of myoglobin caused discoloration of the muscle, decreased extractability of the myoglobin and released non-heme iron; thereby accelerating lipid oxid ation in sardine and mackerel mu scle during ice storage. It is well-known that HPP has a direct effect on dena turation of globin proteins. Carlez and others (1995) stated that there are two main possible explanations for m eat discoloration due to HPP. The authors suggested that the whitening effect either in the range of 200 and 350 MPa with 10 min holding time was due to denaturation of glob in proteins and heme displacement/release or could be oxidation of ferrous myoglobin to ferric metmyoglobin above 400 MPa. Texture Profile Analysis Results for texture profile analysis (T PA) in terms of hardness, adhesiveness, chewiness, springiness, cohesiveness and gumminess for rainbow trout and mahi mahi, are shown in, Tables 3-3a and 3-3b, respectively. Treating rainbow trout with 450 and 600 MPa pressure gave significantly higher (p<0.05) hardness values than other pressure treated and untreated samples, while no difference was observed between 0.1 and 150 MPa (Table 3-3a). The untreated mahi mahi muscle also had the lowest hardness value of all treated samples (Table 3-3b). The highest pressure gave the highest hardness values. The effect of pressure on the hardness values of trout and mahi mahi fillets could be explained using myofibrillar protein denaturation and aggregation. Cheftel and Culioli ( 1997) stated that myosin from both meat and fish source would

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42 be denaturated by pressure and as a conseque nce form a gel-like texture. Yoshioka and Yamamoto (1998) studied the effect of 50-500 MPa for 10 min on the ultra-structure of carp muscle myofibrils. At 100 MPa, the structure of myofibrils was quite similar to control but the sarcoplasm expanded between myofibr ils. Also the first degradation of thin filaments occurred at 300 MPa and the disappearance of the normal stru cture of myofibrils was observed at 400 MPa and 500 MPa. The authors concluded that HPP a ffects contractile protei ns especially, actin; however, the denaturation of those proteins was at a different degree than the effect of heating due to the difference in ultra-structure change s which might have an influence on both physical properties and taste of fish muscle. In our current research we also found that ch ewiness for both species increased as pressure increased. There was no significant (p>0.05) differe nce between the control and samples treated at pressures 150 and 300 MPa for mahi mahi with respect to springiness. However, the 450 and 600 MPa pressure rainbow trout ha d significantly higher (p<0.05) sp ringiness compared to other treatments. Similar results were reported by Angsupanich and Ledward (1998) who found that treating cod muscle at 400 and 600 MPa for 20 min led to an increase in springiness and subsequent unfolding of actin re sulted in increase in gumminess, hardness and adhesiveness. They stated that the formation of the hydr ogen bonded networks may contribute to these observations. From our observations (Tables 33a and 3-3b), cohesivene ss was significantly higher (p>0.05) at higher pressure for rai nbow trout; however, there was no significant difference between treatments except for 150 MPa having lowest cohesiveness for rainbow trout. Higher pressures showed significant increase in gumminess for both species. Overall, texture profile analysis results show ed that 450 and 600MPa led to significantly (p<0.05) higher hardness, gumminess and chewiness for one sample when compared to control for both species.

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43 There was no significant change in these para meters (p>0.05) between 150 MPa and untreated samples. Conclusion It was found that HPP significantly reduced m icrobial load, with HPP treated samples at 450 and 600 MPa having the most reduction in micr obial growth. This study demonstrated that 300 MPa for rainbow trout and 450 MPa for ma hi mahi are the optimum high pressure processing conditions for contro lling microbial load, lipid oxi dation and color changes. TPA results showed that 450 and 600 MPa led to sign ificantly (p<0.05) higher hardness, gumminess and chewiness when compared to control for both species, while a treatment of 150 MPa resulted in minimum textural changes compared to untreat ed samples. Overall, these results prove the usefulness of HPP in seafood processing while provi ding quantitative parameters in order to help with the application of this process.

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44 Table 3-1. Changes in a* valu e (redness), L* value (lightness) and b* value (yellowness) for rainbow trout muscle after HPP treatment followed by storage for 6 days at 4C. Control was untreated muscle stored unde r the same conditions as treatments. L* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa450 MPa 600 MPa 1 60.7.5 72.6.9 81.7.9 82.9.6 82.0.0 3 60.7.6 72.9.3 82.0.3 82.3.0 82.1.9 6 67.8.1 77.7.0 85.9.3 86.8.0 86.61.0 a* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa450 MPa 600 MPa 1 11.5.6 10.6.3 4.7.0 2.4.8 2.9.9 3 14.9.3 14.0.3 3.3.1 2.2.5 2.0.24 6 14.2.6 11.7.6 0.91.5 0.61.7 -0.63.6 b* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa450 MPa 600 MPa 1 -1.23.2 -0.34.1 -2.5.6 -1.35.1 -0.68.6 3 -2.8.7 -3.2.9 -4.8.3 -1.6.9 0.76.7 6 16.4.1 17.5.3 12.6.1 10.2.7 12.7.6 Values are meansSD for all pixels of the surface of the fillet.

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45 Table 3-2. Changes in a* valu e (redness), L* value (lightness) and b* value (yellowness) for mahi mahi muscle after HPP treatment follo wed by storage for 6 days at 4C. Control was untreated muscle stored under th e same conditions as treatments. L* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa 1 51.0.363.3.666.9.271.2.8 73.4.8 3 51.3.063.85 65.86 70.63.7 74.79.9 6 48.3.764.1.7 66.1.470.6.9 74.9.7 a* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa 1 19.0.1 16.4.7 12.2.7 10.6.8 12.1.4 3 13.5.6 14.6.1 10.9.2 10.9.3 8.4.6 6 19.1.2 13.9.3 9.6.4 10.8.9 6.6.2 b* value for light muscle Storage time(day) 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa 1 6.6.7 9.8.9 10.7.9 11.3.8 12.4.2 3 8.9.6 10.9.0 10.5.7 10.6.0 14.3.0 6 6.1.5 12.5.5 11.45.211.45.7 16.65.7 Values are meansSD for all pixe ls of the surface of the fillet.

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46 Table 3-3. Texture profile anal ysis (TPA) in terms of hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness for (a) Rainbow trout and (b) Mahi mahi muscle after high pressure processing. (a) Rainbow trout Pressure (MPa)Hardness (N)CohesivenessAdhesiveness (J)Springiness(mm)Gumminess(N)Chewiness(N*mm) 0.122.8.6bc0.160.05bc0.00018.0a3.49.9ab3.68.5bc12.4.7bc 15017.6.4c0.170.04c0.00034.0a2.76.4b3.01.1c8.4.3c 30029.1.0b0.190.06b0.0024 .0b3.56.4ab5.90.8b21.7.7b 45040.55.8a0.25.04a0.0014.0ab4.250.07a10.21.1a43.317.8a 60040.1.9a0.220.06a0.00180.0b4.13.5a8.58.7a35.6.6a (b) Mahi mahi Pressure (MPa)Hardness (N)CohesivenessAdhesiveness (J)Springiness(mm)Gumminess(N)Chewiness(N*mm) 0.126.3.3d0.300.08a0.00066.0a3.57.3b7.30.4c26.1.7c 15044.9.1bc0.170.04b0.00067.0a3.36.3b7.63.1c25.6.3c 30048.73.2b0.28.07a0.00095 0.0a3.630.5b13.63.4b49.417.7b 45039.3.7c0.290.1a0.00062.0a4.11.5a11.1.7b45.5.8b 60068.8.1a0.280.04a0.00201.0b4.46.4a19.6.5a87.2.2a Values are Means standard deviations, n=8, different letters within a column indicate significant differences at P < 0.05 separated by Tukeys HSD.

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47 Figure 3-1. The 600 MPa process sh owing temperature and pressure profiles. All other pressures showed lower results for pressure and temperature than those presented here.

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48 (a) a a a a b b b c c c d d c dd 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Day 1D a y 3D a y 6log cfu/g 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa (b) a a a a b a b c b c d c c dc 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Day 1D a y 3D a y 6log cfu/g 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa Figure 3-2. Total aerobic plate count for (a) ra inbow trout and (b) mahi mahi after 15 min high pressure processing followed by storage at 4 C for 6 days. Different letters within each day indicate significant differences at P < 0.05 separated by Tukeys HSD.

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49 (a) a d c a c c a cd c b b b b a a 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Day 1D a y 3D a y 6TBARS (mol MDA/kg) 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa (b) c d d ab b c bc a a ab c b a c b 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 Day 1D a y 3D a y 6TBARS (mol MDA/kg) 0.1 MPa 150 MPa 300 MPa 450 MPa 600 MPa Figure 3-3. Changes in lipid oxidation (sec ondary oxidation products ) as TBARS for (a) rainbow trout dark muscle and (b) mahi mahi after HPP followed by storage for 6. Different letters within each day indicate si gnificant differences at P < 0.05 separated by Tukeys HSD.

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50 (a) (b) Figure 3-4. Images for (a) rainbow trout and (b ) mahi mahi at differe nt pressures after HPP treatment and storage for 6 days at 4 C.

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51 CHAPTER 4 EFFECT OF HIGH PRESSURE PROCESSING AND HEAT TREATMENT ON THE QUALIT Y OF ATLANTIC SALMON Introduction Recognition of the health benefits associated with the consumption of omega-3 fatty acids from seafoods is one of the most promising developments in nutrition research in the past 20 years. Research has provided a wide array of explanations for the positive influence of omega-3 fatty acid. Omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have direct effects on the heart musc le itself, increasing blood flow, decreasing arrhythmias, improving arterial compliance, decr easing the size of the infarct, and reducing several chemical and cellular processes that co mpromise heart function (Nestel, 1990). As a result, seafood remains a hea lthy, attractive choice to cons umers. NOAA Fisheries Service reported that the consumption of seafood in the U.S. has increased 16.5 pounds per person in 2006 (NOAA, 2007). In general, seafoods are highly perishable with a 14 day shelf-life for a fresh or thawed product. Usually beyond 7 days of cold storage the product is considered being of a lower grade and frequently sold at reduced cost or discar ded. Moreover, seafoods are more susceptible to post-mortem textur e deterioration than meats from land animals (Ashie and others 1996). Processing techniques that can extend the sh elf-life of seafood over 14 days and also can dramatically change the sensor y attributes and characteristics of the product beyond the fresh quality demanded by consumers. Heat processing can lead to inactivatio n of microbial growth and lipolytic enzymes. However, it may damage vitamins, flavor compounds and polyunsaturated fatty. High pre ssure processing (HPP) has been applied to foods as a preservation method with its major advantag e being the maintenance of the fresh quality attributes of foods. High pressure processing is a promising seafood preservation method. This novel technology reportedly provides long shelflife and minimum quality loss since it does not

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52 have many of the undesirable changes that are associated with thermal processing. There is limited information on the influence of high pres sure on the oxidative stability and quality changes of fish muscle. The major purpose of H PP is to enhance the safety of mostly raw seafoods by inactivating microorganisms and pa rasites without changing sensorial quality attributes (Cheftel and Culioli, 1997). Color of meat and muscle product plays an im portant role in the consumer perception of meat quality (Jeremiah and others 1972). St udies on seafood products have shown that consumers associate color with the freshness of a product having better flavor and higher quality (Gormley, 1992). Many researchers claim that HPP has less impact on color compared to thermal processing treatments. Applications of HPP in food processing and preservation fo llow the premise that it can destroy microorganisms while heat-labile compounds undergo limited degradation compared with heat processing (Hayashi and others 1989). High pressure processing can diminish the microbial load without any addition of chemical substances, while flavor, vitamins, color and other properties of foods are presumably unc hanged, or changed only to a small extent (Hendrickx and others 1998; Ohshima and others 1993). One major problem associate with seafoods is lipid oxidation. Various compounds can intiate and mediate oxidation in seafoods, including heme proteins, which on pressurization can become denatured and more pro-oxidative. Limited information is available on HPP of seafood, and more specifically, its effect on lipid oxidation and fatty acid profile. Dark muscle has not only a higher amount of unsaturated lipids than light muscle but also a higher amount of prooxidants such as iron and heme proteins (Hul tin, 1992). The lipids in dark muscle are more vulnerable to lipid oxidation than light muscle because dark muscle has more unsaturated

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53 membrane lipids. Since fish has a high con centration of polyunsaturat ed lipids, oxidative changes induced by HPP could be very significant and studies in this ar ea are very limited. The oxidative quality changes during processing and following storage di rectly affect the quality of seafood products. The aim of this study was to i nvestigate the effect of different pressures or cooking on the quality changes (color, lipid oxidation, fatty aci ds profile, texture and total plate count) for Atlantic salmon fillets during cold storage. Materials and Methods Thirte en Atlantic salmon ( Salmo salar ), with average length and weight of 81.6.1 cm and 5.5.2 kg, respectively, were purchased from a local seafood supplier (Save-on-Seafood, St Petersburg, FL) within two days of harvest and the fish was transported to the laboratory in ice. The fish was filleted and skinned, and fillets we re vacuum packaged using FoodSaver Vacloc vacuum bags (Jarden Co. Rye, NY). All equipm ent was sterilized by using bleach followed by ethanol before and during the entire experiments to minimize contamination. High Pressure Processing The high-pressure equipm ent consisted of a St ansted laboratory scale unit (Stansted Fluid Power, Stansted, Essex, UK) with a pressuri zation chamber of 114 mm diameter and 243 mm height, providing a usable volume of approximately 2.5 L. Skinned fillets from fresh fish were vacuum packaged and treated at different pressures 150 and 300 MPa for 15 min initially at room temperature. Initial, maximum and final vessel temperature was r ecorded at 19.4, 32.6, and 16.1C, respectively. After treatment samples were stored for 6 days at 4C. Samples were removed aseptically from vacuum bag and placed into oxygen permeable bags for storage study.

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54 Cooking Treatment Skinless va cuum packed fillets were placed in to a water bath containing boiling water. A USB data logger model DAQ-56 (Omega Engineeri ng, Stamford, CT, USA) was connected to a T-type thermocouple probe placed into center of fillets for temperature measurements. The thermocouple probe was calibrated using either boiling water or ice mixed water in the temperature range of 0 to 100C. When the center temperature of fillets reached 72C, all fillets were removed and placed in ice to cool down. The temperature of samples was recorded during both cooking and cooling. After treatment, sample s were removed aseptically from the vacuum bags and placed into oxygen permeable bags and st ored for 6 days at 4C. Temperature profile of Atlantic salmon fillets during cooking and cooling was shown in Figure 4-4. Microbial Analysis Total aerobic m icrobial growth before and after HPP treatment was determined using PetrifilmTM (3M Laboratories, St. Paul MN) acco rding to the AOAC Official Method 990.12 (AOAC., 1995). Analysis was done on 10 g fish mu scle mixed with 90 ml sterile pre-filled dilution vials of 0.3 mM monopota ssium phosphate buffer solution at pH 7.2 (Hardy Diagnostic, Santa Maria CA). The solution was then mixed in a stomacher for 1 min and pH adjusted to 7.2 with 1N NaOH and then serially diluted (10-1-10-7). For inoculation, 3M PetrifilmTM was placed on a sterile flat surface and 1.0 ml of the sample wa s placed at the center of the film and spread by a sterile plastic spreader to an area of ~ 20 cm2. Duplicate inoculations were conducted for each dilution and no more than 10 plates were stacked at 35.5C for an incubation time of 48 hours. Color Analysis The surface of color for treated and u ntreated Atlantic salmon muscle was measured during storage by a color machine vision system, cons isting of a light box and a CCD color camera

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55 connected to a computer with a firewire conne ction. A color machine vision software program was used to capture images, and to obtain colo r results based on L* (lig htness), a* (redness), and b* (yellowness) values. Fish fillets were placed in the light box and the digital camera captured a picture of the fillets for each analysis time point. The machine vision system was calibrated by using a standard red plate (L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). Average L*, a*, b* values of whole fillet surface were calculated using a color analysis program (Luzuriaga and others 1997; Yoruk and others 2004; Yagiz and others 2007). The reason for choosing the color machine vision system is discussed in Appendix. Texture Profile Analysis Texture profile analysis (TPA) (Bourne, 1978) was perform ed as described by Yagiz and others (2007). Briefly, high pressu re treated Atlantic salmon fille ts were cut into rectangular shapes with dimensions 2x2x1.5 cm. Rectangular cu ts were taken from either side of the midsection of fillets. All samples were dried with filter paper after treatment and stored at 4C prior to TPA, which was performed using an Instron Universal Testing Instrument, model 4411 (Canton, MA) at room temperature. Samples were stored at 4C for not mo re than 1 h prior to TPA analysis. Eight replicates for each treatment were compressed twice to 70% of their original height at 100 mm/min speed and 100 Newton co mpression load using a cylindrical-shaped probe (38 mm in diameter). Texture analysis parameters (hardness, adhesiveness, chewiness, springiness, cohesiveness and gumminess) were calculated usi ng Blue Hill Software (Norwood, MA). Lipid Oxidation Analysis Lipid oxidation analy sis was performed as described by Raghavan and Hultin (2005), measuring secondary products of oxidation in dark muscle. Dark muscle tissue (5 g) was blended with 15 ml of TCA extracting solution (7.5% tric hloroacetic acid in wate r, 0.1% propyl gallate

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56 and 0.1% EDTA) using a Waring commercial bl ender (Waring Products Division, Dynamics Corp. of America, CT) for 30 s in a plastic b eaker, the suspension filtered using Whatman #1 filter paper and then 2 ml of suspension was mixe d with 2 ml of TBA (thiobarbituric acid) in a screw cap tube. The tube was vortexed for 10 s, and placed into boiling water for 40 min. Finally, the tube was placed in ice for 5 min, and absorbance of samples was measured at 530 nm. A standard plot was prepared using tetrae thoxypropane (TEP). As each mole of TEP would yield one mole of malonaldehyde, the results were expressed as micromoles of malonaldehyde (MDA) per kg tissue. All analyses were done in six replicates. Lipid Extraction Lipids were extracted from th e dark muscle using the m et hod of Bligh and Dyer (1959). Dark muscle tissue (1 g) was blended with 1 mL water using a Waring commercial blender (Waring Products Division, Dynamics Corp. of Am erica, Torrington, CT) for 30 s in a plastic beaker, and sample transferred to 10 ml round bottom screw cap centrifuge tube adding 3.75 ml of a mixture of chloroform:methanol (1:2) and vortexed for 10 min. Then 1.25 ml chloroform was added with mixing for 1 min and then 1.25 ml of water was added with mixing for another minute before centrifugation for 10 min at 6500 rp m. The lower phase was collected with a Pasteur pipette into a pre-weighed glass tube. The chloroform phase (1.88 ml) was added to a centrifuge tube, vortexed, centrifuged at 6500 rpm for 10 min and the lower phase collected into a pre-weighed glass tube. Chloroform was remo ved under a nitrogen gas stream by using a NEVAP Nitrogen Evaporator (Organomation Associ ates Inc., Berlin, MA). The weight of each sample was recorded and percent lipids determined gravimetrically. The oil recovered was flushed with nitrogen an d stored in amber vials at -80C until analysis.

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57 Preparation of Fatty Acid Methyl Esters Determ ination of fatty acid methyl esters (FAME) was performed as described by Maxwell and Marmer (1983). Oil (20 mg) was placed into a 10 mL round bottom capped centrifuge tube. Oils were dissolved in 1.9 mL iso-octane (Sigma grade 99%, St. Louis, MO) with 100 L of 10 mg/ml tricosanoic acid methyl ester (C23:0) (Supelco, Bellefonte, PA) used as an internal standard. Then 200 L of 2N KOH in methanol (1.12 g/10 ml ) was added to the centrifuge tube. After vortexing for 60 sec, the solution was cen trifuged at 3,400 rpm fo r 5 min, and the lower layer discarded. This procedure was repeated tw ice using 0.5 mL of a saturated solution of ammonium acetate in water, followed by 0.5 mL of deionized water. Fatty acid methyl esters in iso-octane were dried with the addition of approximately 200-300 mg of anhydrous sodium sulfate for 20 min, and centrifuged at 3,400 rpm for 20 min. Fatty acid methyl esters were transferred into 2 mL screw capped amber GC vials (National Scien tific, Rockwood, TN) for chromatographic analysis. Gas Chromatography (GC) Analysis FAMEs were analyzed with a HP 6890 ga s chrom atograph, equipped with a flame ionization detector and an AT-Silar-100 cyano silicone capillary column (30 m x 0.25 mm x 0.2 m) (Alltech Assoc. Inc, Nicholasville, KY). Splitless injection was used. Operation conditions were as follows: in jection port temperature, 240C ; detector temperature, 250C; initial oven temperature, 120C for 2 min, ri sing to 200C hold for 10 min, and rising to 210C hold for 4 min, and then rising to 240C hold for 5 min at a rate of 4C/min. The carrier gas was helium (1 ml/min). Retention times and peak ar eas were computed automatically by Turbochrom Workstation 6.1.1 (Perkin Elmer, MA, USA). Compounds were tentatively identified by comparison with the retention times of known standards. All standards used in the identification of peaks were purchased from Supelco (Bellefo nte, PA). The standards used were: Supelco 37,

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58 Marine Oil PUFA#1, and Menhaden Oil PUFA#3, and tricosanoic acid methyl ester (C23:0) used as an internal standard. Six fillets were used for each treatment for e ach day with a duplicate GC run. Statistical Analysis Color data (L*, a*, b* values) was reported as m ean and standard deviation of all pixels for the whole su rface of the fillets. Texture, microbial analysis, fatty acids profile and lipid oxidation data were analyzed by analysis of variance (ANOVA) and the m ean separations were performed by LS Means Tukeys HSD (P<0.05) using the JMP 5 software (SAS Institute Inc., Cary, NC). Results Microbial Analysis Microbial evaluation of pressu re or heat treated Atlantic salmon sa mples during 6 days aerobic storage at 4oC is presented in Figure 41. The initial microbial load of samples was 3.56.17 log cfu/g. The microbial load of untreat ed sample reached 6.16.11 log cfu/g after 6 days of storage. Pressurizing the fillets at 150 MP a for 15 min resulted in a 3 log cycle and 2 log cycle reduction in microbial counts at day 0 a nd 6, respectively. Samples pressurized at 150 MPa had significantly (p<0.05) lower microbiological counts than untre ated samples during storage at 4oC. Pressurizing samples at 300 MPa for 15 min led to total microbial reduction after HPP treatment. The 300 MPa treatments were very eff ective and no bacterial growth was detected for this treatment even after 6 days storage. Cooking also reduced aerobic microbial load to undetectable levels and no growth was seen during the entire storage period at 4oC. Both samples treated at 300 MPa and cooked samp les had significantly (p<0.05) lower microbiological counts than untreated samples and samples treated at 150 MPa.

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59 Color Analysis Color evaluation was perform ed using a color machine vision system where average L* (lightness), a* (redness) and b* (yellowness) valu es of the surface of dark and light muscle was measured (Figure 4-2and Table 4-1). As the pressure level increased, the sample became opaque and had a cooked appearance for both dark and li ght muscle (Figure 4-2). Table 4-1 shows the changes in L*, a* and b* values for pressure or heat treated and untreated samples during 6 days of storage at 4C. L* value for dark and light mu scle increased slightly as a function of increased pressure or heat treatment; however, there wa s little difference betw een L* values during storage. The 300 MPa treatment and cooking show ed higher L* and b* values but lower a* values for both light and dark muscle compared to control and the sample s treated at 150 MPa. Texture Profile Analysis Texture profile analysis (TPA) results (hardness, cohesiveness adhesiveness, springiness, and gumm iness) for HPP treated, cooked and untr eated samples are shown in Table 4-2. It was observed that as the pressure level increase d, hardness, gumminess and chewiness parameters increased, adhesiveness decrea sed compared to untreated an d cook samples. The highest pressure gave samples with the highest har dness, gumminess and chewiness values. The 300 MPa treatment led to significantly (p<0.05) higher hardness values than the other treatments. There was no significant (p>0.05) difference be tween samples treated with 150 MPa pressure and untreated samples in terms of hardness, adhesiveness, gumminess and chewiness. However, it was found that untreated samples were significan tly (p<0.05) different in terms of springiness and cohesiveness than samples treated with 150 MPa pressure. Cooked samples had lower hardness, cohesiveness, gumminess and chewine ss values compared to pressure treated and untreated samples (Table 4-2).

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60 Lipid Oxidation Analysis Lipid oxida tion was measured in the dark mu scle section of the salmon fillets during storage at 4C by using the TBARS method to m onitor levels of secondary oxidation products formed, mainly malondialdehyde (MDA) (Figur e 4-3). TBARS values for all treated and untreated samples significantly increased during the six day storage period at 4C. Lipid oxidation of all samples increased from 3.78 mol MDA/kg muscle to almost 116 mol MDA/kg after 6 days of storage. The increase in standard deviation with increasing storage time reflected the biological variation between the indi vidual fish sample and the analysis of whole fish. At day 0, there was only a significant (p<0.05) difference in TBARS found between samples treated at 150 MPa and untreated and co oked samples. On the second day of storage cooked samples had higher ((p<0.05) levels of TBARS compared to pressure treated and untreated samples. At this time point, no signi ficant difference (p>0.05) was observed between 150 and 300 MPa treatments. In addition, untreat ed samples showed no significant difference (p>0.05) in TBARS values than samples treate d at 150 MPa or cooked samples. At day 4 and day 6, no significant (p> 0.05) difference in TBARS was found among untreated, 150 MPa treated and cooked samples. The 300 MPa treatm ents did however have significantly (p<0.05) lower oxidation values than the other treatments at day 4 and 6 (Figure 4-3). Fatty Acids Analysis Fatty acids profile of pressure treated, cooke d and untreated A tlantic salmon dark muscle during 6 days of storage at 4 C is shown in Tables 4-3 to 4-6. Th e source of total saturated fatty acids mainly came from 14:0, 16:0 and 18:0. Th ere was no significant (p>0.05) difference in total saturated fatty acids composition (g fatty acids/100 g total fatty acids) for untreated and pressure treated (150 MPa, 300 MPa) samples. However, cooked samples were significantly (p<0.05) lower in total sa turated fatty acids than pressure treated and untreated samples. All

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61 samples had major monoenes, including 16: 1n-7, 18:1n-7, 18:1n-9, 20:1n-9 and 22:1n-11. No significant (p>0.05) difference in 18:1n-7, 18: 1n-9 at day 0, 2 and 6 was found among all sample, regardless of treatment and storage tim e. The pressure levels 150 MPa and 300 MPa did not lead to significant differe nces in 20:1n-7 and 22:1n-11 at day 0, 2 and 4 compared to untreated samples. However, cooked samples ha d significantly (p<0.05) hi gher levels of these monounsatured fatty acids than both pressure tr eated and untreated samp les during the entire storage period. Total n-6 polyunsaturated fatty acids (PUFA) were mainly composed of 18:2n-6 and 20:2n-6. Cooking samples led to significant (p<0.05) decrease in total n-6 PUFA compared to HPP treatment or no treatment. Several n-3 PUFA s were detected in the samples: 18:3n-3, 18:4n3, 20:5n-3 (EPA), 22:5n-3 (DHA), 22:6n-3. The majo r individual fatty acids making up the total n-3 PUFAs were 20:5n-3 (EPA), 22:5n-3 and 22:6n-3 (DHA). Cooking significantly (p<0.05) reduced the amount of EPA from 9.5 to 7.0 g/ 100 g at day 0. The level of EPA remained significantly low for the cooked samples compared to other treatment duri ng the entire storage period. On the other hand, HPP treatment did not change the amount of EPA at day 0 (Table 43). Cooking also significantly decreased the amount of DHA from 16.8 to 12.7 g/100 g, unlike HPP (Table 4-3), which remained lower than before cooking during the entire storage. There were no significant differences in the level of 22:5n-3 fatty acids at day 0, 2 and 4 between HPP treated and untreated samples. However, it wa s found that cooking signif icantly reduced levels of 22:5n-3 fatty acids, below that of the other tr eatments, and levels remained reduced during the entire storage time. Untreated and pressure treated samples did not show any significant difference in the levels of 22:5n-3 at day 0, 2, 4 and 6 except for 300 MPa which did show a significant (p<0.05) difference from the untreated and 150 MPa pressure treated sample at day 6.

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62 Discussion One of the important criteria for quality eval uation of fresh and frozen seafood products is the initial total m icrobial count As stated by the Internationa l Commission of Microbiological Standards for Foods (ICMSF, 1978), the maximum acceptable microbial limit in fresh and frozen fish is 7 log cfu/g. The current study indicated that the initial microbial count for untreated Atlantic salmon sample was about 3.5 which is an acceptable level to ICMSF. The 150 MPa treatment with a 15 min holding time resulted in about a two log reductio n in microbial counts. As the pressure increased to 300 MPa, with same holding time, a total reduction in aerobic microorganisms was observed and no microbial growth was seen duri ng all 6 days of refrigerated storage. Similarly, no microbial activity was found for cooked samples after cooking and during storage (Figure 4-2). The 300 MPa treatment was therefore equally as e ffective as cooking in killing aerobic microorganisms. The effectivene ss of HPP for reducing or inactivating microbial growth of seafoods has been re ported, including Sea Bass (Chere t and others 2005), Oyster (He and others 2002), octopus (Hurtado and others 2001), albacore tuna (Ramirez-Suarez and Morrissey, 2006), mahi mahi and rainbow trout (Y agiz and others 2007). Various authors have suggested that the mechanisms for the inactiv ation of microbial grow th by HPP could be denauturation/inactivation of key proteins and enzymes, and damage to cell membrane, releasing intra cellular constituents, which could play a role in microbial cell inactivation (Carlez and others 1995; Smelt, 1998). The higher the pressure the more profound these effects would be, in agreement with the results seen here for Atlantic salmon. Color and appearance of seafoods are very im portant in terms of c onsumer perception of seafood quality, and are dominant factors in consumer purch asing decision. Although seafood texture is a parameter normally not used by ma ny consumers in their buying decision of seafood, it is very important when seafoods are consumed. The highest pressure level (300 MPa) resulted

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63 in a cooked appearance, increasing 22% in L*valu e for light muscle and 32% for dark muscle and decreasing 33% in a* value for light muscle and 13% for dark muscle at the end of the storage period (Figure 4-2and Table 4-1). An increased L* value and decreased a* value as pressure is increased has been reported for diffe rent seafood species such as blue fish (Matser and others 2000), cod muscle (Angsupanich and Ledward, 1998), mackerel (Ohshima and others 1993), salmon (Amanatidou and others 2000), sheaphead (Ashie and others 1996), and turbot (Chevalier and others 2001). The mechanisms of those changes are not entirely clear. However, Carlez and other (1995) stated th at the reason for cook appearance, decreasing in a* value or increasing L* value could be either denaturation of proteins when pre ssures of 200-300 MPa are applied for 10 min, or could be due to oxida tion of ferrous myoglobi n to ferric metmyoglobin due to heme displacement/release above 400 MPa. In terms of textural changes of the salmon fillet pieces, HPP caused a significant increase in hardness, gumminess, chewiness and springiness over both control and cooked samples (T able 4-2). These textural parameters were increased as the pressure level increased. The r eason for these changes co uld be denaturation of proteins and cellular damage due to HPP. Since H PP led to increases in all textural parameters tested compared to cooking, except for adhesivene ss, it can be concluded that pressure treatment led to either more denaturation than cooking or a different type of dena turation, resulting in a unique texture for the pressurized samples. The dark muscle of Atlantic salmon had a large increase in lipid oxidation for all treated samples and control during 6 days refrigerated storage. It was observed that there was no significant (p>0.05) difference am ong the untreated, 150 MPa treate d and cooked samples at day 4 and 6, however, the 300 MPa had significantly (p<0.05) lower TBARS values compared to other samples (Figure 4-3). It is generally assumed that as the pressure level increases, lipid

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64 oxidation increases. The effect of HPP on lipid oxi dation could depend on pre ssure level, holding time, fish species and type of muscle. For example, Ramirez-Suarez and Morrissey (2006) studied the effect of different pressure levels (275-310 MPa) and different holding times (2-6 min) on oxidation of albacore t una muscle. They stated that untreated samples had higher TBARS values than all of th e HPP treated samples during a 3 month storage period. The pressure levels used in the current study are kno wn to denature proteins, as in cooking. Heme proteins are key catalysts of lip id oxidation in fish muscle, and would likely have been denatured at the pressure levels used. Even though TBARS values were higher for the 150 MPa treatment at day 4 and 6 compared to control, they we re not significantly higher. Changes in lipid membranes are also to be expected at the pressu res used, and one might expect perturbations in membranes could make them more susceptible to oxidation. Its interesting that no difference was seen at 150 MPa in terms of oxidation and those samples were actually less susceptible to oxidation at 300 MPa than all the other treatments. Research on acid treatment of fish muscle has shown that significant changes in cell membrane structure may in fact make them less susceptible to lipid oxidation even though heme proteins are significantly denatured with the same treatment (Hultin, 1994). Its possible this occurred with the high pressure treatment. In addition, salmon have higher amounts of astaxanthin compared to many seafood products. It has been stated that the antioxidation capacity of as taxanthin is 10 times greater than that of carotene, and up to 500 times greater than vi tamin E (Shimidzu and others 1996). During and after pressure treatment the changes in the mu scle structure might make astaxanthin become more available to protect lipids from lipid oxidation. Fatty acid profiles of cooked Atlantic salmon dark muscle showed significantly lower amounts of total saturated, n-3 PUFA and n-6 PUFA and significantly higher amounts of

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65 monoenes than HPP treated samples during th e entire storage period. However, the most important finding of this study was that there was no significant difference between untreated and HPP treated samples in terms of total satu rated, monoenes n-3 PUFA and n-6 PUFA fatty acid profiles (Table 4-6). This demo nstrates that HPP is a very mild process in terms of its effect on fatty acids. Seafood lipids are characterized by a high le vel of PUFA such as EPA and DHA fatty acids (Ackman, 1994). It was found that the pred ominant amount total n-3 PUFA came from EPA and DHA in Atlantic salmon dark muscle for all samples. There were no significant changes for amount of EPA and DHA for untreat ed samples and HPP treated samples during storage. However, cooking resulted in significant loss of EPA and DHA compared to untreated and HPP treated samples (Table 4-3 to 4-6). It is well known that HPP does not affect covalent bonds; on the other hand, cooking can break covale nt bonds. These findings could be correlated experimentally that HPP processi ng does not affect the polyunsatur ated fatty acids while cooking does so. Conclusion It was found that HPP and cooking significantly reduced m icrobial growth. The 150 MPa treatment had a lesser effect on the color compared to cooking and 300 MPa. While cooking and 150 MPa led to similar oxidation development as untreated control, the 300 MPa treatment effectively reduced the samples susceptibility to oxidation. Cooking sign ificantly reduced the amount of total PUFA n-6 and PUFA n-3, in cluding EPA and DHA fatty acids, however, HPP did not change the level of those fatty acids. Overal l, these results prove the usefulness of HPP in seafood processing while providing qua ntitative parameters in order to help with the application of this process.

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66 Table 4-1. Changes in a* valu e (redness), L* value (lightness) and b* value (yellowness) for Atlantic salmon dark and light muscle af ter HPP treatment followed by storage for 6 days at 4C. Control (0.1 MPa) was untreated muscle stored under the same conditions as the treated samples. L*-value L*value Storage day0.1 MPa150 MPa300 MPaCook Storage day0.1 MPa150 MPa300 MPaCook 2 43.411.047.010.354.78.454.410.02 46.28.243.29.168.44.775.04.9 4 44.69.748.19.355.26.856.78.94 46.77.444.08.868.54.676.94.4 6 43.411.449.010.555.87.757.510.06 46.07.344.18.867.94.076.13.6 a* -value a* -value Storage day0.1 MPa150 MPa300 MPaCookStorage day0.1 MPa150 MPa300 MPaCook 2 15.06.5 11.8.511.44.55.7.72 29.7 .226.25.424.63.912.9.8 4 13.46.411.66.711.36.34.22.84 30.85.126.55.826.24.110.43.5 6 12.97.711.36.810.15.83.22.66 29.05.425.86.026.03.810.13.1 b*-value b*-value Storage day0.1 MPa150 MPa300 MPaCook Storage day0.1 MPa150 MPa300 MPaCook 2 10.62.710.12.612.22.712.13.12 17.02.814.32.619.62.519.83.1 4 11.02.310.82.812.72.712.93.14 17.22.714.22.619.22.520.83.3 6 10.83.210.83.012.52.712.93.46 16.92.414.12.619.22.121.22.6 Light muscle Dark muscle Values are meansSD for all pixe ls of the surface of the fillet.

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67 Table 4-2. Texture profile anal ysis (TPA) in terms of hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness fo r Atlantic salmon muscle after HPP treatment or cooking. Control ( 0.1 MPa) was untreated muscle. TreatmentsHardness (N)CohesivenessAdhesi veness (J)Springine ss(mm)Gumminess(N ) Chewiness(N*mm) 0.1 MPa19.4.9bc0.140.05b0.000830. 00042ab3.3.6b2.7.4bc9.35.9b 150 MPa20.5.5b0.20.04a0.000230.00016b 4.5.2a4.1.9ab18.43.5ab 300 MPa25.5.4a0.180.05ab0.001250. 00116a3.9.1ab4.6.6a19.111.8aCook13.0.3c0.130.03ab0.00150.0007a3.9.2ab1.6.7c6.43.9ab Values are Means standard deviations, n=8, different letters within a column indicate significant differences at p < 0.05 separated by Tukeys HSD.

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68 Table 4-3. Fatty acid compositions (g fatty acids/100 g total fatty aci ds) of Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 0 at 4C. Control (0.1 MPa) was untreated muscle stored under the same conditions as treated samples. 0.1 MPa 150 MPa 300 MPa Cook 14:00 4.6.3a 4.6.2a 4.5.4a 4.4.4a 16:00 16.8.5a 16.9.4a 16.6.1a 13.1.9b 18:00 4.8.1a 4.7.1a 4.8.2a 2.7.6b Total saturated 27.7a 27.5a 26.7.6a 20.8.5b 16:1n 7 5.9.2b 5.6.2bc 5.5.4c 6.4.5a 18:1n 9 14.4.3a 14.6.9a 14.9.6a 14.8.5a 18:1n 7 5.3.3a 5.3.3a 5.1.6a 5.1.3a 20:1n 9 1.1.1b 1.3.4b 1.4.2b 7.1.7a 22:1n 11 1.9.1b 2.7b 2.2.3b 13.9.6a Total monoenes 30.1.6b 30.4.5b 30.7.3b 48.9.2a 18:2n 6 6.8.3a 6.3.3a 5.6.6b 3.7.8c 20:2n 6 0.5a 0.5.1a 0.5a 0.4b Total n6 PUFA 8.3.4a 7.8.4a 7.6b 4.7.9c 18:3n 3 0.4.1a 0.3.1b 0.2b 0c 18:4n 3 1.2.1b 1.2b 1.2.1b 1.5.1a 20:5n 3 (EPA) 9.5.3a 9.2.3a 9.4.6a 7.5b 22:5n 3 4.6.2a 4.5.2a 4.6.1a 3.2.4b 22:6n 3 (DHA) 16.8.8a 17.7.2a 18.2.2a 12.7.1b Total n3 PUFA 32.5a 32.8.3a 33.7.2a 24.4.9b n3/n 6 3.9.2b 4.2.2b 4.9.5a 5.2.5a Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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69 Table 4-4. Fatty acid compositions (g fatty acid s/100 g total fatty acids) of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 2 at 4C. Control (0.1 MPa) was untreated muscle st ored under the same conditions as treated samples. 0.1 MPa 150 MPa 300 MPa Cook 14:00 3.4.4a 3.2.3ab 3.2.3c 3.4.3bc 16:00 14.8.7a 14.6.7a 13.4.3b 11.5.1c 18:00 4.8.1a 4.8.1a 4.4.9a 2.8.9b Total saturated 23.7a 23.1ab 21.6.2b 18.1.9c 16:1n 7 5.2.4a 4.8.3ab 4.7.4b 5.3.6a 18:1n 9 15.5.7a 15.4.4a 15.5.6a 14.9.8a 18:1n 7 4.2.1a 4.3.1a 4.3.2a 4.1.2a 20:1n 9 2.6.1b 3.1.5b 5.1.1b 11.3.7a 22:1n 11 1.5.3b 1.8.4b 3.2.5b 10.2.8a Total monoenes 30.6.2b 31b 34.6.9b 47.4.7a 18:2n 6 6.8.1a 6.4.5a 5.2.9b 3.8.9c 20:2n 6 0.6ab 0.6a 0.5bc 0.5c Total n6 PUFA 8.6.2a 8.3.5a 6.9.9b 6.8.1b 18:3n 3 1.2a 1.2.1ab 1.2b 0.7.2c 18:4n 3 1.4.1b 1.3b 1.4.1b 1.6.2a 20:5n 3 (EPA) 10.3.4a 10.1.2a 9.8.1a 7.6.9b 22:5n 3 5.1.3a 5.2a 4.9.8a 3.5.7b 22:6n 3 (DHA) 17.5a 18.3.9a 18.5.2a 13.3.8b Total n3 PUFA 35.6.9a 36.9a 35.5.1a 26.8.3b n3/n 6 4.2.3b 4.4.3b 5.2.5a 4.7b Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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70 Table 4-5. Fatty acid compositions (g fatty acid s/100 g total fatty acids) of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 4 at 4C. Control (0.1 MPa) was untreated muscle st ored under the same conditions as treated samples. 0.1 MPa 150 MPa 300 MPa Cook 14:00 3.8.5a 4.4a 4.3.4a 4.2.5a 16:00 15.9a 16.2.7a 16.2.8a 12.2.4b 18:00 4.5a 4.7.1a 4.6.1a 2.4.1b Total saturated 23.9.1a 25.5.1a 25.8.2a 19.1.9b 16:1n 7 5.4.4b 5.4.3b 5.4.3b 6.2.4a 18:1n 9 14.9.4a 14.8.5a 14.9.7a 14.1.6b 18:1n 7 4.1.3a 4.4.2a 4.3.3a 4.3.1a 20:1n 9 4.3.6b 2.7.4b 3.4b 12.3.7a 22:1n 11 3.4.7b 2.1.2b 2.1.3b 12.8.6a Total monoenes 33.7.6b 31.9b 31.4.3b 51.2.7a 18:2n 6 6.3.3ab 6.5.4a 5.6.5b 3.4.1c 20:2n 6 0.5ab 0.6.1a 0.5b 0.4c Total n6 PUFA 8.1.3a 8.2.5a 7.6b 4.5.5c 18:3n 3 1.1.3b 1.1.2b 1b 1.7.3a 18:4n 3 1.4.1b 1.3bc 1.3.1c 1.6.1a 20:5n 3 (EPA) 9.4.2a 9.5.4a 9.1.4a 6.6.3b 22:5n 3 4.5.8a 4.6.3a 4.5.2a 2.9.1b 22:6n 3 (DHA) 16.2.6b 16.9ab 18.1.6a 11.5.3c Total n3 PUFA 32.7.6a 33.5.2a 34.8a 24.2.3b n3/n 6 4.1.4b 4.1.3b 4.9.6a 5.5.7a Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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71 Table 4-6. Fatty acid compositions (g fatty acids/100 g total fatty acids) of the Atlantic salmon dark muscle after HPP treatment or cooking followed by storage for day 6 at 4C. Control (0.1 MPa) was untreated muscle stored unde r the same conditions as treated samples. 0.1 MPa 150 MPa 300 MPa Cook 14:00 5.6.5a 5.4.4a 4.7.4a 5.1.4a 16:00 16.7.6b 18.1a 16.8.8b 13.4.5c 18:00 3.9b 4.5.1a 4.6.1a 2.4.1c Total saturated 26.9.4b 28.9.4a 27.2b 21.5.8c 16:1n 7 6.8.7a 6.2.4b 5.6.4b 6.9.6a 18:1n 9 14.4.4a 14.5.3a 14.3.7a 14.3.6a 18:1n 7 4.6.4a 4.7.5a 4.9.5a 4.6.2a 20:1n 9 3.7.5b 1.6.4c 1.5.2c 8.1.6a 22:1n 11 5.5.3b 2.1.3c 2.1.1c 14.8a Total monoenes 36.6.5b 30.5.6c 30.3c 49.6a 18:2n 6 5.7.7ab 6.4.6a 5.3.4b 3.5.1c 20:2n 6 0.4.1a 0.4.1a 0.4.1a 0.4.1a Total n6 PUFA 7.1.1ab 7.9.6a 6.6.5b 4.4.2c 18:3n 3 0.4.3a 0.4.1a 0.3a 0b 18:4n 3 1.4.2b 1.2c 1.2.1c 1.6.1a 20:5n 3 (EPA) 8.3.3b 8.9.3ab 9.5.5a 6.7.2c 22:5n 3 3.9.9b 4.2.2ab 4.6.2a 3.1c 22:6n 3 (DHA) 13.8.6c 15.9.5b 18.7.8a 12.2.1c Total n3 PUFA 27.8.9b 30.8.8b 34.3.7a 23.5.1c n3/n 6 4.1.7b 3.9.5b 5.2.5a 5.4.4a Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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72 a a a a b b b b c c c c 0 2 4 6 8 20246log cfu/gtime (day) 0.1 MPa 150 MPa 300 MPa Cook Figure 4-1. Total aerobic plate count for A tlantic salmon after 15 min of high pressure processing (150 and 300 MPa) followed by storage at 4 C for 6 days. Values are means standard deviations (n=3). Diffe rent letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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73 Figure 4-2. Images for Atlantic salmon dark and light muscle at different pressures after high pressure processing treatment (150 and 300 MPa for 15 min) and subsequent storage for 6 days at 4 C.

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74 b c a a a bc a a ab b b b b a a a 0 20 40 60 80 100 120 140 0246 mol MDA/kgTime (day) 0.1 MPa 150 MPa 300 MPa Cook Figure 4-3. Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances (TBARS) for dark muscle of Atlantic salmon after high pressure processing (150 and 300 MPa) followed by storage for 6 days at 4C. Values are means standard deviations (n=6). Diffe rent letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD. Figure 4-4. Temperature profile of Atla ntic salmon during he ating and cooling.

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75 CHAPTER 5 EFFECT OF IRRADIATION TREATMENT ON THE QUALITY OF FRES H ATLANTIC SALMON MUSCLE Introduction The consumption of seafood in the U .S. has increased 16.5 pounds per person in 2006 (NOAA, 2007). Seafoods are highly perishable with a 14 day shelf-life for a fresh or thawed product. Usually beyond 7 days of cold storage th e product is considered being of a lower grade and frequently sold at reduced cost or discar ded. Moreover, seafoods ar e more susceptible to post-mortem texture deterioration than meats from land animals (Ashie and others 1996). Processing techniques that can exte nd the shelf-life of seafood past 14 days can dramatically change the sensory attributes and characteristics of the product beyond the fresh quality demanded by consumers. The irradiation is a col d process treatment due to only a few degrees temperature rise in foods from the radiation energy absorbed, even at a sterilization dose. Therefore, radiation treatment causes minimal ch anges in appearance and provides good nutrient retention. Radiation does not leave any chemical residue and thus can substitute for chemical fumigation; thereby reduci ng the need for chemical substances and allowing for the treatment of products with a wide range of sizes and shapes (Nawar, 1995). In genera l, there are two main advantages of using low dose ionizi ng radiation (< 3 kGy) as a pr ocessing method. Firstly, it will reduce or eliminate the microorganisms responsib le for spoilage and subsequently extend the fresh-storage shelf-life. Secondly, the low dose i rradiation also has the ability to reduce or eliminate specific pathogenic bacteria co mmonly associated with seafood (Grodner and Andrews, 1991). Color of meat and muscle product plays an im portant role in the consumer perception of meat quality(Jeremiah and others 1972). Studies on seafood products have shown that consumers associate color with the freshne ss of a product having better fla vor and higher quality (Gormley,

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76 1992). The irradiation treatment could have a damaging effect on the color of the seafood depending on amount of dose. Astaxanthin is one of the main carotenoi d pigments found in seafood including salmon, trout, shrimp, lobster and fish egg (Torrissen, 1 989). The intensity of red hue in the muscle of shrimp, salmon, rockfish, and snapper is directly li nked to the grading or pr icing of those species (Sacton, 1986). Seafood muscles mostly consis t of polyunsaturated fatty acids (PUFA), especially highly unsaturated fatty acids (HUFA) of the omega-3 and omega-6 family, are susceptible to quick oxidative de terioration. Oxidation of oils generally depends on their degree of unsaturation, as represente d by double-bond index or methylen e bridge index (Senanayake and Shahidi, 2002). It has been stated that astaxanthin has an antioxi dant activity 10 times greater than other carotenoids such as zeaxanthin, lutein, canthaxantin, and -carotene; and 100 times more than -tocopherol. Hence, astaxanthin has b een dubbed a super vitamin E (Miki, 1991). Astaxanthin acts as an antioxidant by que nching singlet oxygen and free radicals. This powerful antioxidant ability of astaxanthin co mes from its unique molecular structure of 11 conjugated double bonds and two hydroxyl groups. Seafoods have high amount of lipids, especially polyunsaturated fatty acids, compared to other muscle foods. The oxidative quality ch anges during processing and following storage directly affect the quality of s eafood products. Irradiation of lipids at high dose in the presence of oxygen can result in the formation of lipid hydro peroxides, which is not dangerous but have undesirable odors and flavors (rancidity). In add ition, the unsaturated fatty acids are more prone to develop rancidity. Lipid oxidation can be significantly redu ced by freezing, and/or by oxygen removal before irradiation treatments (Miller, 2005).

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77 Irradiation processing of food products is a widely studied field of research and is currently being practiced on several commercial food products, worldwide (Hayashi, 2007; Hileman, 2007). Microbial safety is a potential problem associated with seafood products. Recently, Irradiation treatment has been recognized as a pr omising method to enhance shelf-life and safety of products introduced to consumers. The purpose of food irradiation is to enhance the shelf-life and inactivate microorganisms or d ecrease their level in the product. The objective of this study was to investigate the effect of different irradiation treatment doses (1, 1.5, 2 and 3 kGy) on the quality cha nges (color, lipid oxidati on, fatty acids profile, astaxanthin analysis, total plate count and sensory analysis) of fresh Atlantic salmon fillets 6 days during storage at 4 C. Materials and Methods Twenty whole, gutted Atlantic salm on ( Salmo salar ), with average length and weight of 69.0.5 cm and 4.0.2 kg, respectively, were purchased from local seafood supplier (Save-onSeafood, St Petersburg, FL) within two days of harvest and the fish was transported to the laboratory in ice. The fish was filleted and skinless fillets were vacuum packaged using FoodSaver Vacloc vacuum bags (Jarden Co. Rye, NY).All equipment were sterilized by using bleach followed by ethanol before and during al l experiments to minimize contamination. Irradiation Treatment Skinned Atlantic salm on fillets from fresh fish were vacuum packaged. All fillets were placed in a cooler with ice a nd transported to the Florida Ac celerator Services and Technology linear accelerator (Gainesv ille, FL, U.S.A). During the irradiat ion, samples were placed into an open corrugated box, located at the cen ter of a tray in a single laye r. The tray was placed on the linear accelerator conveyor belt, and irradiated using a 5.2 MeV Linatron (Varian, Palo Alto, CA) linear accelerator at room temperature. Th e samples were subjected to 0, 1, 1.5, 2 and 3 kGy

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78 dosages of irradiation. In orde r to determine the applied ir radiation dose, minimum four dosimeters (PMMA Amber 3042, Harwell, UK) were pl aced evenly at the surface of two fillets; one at the top and one at the bottom of each fille t. Samples were exposed to ambient temperature during irradiation for a maximum of 12 min. Contro l consisted of non-irradiated samples kept on the ice all the time. Upon completion of all irradiation treatments, samples were placed into an ice cooler. Samples were removed aseptically from the vacuum bag and placed into oxygen permeable bags and then stored for 6 days at 4C. Dosimeter Calibration It is im portant that the delivered dose from the irradiation source to samples must be determined accurately and also all radiation sources must be calibrated periodically using traceable methodology systems to prevent overdose or low dose treatment of samples. A single beam 5.2 MeV electron beam linear accelerator, locat ed at the Florida Accelerator Services and Technology facility (Gainesville, FL., U.S.A. ), was calibrated in cooperation with Food Technology Service Inc. (FTSI, Mulberry, FL, U.S.A). The calibration methods from ASTM E 1276-93 standards (ASTM, 1993) was used to calib rate Harwell Amber type PMMA dosimeter (Harwell, UK) against NIST approved referen ce standard alanine film dosimeter (Kodak BioMax, Bruker biospin, U.S.A.) in the absorb ed dose range of 1 kGy to 10 kGy. Briefly, one dosimeter packet including three alanine refe rence dosimeters and five Harwell Amber PMMA dosimeters was used for each calibration point. Th e packet contents were sandwiched between two polystyrene sheets with a 5 mm thickness du ring irradiation process. Specific absorbance, k-1 (ratio of the optical absorbance at 603 nm divided to the optical path length or thickness of PMMA dosimeter) for PMMA dosimeter was calcu lated after spectrophotometrically measuring optical density of the Harwell Amber PMMA dos imeter at 603 nm, and recording its thickness. The reference alanine dosimeters were transpor ted to FTSI facility to measure the actual

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79 absorbed dose. The 2nd order polynomial calibrati on curve was obtained fo r absorbed dose from alanine dosimeter versus specific absorbance from PMMA dosimeter, shown in Figure 5-2. The absorbed doses for fresh treated salmon samples were determined for microbial, lipid oxidation and fatty acids analysis as 1.1, 1.5, 2.3 and 3.4 kGy, for color and astaxanthin analysis as 1.2, 1.7, 2.3, and 3.6, kGy and for sensory evaluation as 1.0, 1.5, 2.2 and 3.2 kGy. Microbial Analysis Total aerobic m icrobial growth before and afte r irradiation treatment was determined using PetrifilmTM (3M Laboratories, St. Paul MN) acco rding to the AOAC Official Method 990.12 (AOAC., 1995). Total aerobic coun t was performed on 10 g fish muscle mixed with 90 mL sterile pre-filled dilution vial s of 0.3 mM monopotassium phospha te buffer solution at pH 7.2 (Hardy Diagnostic, Santa Maria CA). The solution was then mixed in a stomacher for 1 min and the pH adjusted to 6.6 7.2 with 1N NaOH and then serially diluted (10-1 10-5). For inoculation, 3M Petrifilm TM was placed on a sterile flat surface and 1.0 mL of the sample was placed at the center of the film and spread by a sterile plastic spreader to an area of ~20 cm2. Duplicate inoculations were conducted for each dilution an d no more than 10 plates were stacked at 35.5C for an incubation time of 48 hours. Color Analysis The surface of color of treated and un treated Atlantic salmon muscle was measured during storage by the machine vision system, consistin g of a light box and a CCD color camera connected to a computer with a firewire connec tion. A software program developed was used to capture images, and to obtain color results base d on L* (lightness), a* (redness), b* (yellowness) values and E (Luzuriaga and others 1997; Yoruk and others 2004; Yagiz a nd others 2007). Fish fillets were placed in the light box and the digita l camera captured a picture of the fillets for each analysis time point. Machine vision system wa s calibrated by using a standard red plate (

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80 L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). Average L*, a*, b* values and E of surface dark and light muscle were calc ulated using a color analysis program. The reason for choosing the color machine vi sion system is discussed in Appendix. HPLC Procedure for Quantification of Astaxanthin Salm on muscle tissue (1 g) was blended with 1 vol water using a Waring commercial blender (Waring Products Division, Dynamics Corp. of America, CT) for 1 min in a disposable glass tube. Two mL of the homogenate was mixed w ith 6 mL of an ethanol-water (2:1) mixture. Five mL of hexane was then added and the mixt ure was shaken for 5 min. The hexane phase was collected in a clean disposable glass tube. The hexane was evaporated using nitrogen. One mL mobile phase (Acetonitrile: dichloromethane: meth anol, 7:2:1) was added to the glass tube. The mixture was filtered through a 0.45 m filter into amber glass vial s under nitrogen prior to HPLC injection. Astaxanthin analysis was conducted usi ng a Dionex HPLC system with a PDA-100 photodiode array detector and a 250 mm 4.6 mm Acclaim 120 A C18 reverse-phase column (Dionex, Sunnyvale, CA) with a C18 guard column (2 mm x 4 mm). An isocratic solvent delivery of acetonitrile:dichloromethane:methanol (7:2:1, vol/vol) was run at 1.5 mL/min with detection at 445 nm. Sample injection volume was 50 L. Se veral standards including astaxanthin, lutein, zeaxanthin (Fluka, Switzerland) were run to identify sample peaks. Only one peak was obtained in the HPLC chromatogram. After comparison of re tention time with the standards, the peak was identified as astaxanthin. The amount of asta xanthin in salmon muscle was quantified using calibration curves obtained from different concen tration of astaxanthin standard. Three fillets were used for each treatment for each day with duplicate HPLC injections.

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81 Lipid Oxidation Analysis Measurement of thiobarbituric acid reactive substances (TBARS) TBARS were perform ed based on a modificatio n of Lemon (1974) according to Raghavan and Hultin (2005), by measuring secondary products of oxidation in dark and ligth muscle. After weighing approximately 1 g of the Atlantic salmon muscle in a disposable glass tube, sample was extracted with 3 mL of 7.5% TCA solution by homogenization with a Biohomogenizer at high speed for 1 min. The sample was centrif uged at 2000 rpm in an Eppendorf 5702 centrifuge (Brinkmann Instruments Inc., Westbury, NY) for 10 min. A 2 mL aliquot of the supernatant was mixed with 2 mL of 0.02 M TBA solution and heat ed in a boiling water bath for 40 min. Sample was cooled under cold running water. The color developed was spectrophotometrically measured at 530 nm. A standard plot was prepared using tetraethoxypropane (TEP). As each mole of TEP would yield one mole of malonaldehyde, the results were expressed as micromoles of malonaldehyde (MDA) per kg tissue. All experiments were done in triplicate. Measurement of lipid hydroperoxides Lipid hydro peroxides were measured according to the method of Raghavan and Hultin (2005) by measuring primary products of oxidation in Atlantic salmon dark and light muscle. Approximately 1 g of Atlantic salmon muscle was transferred to a disposable glass tube, homogenized for 1 min with 10 mL of chlorofo rm/methanol (2:1) usi ng a Biohomogenizer, and mixed with 3 mL of 0.5 % NaCl solution. The mi xture was first vortexed for 30 sec and then centrifuged at 2000 rpm in an Eppendorf centr ifuge (Brinkmann Instruments Inc., Westbury, NY). The chloroform phase was removed, and a 2 mL volume of the chloroform phase was made to 10 mL using chloroform/methanol (2:1). Ammonium thiocyanate and ferrous chloride were prepared as in Shantha and Decker (1994). A 25 L aliquot of each reagent was added and vortexed for 10 s. The samples were incubate d for 10 min at room temperature, and the

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82 absorbance was measured at 500 nm. A sta ndard curve was prepared using cumene hydroperoxide. All experiments were done in triplicates. Lipid Extraction for Fatty Acids Methyl Esters Lipids were extracted from Atlantic salm on li ght and dark muscle using a modified method of Bligh and Dyer (1959). Salmon muscle ti ssue (1 g) was blende d with 4 mL of a chloroform:methanol (1:2) mixture using a Waring commercial blender (Waring Products Division, Dynamics Corp. of America, CT) for 1 min in a disposable glass tube, 1.25 mL of chloroform was added and vortexed for 5 min, and then 2.25 mL of 0.5% KCl solution was added and mixing for 1 min before centrifugatio n. Samples were centrifuged at a speed of 6500 rpm for 10 min and the lower phase collected through the protein disk with a Pasteur pipette into weighed glass tube. 2 mL of chloroform was then added to the remaining part of the mixture, vortexed, centrifuged and the lowe r phase collected into the pr eviously weighed glass tube. Chloroform was removed under a nitrogen gas st ream using N-EVAP 112 Nitrogen Evaporator (Organomation Associates Inc., Berlin, MA, and U.S.A). The weights were recorded and percent lipids determined gravimetrically. Oils were flushe d with nitrogen and stor ed in amber vials at 80C until analysis. Preparation of Fatty Acid Methyl Esters Fatty acid methyl esters (FAME) preparat ion was perform ed according to a modified AOAC official method 991.39 (AOAC ., 2000). Ca. 20 mg of oil was accurately weighed into a 20 ml round bottom screw cap centrifuge tube w ith a Teflon liner cap containing 100 L (10 mg/mL) of both methyl tricosanoate (C23:0) and heptadecanoic acid (C17:0) (Fluka, Buchs, Switzerland) as internal standards. After ad ding 1 mL 0.5 M methanoic NaOH solution, mixture was flushed with nitrogen, vortexed and heated for 5 min at 100 C in an Isotemp dry bath (Fisher Scientific, USA). The mixtur e was cooled, and 2 mL of 12% BF3 in methanol (Fisher

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83 Scientific, USA) was added and heated at 100 C for 3 min. The mixture was cooled, and 1 mL isooctane was added and heated at 100 C for 1 min. 5 ml of saturated Sodium Chloride (NaCl) solution was added to mixture, agitated and cooled to room temperature. The isooctane layer was transferred to a clean glass tube, a small amount of anhydrous Na2SO4 was added. Fatty acid methyl esters were then transferred into 2 mL screw thread amber GC vials (National Scientific, Rockwood, TN) for chromatographic analysis. Gas Chromatography (GC) Analysis FAME was analyzed on a GC HP 6890, equipped with a flam e ionization detector and AT-Silar-100 cyano silicone capillary column (30m x 0.25mm x 0.2 m) (Alltech Assoc. Inc, Nicholasville, KY). Split injection with a split ra tio of 60:1 was used. Operation conditions were as follows: injection port temperature and detector temperature were 250C; initial oven temperature was 140C for 2 min, gradually heated to 235C at a rate of 4C /min and held for 10 min. The carrier gas was helium (1 ml/min). Re tention times and peak areas were computed automatically by a Turbochrom Workstation 6.1. 1 (Perkin Elmer, MA, and U.S.A). Compounds were tentatively identified by comparison with the retention times of known standards. All standards used in the identification of peaks we re purchased from Supelco (Bellefonte, PA). The standards used were Supelco 37, Marine Oil PUFA#1, and Menhaden Oil PUFA#3. Methyl tricosanoate (C23:0) and heptadec anoic acid (C17:0) were used as internal standards. Three fillets were used for each treatment fo r each day with a duplicate GC injection. Sensory Evaluation After irradiation treatm ent of fresh salmon sa mples (0, 1.0, 1.5, 2.0, 3 kGy), they were kept on ice for taste panels. Samples were cut into cubes of 2x2x2 cm dimension before serving in a small clear cup and lid. Each cup had a three digi t random coded sticker. All samples kept on ice until they were served to panelists. Sensory evaluation of irradiated Atlantic salmon was

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84 conducted at the Food Science and Human Nutritions taste panel facility (U niversity of Florida, Gainesville, FL). The taste panel facility contains 10 private booths equipped with a monitor, mouse and keyboard for data entry. Seventy five untrained panelists evaluated the acceptability of irradiated Atlantic salmon fillet. When ten panelists at a time entered the room, they were directed to an individu al booth. They were met by a panelist coordinator who asked them to signin, gave a brief explanation of the evaluation pr ocedure and required the pa nelist to sign the IRBapproved consent form by the University of Florida. Untrained paneli sts (75) evaluated the irradiated fresh Atlantic salmon fillets based on color, odor and overall acceptability using a 9point hedonic scale, 1 being extremely dislik e and 9 being extremely like. Five fresh Atlantic salmon samples were given to each panelist; control (0 kGy), 1, 1.5, 2 and 3 kGy treated. The samples were randomly assigned three digit codes and they were placed in different orders on a white tray. The panelists were asked a few demographic questions, and then asked to rate samples based on color, odor and overall acceptability attributes of samples. The panel design was first rate the color attribute of samp les without removing the clear lid from the cup, then removing the lid from the cup and rate th e odor attribute, and as a final rating overall acceptability. The panelists mark on the screen the number to indicate intensity ratings for each attribute. The Compusense software (Version 5.2, Compusense Inc., Ontario, CA) was used to design, conduct the test, and coll ect and analyze the data. Mean hedonic ratings (n=75) for fresh Atlantic salmon fillets were analyzed by analysis of variance and Tukeys HSD test (p<0.05) for mean separations. Statistical Analysis Color data (L*, a*, b* values), m icrobial anal ysis, fatty acids profile lipid oxidation data (TBARS, PV), astaxanthin analysis and sensor y were analyzed by analysis of variance (ANOVA) and the mean separations were perf ormed by LS Means Tukeys HSD (P<0.05) using

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85 the JMP 5 software (SAS Institute Inc., Cary, NC ). Sensory data were an alyzed by analysis of variance and Tukeys HSD test (p<0.05) for mean separations using Compusense software (Version 5.2, Canada). Results Microbial Analysis Total aerobic count for irradi ation treated fresh Atlantic salmon sa mples during 6 days aerobic storage at 4oC is presented in Figure 5-1. The init ial microbial load of samples was 1.8.6 log cfu/g. The microbial load of untreat ed samples gradually increased and reached 6.3.3 log cfu/gr after 6 days of storage. All irradiation treatm ents including 1, 1.5, 2 and 3 kGy resulted in approximately 2 log cycle and 6 log cycle reduction in microbial counts at day 0 and day 6, respectively. Irradiation treatment at 1 kGy and above significantly (p<0.05) reduced aerobic microbial load to undetectable levels a nd no growth was seen during the entire storage period at 4 oC. There was no significant (p>0.05) di fference among irradiation treated fresh Atlantic salmon samples. Color Analysis Effect of irradiation treatm ent on color of fr esh Atlantic salmon light and dark muscle is shown in Figure 5-3. As the irradiation dose leve l increased, the samples discolored compared with untreated samples. Color evaluation wa s performed by using a color machine vision systems where average L* (lightness), a* (redness), b* (yellowness) values and E of the whole surface of dark and light muscle was measured du ring 6 days of storage at 4C (Table 5-1 and Table 5-2). At the beginning of the storage study, it was found that increasing the level of irradiation dose from 1, 1.5, 2, 3 kGy for light musc le resulted in decreased a* values of 12, 27, 41, 56 %, respectively. The 1.5 kGy treatment and higher doses had significantly lower (p<0.05) a* value than light muscle control but no si gnificant difference was seen between 1 kGy

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86 treatment and control. Although not statistical significant (p>0.05), L* value increased by increasing irradiation dose at da y 0 and day 4 for the light muscle Irradiation dose level at 2 kGy and 3 kGy had a significantly lower b* value than cont rol and light muscle treated at 1 kGy during the entire storage time. The E value was increased as an increase in irradiation dose level (Table 5-1). For the dark muscle, it was found that there was no significant difference between L*and b* values for untreated and trea ted samples during entire storage time. However, 2 kGy and higher doses resulted in signi ficantly lower (p<0.05) redne ss and significantly higher E values than control after second day of storage time (Table 5-2). Storage time did not have a significant effect on L*, and b* values for irradiated and untreated light muscle (Table 5-1). However, irradiation had significant effect on a* value for leading to decrease 50% in a*value irradiation treated and untreated dark muscle at the e nd of 6 days storage at 4 C (Table 5-2). Astaxanthin Analysis Astaxanthin analysis was perform ed on both light and dark muscle of Atlantic salmon using HPLC (Figure 5a-b). When the light muscle was subjected to differe nt irradiation doses, a significant decrease (p<0.05) in amount of astaxanthin was detect ed, with a maximum value of 3.38 mg/kg for control which decreased to 0.36 mg/ kg for the 3 kGy treatment at day 0 (Figure 5-4a). Although the amount of astaxanthin was not statistic ally significant di fferent (p>0.05) between control and the 1 kGy treatment for light muscle, the treatment dose level 1.5 kGy and above led to significantly lower (p<0.05) amount of astaxanthin than both control and 1 kGy treatment at day 0. Storage time did not have any effect on astaxanthin in light muscle between treated and untreated samples (Figure 5-4a). The effect of irradiation on the amount of astaxanthin in dark muscle followed a similar trend as light muscle did (Figure 5-4b). Asta xanthin amount in dark muscle significantly

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87 (p<0.05) decreased as the dose level increas ed, from 1.0 mg/kg to 0.45 kg on day 0. Although no significant difference was seen between contro l and the 1 kGy treatment in the amount of astaxanthin in dark muscle at day 0, there wa s a significant difference between them on the consecutive storage days. It was found that the amount of astaxanthin in light muscle was 3 to 5 times greater than that of dark muscle during 6 days storage at 4 C (Figure 5a-b). Correlation between Amount of Astaxanthin and a*Value It was found that at increased level of irradi ation dose there was a co rrelation between the average am ount of astaxanthin and the average a* value (redness) for each storage day, shown in Figures 5-5 and 5-6. Increasing irradiation dose resulted in decrease in both the amount of astaxanthin and a* value of light muscle. The lowest amount of astaxanthin and a* value was obtained at 3 kGy. The square of correlation coefficient (R2) between amount of astaxanthin and a* value for light muscle at day 0, 2, 4, 6 was 0.99, 0.92, 0.99 and 0.96, respectively (Figure 55). The effect of dose level on dark muscle al so showed correlation between astaxanthin amount and redness. Increasing dose level, resulted in loss of astaxanthin and reduction in redness in dark muscle. The R2 ranged between 0.78 and 0.92 for the 6 day storage period. The highest correlation between amount of astaxanthin and a* value for dark muscle was obtained at day 0 (Figure 5-6). Lipid Oxidation Analysis Lipid oxida tion was measured in the light and dark muscle section of the salmon fillets during storage at 4C by using the Peroxide Valu e (PV) to monitor levels of primary oxidation products formed and the TBARS method to monito r levels of secondary oxidation products formed (Figure 5-7ab, Figure 5-8ab).

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88 Figure 5-8ab shows the effect of irradiation on lipid peroxides formation in light and dark muscle, Although low levels of lipid peroxides formed in both trea ted and untreated light muscle at day 0 and 4, control had significantly (p<0.05) higher amount of lipid peroxides than the treated samples. Lipid peroxide formation in light muscle remained low after 6 days of storage at 4oC. There was only a small, but not statistica lly significant difference between untreated and treated light muscle at day 2 (Figure 5-8a). Higher lipid oxidation based on form ation of lipid peroxide values were observed at day 2 through day 6 for both treated a nd untreated dark muscle samp les. However, no significant (p>0.05) difference was found between treated and untreated dark muscle samples based on formation of lipid peroxide values through storage of 6 days at 4oC (Figure 5-8b). A large standard deviation was seen through the 6 days of storage for almost all the treated and untreated samples. The reasons could be natural variati on between different fish. The 1.5 kGy and higher doses had significant lower PV values compared to control at day 0. TBARS values of light and dark muscle for treated and untreated samples significantly (p<0.05) increased during the 6 day storage period at 4C. Lipid oxidatio n of all light muscle samples increased on storage, with a minimum of 3.73 mol MDA/kg muscle to a maximum of 15.8 mol MDA/kg after 6 days of storage. The standard deviati ons for TBARS were considerably high, indica ting there was a great amount of vari ation in individua l fish. There was no significant (p>0.05) difference between irradiated and untreate d light muscle in terms of secondary oxidation products for each storage period (Figure 5-7a). The TBARS values of dark muscle increased during storage, with an initial value of 6.5 mol MDA/kg muscle to a maximum value of 141.9 mol MDA/kg muscle. No significant (p>0.05) difference was found between treated and untreated dark muscle samples during each

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89 storage time point (Figure 5-7a). It was found that the dark muscle had almost 9 times higher TBARS values than light muscle at the end of storage presumably due to higher lipid contents and heme content. Fatty Acids Analysis Fatty acid profile of irradiat ed and untreated Atlantic salm on light and dark muscle during 6 days of storage at 4 C is shown in Tables 53 to 5-10. The major total saturated fatty acids in the light muscle were 14:0, 16:0 and 18:0. There was no significant (p>0.05) difference in total saturated fatty acid composition (g fatty acids/100 g total fatty acids) for e-beam treated (1 kGy, 1.5 kGy, 2 kGy and 3 kGy) and untreated light muscle samples dur ing the entire storage time (Tables 5-3 to 5-6). However, the 3 kGy treatment gave samples of significantly (p<0.05) lower levels of 16:0 than control at day 0, while no significant difference was detected for the following storage days. All samples had majo r monoenes, including 16:1n-7, 18:1n-7, 18:1n-9, 20:1n-9 and 22:1n-11. Although no si gnificant (p>0.05) differen ce in total monoenes both treated and untreated light muscle samples at day 4, 6 (Tables 5-5 to 5-6),the irradiation levels of 3 kGy showed significant difference in 16:1n-7, 18:1n-7, 20:1n-9 compared to control at day 0 (Table 5-3). Total n-6 polyunsatur ated fatty acids (PUFA) were mainly composed of 18:2n-6 and 20:2n-6 and 22:4n6 in light muscle. Although no signi ficant (p>0.05) difference was detected in light muscle between treated samples and control at day 2, 4 and 6 (Tables 5-4 to 5-6), control showed significant (p<0 .05) difference in the amount of 18:2n6 and total n-6 PUFA compared to the 3 kGy at day 0 (Table 5-3). Several n-3 PUFA s were detected in the light muscle samples, 18:3n-3, 18:4n-3, 20:5n-3 (EPA), 22:5n-3, 22:6n-3( DHA). The major individual fatty acids making up the total n-3 PUFAs were 20:5n-3 (EPA), 22:5n-3 and 22:6n-3 (DHA). The 3 kGy dose significantly reduced both the amount of EPA from 9.2 to 7.7 g/100 g and DHA from 14.4 to 9.1 g/100 g at day 0 (Table 5-3). Although not statistically signifi cant, the level of DHA

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90 remained low for the 3 kGy compared to the othe r treatments and control during the rest of the storage (Tables 5-4 to 5-6). No significant difference was seen in total n-3 PUFA among the 0, 1, 1.5, 2 kGy treatment during the entire storage pe riod, however, the 3 kGy did show significantly (p<0.05) lower amount of total n3 PUFA compared to control in light muscle at day 0 and 2 (Tables 5-3 to 5-4). The fatty acids composition of dark muscle for both treated and untreated samples are shown in Tables 5-7 to 5-10. No significant (p >0.05) difference between treated and untreated dark muscle samples in terms of total saturate d, total monoenes, total n-6 PUFA and total n-3 PUFA during 6 days storage at 4oC Tables 5-7 to 5-10. Sensory Evaluation A total of 75 untrained panelists evaluated the irradiated fresh Atlantic salmon fillets based on color, odor and overall acceptability using a 9-point hedonic scal e, 1 being extremely dislike and 9 being extremely like (Figure 5-9). It was observed th at an increase in irradiation dose resulted in a decrease in hedonic scores of color, odor, and overall a cceptability of Atlantic salmon fillets. Fillets treated with 2 and 3 kGy rated significantly lower (p<0.05) than untreated fillets and those treated with 1 and 1.5 kGy in color attributes. Control was rated highest score in color evaluation, but not signifi cantly different than 1 and 1.5 kGy. The 3 kGy was the lowest score of 4 in color attribute. The general trend seen was an increase in odor acceptability as irra diation dose decreased. Control had the highest odor scor e with 5.5, just slightly above the acceptability line. The odor score decreased to 3.0 for 2 kGy and 3 kGy. No significant (p>0.05) odor difference was observed between 1 and 1.5 kGy. Odor scores of all treated samples remained below the acceptability level score (5.0).

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91 The overall acceptability of fillets trea ted with 1 and 1.5 kGy showed no significant (p>0.05) difference. Significant (p<0.05) differen ce occurred in overall acce ptability scores of 1 and 1.5 kGy, compared with that of untreated sa lmon samples. Panelists found that 2 and 3 kGy were not acceptable based on overall acceptability. The overall acceptability for 3 kGy treated sample scored lowest (3.0) compared to othe r treated and untreated samples (Figure 5-9). Discussion Irrad iation treatment will reduce or eliminate the microorganisms responsible for spoilage and subsequently extend the fresh-storage shelflife of seafoods. The low dose irradiation also has the ability to reduc e or eliminate specific pathogenic ba cteria commonly associated with seafood (Grodner and Andrews, 1991). In the cu rrent study, it was f ound that irradiation treatment of 1 kGy and higher si gnificantly eliminated microbial growth during 6 days at 4 oC (Figure 5-1). Although control reached 6.3 log cfu/g at the end of 6 days, it did not exceed quality evaluation criteria for fresh and fro zen seafood products, or the maximum acceptable microbial limit as set by the International Co mmission of Microbiologic al Standards for Foods (ICMSF, 1978). The effectiveness of irradiation treatment for reduc ing or inactivating microbial growth of seafoods has been reported for se veral species, including oys ter (Novak and others 1966), cod (Thibault and Charbonneau, 1991), crab (C hen and others 1996), lobster (Dagbjart.B and Solberg, 1973), sweet-lip, red emperor, m ackerel, mullet, whiting, barramundi, crabmeat, prawn (Poole and others 1994) and sea bream (C houliara and others 2004). These researchers have also demonstrated reduction in microbial levels similar to those found in this study. The inactivation of living cell by irradiation is either damage to DNA or to cause damage to the membrane and other structures of living cells as a result of causing sublet hal injury (Diehl, 1995; Venugopal and others 1999).

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92 One of the most important criteria for c onsumer perception of seafood quality and consumer purchasing decision is color. Astaxanthi n is a major carotenoid in salmon muscle that provides with its characteristic s orange color. An increased irradiation dose level caused discoloration of Atlantic salmon li ght muscle (Figure 5-3). Irradiat ed samples led to a significant decrease in both a* value and b* value. The a* and b* value of untreat ed salmon light muscle was 31.4 and 33.5, while that of muscle irradi ated with 3 kGy was 13.8 and 27.2 respectively. The storage time did not have any significan t (p>0.05) effect on L*, a* and b* value for irradiation treated and untreated light muscle (Table 5-1). In salmon dark muscle, there was no significant (p>0.05) difference in the L*, a* a nd b* values of untreated and treated samples immediately upon irradiation (day 0). However, after 6 days of storage at 4C there was a significant decrease (p<0.05) in a* value. The a* value of untreated salmon dark muscle decreased from 16.7 to 7.7 while that of 3 kGy i rradiated samples decreased from 14.4 to 4.4. There was also a significant (p<0.05) difference in the a* and b* values of salmon light and dark muscle. In general, the a* and b* values of da rk muscle were lower than those of the light muscle. Similar results were reported in fully cooked salmon and catfish fillets by McKenna and others (McKenna and others 2003). In salmon, the color could be attributed to both carotenoids such as astaxanthin, as well as to heme pigments. The amount of astaxanthin in salmon light muscle was approximately 3 times higher than that of dark muscle (Figure 5-4a and 5-4b) while the amount of heme (arising from the heme proteins in hemoglobin and myoglobin) in dark muscle would be far greater than the amount in light muscle (Richards and Hultin, 2002). Hence, in our current study, the color of salmon light musc le could be primarily due to astaxanthin while the color of dark muscle would be due to th e heme pigments. In our studies, we found a significant loss in the amount of astaxanthin in both salmon li ght and dark muscle due to

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93 irradiation (Figure 5-4a and 54b). Irradiation could lead to lo ss in the color and quality of seafood products. Hammad and others (1992) repor ted a loss in the co lor of smoked salmon upon irradiation. Hence, the loss of a* value in salmon light muscle could be due to the loss of astaxanthin. Salmon dark muscle contains a si gnificantly higher amount of heme proteins and lipids compared to salmon dark muscle (Katikou and others 2001). Irradiation could lead to the oxidation of unsaturated lipids su ch as those found in the dark muscle of salmon (Katusinrazem and others 1992). Hence, the loss in the a* value in salmon dark muscle could in some part be due to the oxidation of lipids. Its however likely that in most part the reduction in a* value was due to oxidation of heme proteins. In our resear ch, we evaluated lipid oxidation in both dark and light muscle of salmon using the primary produc ts of oxidation, i.e., lipid hydroperoxides value (PV), as well as using the sec ondary products of oxidation, i.e., TBARS. In salmon light muscle, we found no significant difference (p>0.05) in both PV and TBARS values (Figure 5-7a and 58a) between treated and untreated samples, as well as for different days of storage at 4C While in salmon dark muscle, there was no significan t difference (p>0.05) in the TBARS value among the different treatments, but a significant diffe rence (p<0.05) was observe d for different storage days (Figure 5-7b). A trend similar to TBARS was also noticed in the a* value of treated and untreated salmon dark muscle, which could imply th at oxidation may play an important role in the color of salmon dark muscle. Endogenous an tioxidants such as astaxanthin (Miki, 1991) would play a vital role in controlling lipid oxid ation in seafood such as salmon. Especially, the presence of antioxidants is important in dark muscle, which contains polyunsaturated triglycerides, membrane lipids (Hultin, 1992) and catalysts of lip id oxidation such as heme pigments (Richards and Hultin, 2002). In our studie s, a decrease in the astaxanthin level (Figure

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94 5-4b) of salmon dark muscle was observed due to both irradiation as well as due to storage (in treated samples), which could imply a decrease in protection against oxidation. While studying the fatty acid composition of salmon light and dark muscle, we found a significant difference (p<0.05) in the PUFA of light muscle due to irradiation, while no significant (p>0.05) difference was observed in the fatty acid composition of red muscle due to irradiation or storage at 4C. Although numerous researches have reported loss of fatty acids due to ionizing radiation (Chen and others 2007; Form anek and others 2003), researches such as Hammer and Wills (1979) reported little or no change in the fatty acid composition of various fat sources containing high amount of antioxidants. In addi tion, Ghadi and Venugopal (1991) investigated the influence of ga mma irradiation up to 5 kGy on lip id oxidation in skin and flesh fractions of Indian mackerel, wh ite pomfret, and seer during ice storage. They found increase in TBA values in both control and irradiated fish, especially in mackerel and seer meat. Armstrong and others (1994) also did not find any change s in fatty acids composition of two Australian marine fish species at doses up to 6 kGy. The aut hors also stated that va riations in fatty acid composition between individual samples were greater than any radiation-induced changes. In our studies, we found little change in the fatty aci d composition of salmon muscle, which could be due to the presence of high amount of the antioxidant, astaxanthin. During oxidation, antioxidants are usually consumed or degraded first, before lipid oxidation would occur. The large decrease in the amount of as taxanthin (Figure 5-4a and 5-4b) due to irradiation indicates that antioxidants are being consumed or sacrificed to prevent the oxidation of lipids. The storage studies in this study were conducted for 6 days at 4C. It is possible that a significant change in the fatty acid content may have been observed for a longer storage time.

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95 As a part of our study, we also did sensory analysis using taste panels for treated and untreated salmon light muscle ba sed on the evaluation of color, odor and overall acceptability. Irradiated samples scored less on color, odor as well as overall acceptability (Figure 5-9). Among the color values from color machine vision syst ems, we did not observe significant difference (p>0.05) in the L* and b* values, while signifi cant difference (p<0.05) was observed among the a* values of treated and untreated samples. He nce, it is possible that during sensory evaluation, the panelists were primarily evaluating the redn ess (a* value) rather than b* and L* values. However, more detailed studies are required to correlate the colo r of samples with the evaluation of consumer panelists. The odor of salmon f illets would arise both from lipid oxidation and rancidity, as well as due to the breakdown compounds of irradiation (Batzer and Doty, 1955; Batzer and others 1957). In our studies, we found no significant difference (p>0.05) in the PV and TBARS values of untreated and irradiation tr eated samples. Hence, the sensory score of the panelists on the odor of salmon fillets might be more due to the breakdown products of irradiation than due to oxidative rancidity. However, we did not determine the type of break down products. Therefore, more future research in this field would shed light on the type of irradiation products in salmon muscle. Conclusion Salmon fillets were treated with various doses of irradiation and were stored at 4C for 6 days. There was a significant decrease in the microbial growth of irradiated fillets. The color (a* value) of salmon light muscle was related to the content of astaxanthin which decreased as irradiation increased, while the co lor of salmon dark muscle was re lated more to the lipid content and the degree of lipid oxidation. Sensory score of irra diated samples were lower in color, odor and overall acceptability compared to untreated sa mples and the sensory score decreased with an increase in irradiation level.

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96 Table 5-1. Changes in L* valu e (lightness), a* value (redne ss), b* value (yellowness) and E of fresh Atlantic salmon light muscle after irradiation treatment followed by storage for 6 days at 4C. L* value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 61.6.4a 61.7.1a 63.7.6a 64.2.3a 65.9.1a 2 61.2.8b 63.3.3ab64.3.4ab 65.1.2ab67.2.5a 4 59.5.4a 61.2.7a 62.8.7a 63.3.3a 64.7.8a 6 59b 61.1.6ab62.2.2ab 63.7.7ab64.7.8a a* value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 31.4.3a 27.5.9ab 23.5bc 18.6.3cd 13.8.4d 2 31.4.9a 26.9.1ab 22.9.4bc 18.1.5cd 13.2d 4 31.9.3a 27.9.7ab 22.8.7bc 18.5cd 13.1.1d 6 32.2.9a 27.5.4ab 22.8.1bc 17.3.1c 12.7.8c b*value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 33.5.7a 34.1.3a 32.1.4ab 29.7.4bc 27.2.2c 2 33.4.2a 33.7.6a 32.6.1ab 29.1.6bc 27.2.9c 4 34.1.4a 35.1.9a 32.7.3ab 30bc 27.7.7c 6 34.5.2a 35.1.7a 33.5.6a 29.7.8b 28.5b E Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 7.2.3bc 4.4.2c 9.3.2bc 14.3.7ab22.8.0a 2 6.6.8bc 5.7.6c 9.7.1bc 15.2.9ab23.8.1a 4 6.0.8bc 4.3.4c 9.0.6bc 14.4.0ab22.7.0a 6 5.7.9c 4.5.8c 8.6.0bc 15.2.1ab23.0.1a Values are means standard deviations (n=3). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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97 Table 5-2 Changes in L* value (lightness), a* va lue (redness), b* value (yellowness) and E of fresh Atlantic salmon dark muscle after irradiation treatment followed by storage for 6 days at 4C. L* value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 60.9.2a 62.7.5a 62.5.1a 60.5.9a 59.6.3a 2 61.7.1a 63.9a 63.2.8a 61.9.3a 61.2.3a 4 60.7.9a 62.2.8a 60.2.2a 59.7.9a 58.8.4a 6 61.6.7a 62.7.8a 61.9a 61.6a 60.2.8a a* value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 16.7.7a 15.4a 14.8.8a 15a 14.4.3a 2 14.7.3a 12.7.9ab11.8.3ab 9.1.9b 10.1.3b 4 10.8.3a 9.8.9a 8.9.4ab 6.5.9b 5.9.2b 6 7.7.2a 7.7.2a 6.4.7ab 5ab 4.4.1b b*value Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 14.1.4a 14.7.7a 14.7a 14.2.9a 13.6.8a 2 14.2.8a 15.3.9a 15.4.1a 15.2.7a 14.9.2a 4 16.6a 16.6.3a 18.4.5a 17.1.1a 17.1.7a 6 17.8.1a 18.2.9a 19.3a 19.2.3a 18.8.3a E Storage day 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 0 5.9.8a 2.6.6a 2.4.7a 3.2.9a 4.3.5a 2 6.2.4a 4.9.4a 5.4.5a 8.8.7a 8.9.6a 4 9.4.7a 7.2.9a 8.8.2a 11.4.8a 12.4.3a 6 12.6.4ab 9.9.6b 11.5.0ab 14.0.6ab14.8.1a Values are means standard deviations (n=3). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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98 Table 5-3. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon light muscle after irradiati on treatment (day 0). 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 2.6.3a 3.2.3a 3.1.4a 3.4a 3.3a 16:00 15.9.6a 14.6.2ab 14.9.3ab 15.2ab 14.2.4b 18:00 4.1.3a 4.1.3a 4.2.2a 4.2a 4.2.3a Total saturated 23.3.1a 22.5.6a 22.8.7a 22.8.2a 22.4a 16:1n 7 3.2.3b 4.4a 3.9.5a 3.7.3ab 4.2a 18:1n 9 16.1.2c 18.5.1ab 18.7ab 17.6b 19.3.7a 18:1n 7 2.7a 2.7.2a 2.7.1a 2.7.1a 2.7a 20:1n 9 2.4.1c 2.7.1ab 2.6.1b 2.5.1bc 2.8.1a 22:1n 11 0.3a 0.3a 0.2.1a 0.3a 0.2.1a Total monoe nes 26.3.7b 30.2.6a 29.4.3a 28.1.1ab 31.1.8a 18:2n 6 17.1b 19.4ab 18.2ab 17.6.9b 20.1.1a 20:4n 6 0.9.4a 0.9.4a 0.9.4a 0.9.4a 0.8.3a 22:4n 6 0.5.2a 0.5.1a 0.5.1a 0.5.2a 0.5.1a Total n6 PUFA 19.1.9b 21.1.2ab 20.3.9ab 19.7.8b 22.2.1a 18:3n 3 1.2.1b 1.4.1a 1.3.1ab 1.3.1ab 1.4.1a 18:4n 3 1.2.1a 1.1a 1.1.1a 1.1.1a 1.2.1a 20:5n 3 9.2.7a 7.9.6b 8.3ab 8.3.8ab 7.7.3b 22:5n 3 4.2.4a 4.3a 4.3a 4.2.2a 3.9a 22:6n 3 14.4.6a 10.5.5ab 11.5.5ab 13.3.5ab 9.1.5b Total n3 PUFA 31.3.6a 26.2.3ab 27.4.6ab 29.3.1ab 24.7.6b n3/n 6 1.6.3a 1.2.1ab 1.4.4ab 1.5.3ab 1.1.1b Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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99 Table 5-4. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon light muscle after irradiation treatment and storage for 2 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 2.9.2a 3.3a 3.1.6a 2.8.3a 3.1.3a 16:00 14.8.1a 15.3a 15.1.5a 15.5.8a 14.7.7a 18:00 4.2.2a 4.3.1a 3.6.3a 4a 4.3.2a Total saturated 22.5.5a 22.9.6a22.4.1a 23.1.1a 22.7.9a 16:1n 7 3.7.1a 3.7.4a 3.8.6a 3.5.2a 3.9.2a 18:1n 9 18.9ab 17.6.9ab 17.9.2ab 16.5.9b 18.7.7a 18:1n 7 2.7.1a 2.7.1a 2.6.3a 2.7.1a 2.7a 20:1n 9 2.6.1a 2.6.1ab 2.6.1ab 2.4.1b 2.7.1a 22:1n 11 0.2.1a 0.3a0.3a 0.3a 0.3a Total monoe nes 29.2.1ab 28.9.4ab 29.1.8ab 27.2.2b 30.3.8a 18:2n 6 19.7ab 18.5.7ab 18.3ab 16.9.3b 19.2.3a 20:4n 6 0.9.4a 0.9.4a 0.9.4a 0.9.4a 0.9.4a 22:4n 6 0.4a 0.5.1a 0.5.1a 0.4.1a 0.5a Total n6 PUFA 21.4ab 20.5.9ab 20.4.8ab 18.8.6b 21.2.3a 18:3n 3 1.3.1ab 1.3.1ab 1.4.1ab 1.2.1b 1.4.1a 18:4n 3 1.1.1a 1.2.1a 1.1.1a 1.1.1a 1.2.1a 20:5n 3 8.2.4a 8.2.4a 8.5a 8.7.2a 8.5a 22:5n 3 4.3a 4.2a 4.1.4a 4.2.3a 4.2a 22:6n 3 11.3.5ab 11.8.5ab 11.7.5ab 14.5.4a 9.9.7b Total n3 PUFA 27.3ab 27.8ab 28.1.6ab 30.9.8a 25.8.1b n3/n 6 1.3.1ab 1.4.1ab 1.4.3ab 1.7.3a 1.2.2b Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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100 Table 5-5. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon light muscle after irradiation treatment and storage for 4 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 3.1a 3.4a 3.1.5a 3.5a 3.1a 16:00 14.8.3a 14.5.6a 14.4.4a15.5.3a 14.6a 18:00 4.4.1a 4.3.2a 4.3.1a 3.9.8a 3.5.7a Total saturated 22.8.3a 22.5.1a 22.5.5a 23.4a 21.1.7a 16:1n 7 3.9.1a 3.8.4a 3.9.6a 3.7.6a 4.1a 18:1n 9 18.3.4a 17.9a 18.8.8a17.3.7a 19.2.7a 18:1n 7 2.6.1a 2.7a 2.6.1a 2.6.1a 2.6.3a 20:1n 9 2.6.2a 2.6.1a 2.7.1a 2.5.3a 2.7.1a 22:1n 11 0.3a 0.3a 0.3a0.3a 0.3a Total monoe nes 29.8.8a 29.4.6a 30.4.8a28.3.9a 31.7.1a 18:2n 6 18.4.7a 18.5a 19.3.2a17.8.6a 19.5.6a 20:4n 6 1.2a 1.3a 0.8.3a 1.3a 0.9.3a 22:4n 6 0.4a 0.5a 0.5.1a 0.4a 0.5.1a Total n6 PUFA 20.6.9a 20.7a21.4a 19.8.5a 21.7.5a 18:3n 3 1.4.1a 1.4a 1.4.1a 1.3.2a 1.4a 18:4n 3 1.1.1a 1.1a 1.2a 1.1.1a 1.2.1a 20:5n 3 7.8.2a 8.5a 8.8a 8.5.2a 7.9.5a 22:5n 3 4a 4.2.2a 4.1.2a 4.2.5a 4.1.1a 22:6n 3 11.3.4a 11.5.5a 9.9a 12.6.9a 9.6.2a Total n3 PUFA 26.9.3a 27.4.9a 25.8.9a28.9.2a 25.5.7a n3/n 6 1.3.2a 1.3.2a 1.2.2a 1.5.5a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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101 Table 5-6. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon light muscle after irradiation treatment and storage for 6 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 3.2.5a 3.1.1a 2.9.4a 3.1.6a 3.3.6a 16:00 14.5.1a 14.4.8a 14.3.5a14.6.5a 14.3.1a 18:00 4.3.3a 4.9a 4.3.1a 3.6.8a 3.4.9a Total saturated 22.6.8a 22.1.6a 22.1a 21.9.5a 21.5.1a 16:1n 7 4.1.4a 4.2.2a 3.8.4a 3.9.7a 4.4.5a 18:1n 9 18.8.8a 19.8.7a 18.7.7a18.7.9a 19.7.4a 18:1n 7 2.6.2a 2.5.3a 2.7.1a 2.6.2a 2.2.5a 20:1n 9 2.7.1a 2.7.1a 2.7.1a 2.7.1a 2.8.1a 22:1n 11 0.3a 0.3a 0.3a0.3a 0.2a Total monoe nes 30.6.2a 31.7.8a 30.3a 30.9.8a 32.8.1a 18:2n 6 18.9.3a 19.7.5a 19.6.1a 19.6a 19.5.2a 20:4n 6 0.9.3a 0.9.2a 0.6.4a 0.6.4a 0.6.4a 22:4n 6 0.5a 0.4.2a 0.5a0.5a 0.5.1a Total n6 PUFA 21.5a 21.8.4a 21.5.4a20.8.1a 21.5.3a 18:3n 3 1.4.1b 1.5.1ab 1.4.1ab 1.4.1ab 1.6.1a 18:4n 3 1.1.1a 1.2a 1.2.1a 1.2.1a 1.2.1a 20:5n 3 7.8.6a 7.7.3a 7.9.7a 7.8.6a 7.7.4a 22:5n 3 4.2a 4.1.2a 4.3a 3.9.3a 4.1.2a 22:6n 3 10.2.4a 8.7.6a 10.3.2a10.9.8a 8.2.2a Total n3 PUFA 25.9.2a 24.4.2a 26.2.1a26.5.4a 24.3.5a n3/n 6 1.2.1a 1.1.1a 1.2.1a 1.3.3a 1.1.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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102 Table 5-7. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon dark muscle after irradiation treatment (day 0). 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 3.4a 3.1.3a 3.2.5a 2.9.2a 3.3a 16:00 13.6.9a 13.7.3a 13.5.8a 13.3.7a 13.4.8a 18:00 4.4.2a 4.5.1a 4.4.1a 4.4.2a 4.3.1a Total saturated 21.4.3a 21.7.5a 21.6.3a 21.1a 21.2a 16:1n 7 3.9.4a 4.4a 4.1.4a 3.9.1a 4.3a 18:1n 9 19.1.8a 18.8.7a 18.9.4a 18.7.2a 19.1.5a 18:1n 7 2.8.2a 2.9.1a 2.9.1a 2.9.1a 2.9.2a 20:1n 9 2.5.5a 2.7a 2.7a 2.7.1a 2.8a 22:1n 11 0.4.2a 0.4.1a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 30.9.1a 31a 31.2.7a 30.7.2a 31.2.5a 18:2n 6 20.5a 19.7ab 19.2.3ab 19.6b 19.8.6ab 20:4n 6 0.9.4a 0.9.3a 0.9.3a 0.9.2a 0.9.3a 22:4n 6 0.5.2a 0.6.1a 0.6.1a 0.7.2a 0.6.1a Total n6 PUFA 22.1.3a 21.3.7a 21.4.3a 21.2.7a 22.6a 18:3n 3 1.2.5a 1.4a 1.5.1a 1.5a 1.5.1a 18:4n 3 1.2.1a 1.2a 1.1.1a 1.1a 1.1.1a 20:5n 3 8.2a 8.3a 8.1.5a 8.2.3a 8.3a 22:5n 3 4.4.4a 4.4.4a 4.3.5a 4.6.2a 4.4.4a 22:6n 3 9.4.4a 10a 9.8.1a 10.5.9a 9.5.2a Total n3 PUFA 25.6a 26.1.6a 25.8.8a 27.7a 25.6.7a n3/n 6 1.2.1a 1.2.1a 1.2.1a 1.3.1a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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103 Table 5-8. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon dark muscle after irradiation treatment and storage for 2 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 2.9.3a 3.2a 3.4a 3.2.6a 2.9.3a 16:00 13.5.6a 13.8.3a 13.7.3a 13.6.6a 13.5a 18:00 4.4.1a 4.5.2a 3.6.8a 4.4.2a 4.3a Total saturated 21.4.9a 21.8.4a 20.9.8a 21.7.1a 20.7.8a 16:1n 7 3.8.2a 3.9.3a 4.3a 4.4a 3.9.1a 18:1n 9 18.8.5a 18.7.5a 15.9.8a 18.7.5a 19.2.4a 18:1n 7 2.9.1a 2.9.1a 5.8.2a 2.9.2a 3.1a 20:1n 9 2.7ab 2.7b 2.8.1ab 2.7.1ab 2.8a 22:1n 11 0.4.1a 0.4.1a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 30.7.6a 30.6.9a 31.5a 30.9.7a 31.4.5a 18:2n 6 19.6.7a 19.6.2a 20.9a 18.9.9a 19.8.7a 20:4n 6 0.9.2a 0.9.2a 0.9.3a 0.9.3a 0.9.3a 22:4n 6 0.6.1a 0.6.1a 0.6.1a 0.6.1a 0.6.2a Total n6 PUFA 21.9.7a 21.8.2a 22.3.9a 21.2.9a 22.6a 18:3n 3 1.4a 1.4a 1.5a 1.4a 1.5a 18:4n 3 1.2.1a 1.2.1a 1.2.1a 1.1.1a 1.1a 20:5n 3 7.8.4a 7.9.2a 8.1.3a 8.1.4a 8.4a 22:5n 3 4.4.3a 4.2.3a 4.4.4a 4.5.6a 4.5.3a 22:6n 3 10.2.8a 10.1.8a 9.7.9a 10.6a 9.6.3a Total n3 PUFA 26.1a 25.8a 25.8.4a 26.2a 25.8.7a n3/n 6 1.2.1a 1.2a 1.2.1a 1.2.1a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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104 Table 5-9. Fatty acid compositions (g fatty acid s/100g total fatty acids) of fresh Atlantic salmon dark muscle after irradiation treatment and storage for 4 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 3.2a 3.1.4a 3.1.3a 3.1.1a 3.2a 16:00 13.8.4a 13.7.8a 13.5.7a 13.4.4a 13.5.4a 18:00 4.6.1a 4.5.1a 4.4.1a 4.4.1a 4.4.1a Total saturated 21.8.5a 21.8.3a 21.6.9a 21.4.6a 21.4.6a 16:1n 7 3.9.2a 4.3a 4.2a 4.1a 4.2a 18:1n 9 18.6.3a 18.9.6a 19.1.5a 19.3a 19.2.7a 18:1n 7 3.1a 2.9.1a 2.9.1a 2.9.2a 3.1a 20:1n 9 2.7a 2.7.1a 2.7a 2.8.1a 2.8a 22:1n 11 0.4.1a 0.4.2a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 30.6.4a 31.1.8a 31.3.5a 31.3.2a 31.5.8a 18:2n 6 18.5.5b 19.3.7ab 19.5.7ab 19.7.6a19.7.8ab 20:4n 6 1.2a 0.9.3a 0.9.3a 0.9.3a 0.9.3a 22:4n 6 0.7.2a 0.6.2a 0.6.1a 0.6.2a 0.6.1a Total n6 PUFA 20.8.5b 21.5.8ab 21.8.6ab 22.6a 21.9.7ab 18:3n 3 1.4.1a 1.5a 1.5.1a 1.5a 1.5.1a 18:4n 3 1.1a 1.1.1a 1.2.1a 1.2a 1.1a 20:5n 3 7.9.2a 7.9.3a 8.5a 8.4a 8.4a 22:5n 3 4.5.3a 4.4.3a 4.4.4a 4.4.4a 4.3.3a 22:6n 3 10.7a 9.6.6a 9.3.2a 9.2.7a 9.3.7a Total n3 PUFA 26.7.1a 25.5.5a 25.4.7a 25.4.1a 25.2a n3/n 6 1.3.1a 1.2.1a 1.2.1a 1.2.1a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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105 Table 5-10. Fatty acid compositions (g fatty acids/100g total fatty acids) of fresh Atlantic salmon dark muscle after irradiation trea tment and storage for 6 days at 4C. 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy 14:00 3.1.3a 3.3a 3.3a 3.2.4a 3.2.3a 16:00 13.4.3a 13.4.7a 13.6.6a13.7.6a 13.7.5a 18:00 4.4.1a 4.5.1a 4.5a 4.4.1a 4.5.1a Total saturated 21.4.5a 21.4.9a 21.6a 21.8a 21.8.7a 16:1n 7 4.4a 4.3a 4.3a 4.1.3a 4.1.2a 18:1n 9 18.9.4a 19.3.6a 19.5a 19.3.3a 19.3.5a 18:1n 7 2.9.1a 3.1a 3.1a3.1a 3.1a 20:1n 9 2.7ab 2.7ab 2.7b 2.7a 2.7ab 22:1n 11 0.4.1a 0.4.2a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 31.1.9a 31.4.7a 31.2.7a31.6.7a 31.6.7a 18:2n 6 19.4a 19.4.7a 19.4.1a19.5.3a 19.2.9a 20:4n 6 0.9.2a 0.9.3a 0.9.3a 0.9.3a 0.9.3a 22:4n 6 0.6.2a 0.7.1a 0.6.1a 0.6.1a 0.6.1a Total n6 PUFA 21.3.5a 21.7.6a 21.7.1a21.8.3a 21.5.8a 18:3n 3 1.5a 1.5.1a 1.5a 1.5.1a 1.5.1a 18:4n 3 1.2.1a 1.2a 1.2.1a 1.1.1a 1.2a 20:5n 3 8.1.1a 8.3a 7.9.5a 7.9.3a 8.3a 22:5n 3 4.4.2a 4.5.4a 4.3.4a 4.3.4a 4.4.3a 22:6n 3 10.8a 9.4.6a 9.6a 9.1.1a 9.5a Total n3 PUFA 26.2.9a 25.5.9a 25.5.7a24.8.5a 25.9a n3/n 6 1.2a 1.2.1a 1.2.1a 1.1.1a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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106 c c b a 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 011 523log cfu/gDose (kGy) Day 0 Day 2 Day 4 Day 6 Figure 5-1. Total aerobic plate count of fresh Atlantic salm on after irradiation treatment followed by storage at 4 C for 6 days. Values are means standard deviations (n=3). Different letters within each da y indicate significant differences at p < 0.05 separated by Tukeys HSD.

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107 y = 12.95x2+ 20.45x + 0.658 R = 0.999 0.0 2.0 4.0 6.0 8.0 10.0 12.0 00.050.10.150.20.250.30.350.4Absorbbed dose ( kGy)Specific absorbance (k-1 ) Figure 5-2. Calibration curve for absorbed dos e from alanine dosimeter versus specific absorbance from PMMA dosimeter

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108 Figure 5-3. Images of fresh Atlan tic salmon light and dark muscle after being treated at different irradiation doses and subsequent storage for 6 days at 4 C.

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109 (a) a a a a a b b b b b bc c b c cd cd c d d d 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0246Amount of Astaxanthin (mg/kg)Time (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy (b) a a a a ab b b b b bc bc bc b c c bc b c d c 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 0246Amount of Astaxanthin (mg/kg)Time (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy Figure 5-4. Amount of astaxanthi n (mg/kg muscle) for (a) fresh light muscle (b) fresh dark muscle of Atlantic salmon fillets before and after irradiation treatment followed by storage at 4 C for 6 days. Three fillets were used for each treatment and each day. Different letters within each day indicate si gnificant differences at P < 0.05 separated by Tukeys HSD. Values are means standa rd deviations (n=6). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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110 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 5.638x + 12.24 R = 0.987 0 5 10 15 20 25 30 35 0.01.02.03.04.0a* valueAmount of astaxanthin (mg/kg) Day 0 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 4.098x + 13.69 R = 0.917 0 5 10 15 20 25 30 35 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 2 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 4.796x + 11.65 R = 0.986 0 5 10 15 20 25 30 35 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 4 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 4.507x + 12.19 R = 0.965 0 5 10 15 20 25 30 35 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 6 Figure 5-5. Correlation between th e amount of astaxanthin and aver age a*-value during different dose of irradiation treatment in fresh light muscle during storage at 4 C for 6 days.

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111 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 3.512x + 13.10 R = 0.919 14 15 15 16 16 17 17 0.00.20.40.60.81.01.2a* valueAmount of astaxanthin (mg/kg) Day 0 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 6.707x + 7.954 R = 0.848 0 5 10 15 20 0.00.20.40.60.81.01.2a* valueAmount of astaxanthin (mg/kg) Day 2 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 8.647x + 4.934 R = 0.779 0 2 4 6 8 10 12 0.0 0.2 0.4 0.6 0.8a* valueAmount of astaxanthin (mg/kg) Day 4 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy y = 9.765x + 3.959 R = 0.808 0 2 4 6 8 10 0.00.10.20.30.40.5a* valueAmount of astaxanthin (mg/kg) Day 6 Figure 5-6. Correlation between th e amount of astaxanthin and aver age a*-value during different dose of irradiation treatment in fresh dark muscle during storage at 4 C for 6 days.

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112 (a) a a a a a a a a a a a a a a a a a a a a 0 5 10 15 20 25 30 0246TBARS (mol MDA/kg)Time (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy (b) a a a a a a a a a a a a a a a a a a a a 0 20 40 60 80 100 120 140 160 180 200 0246TBARS (mol MDA/kg)Time (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy Figure 5-7. Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances (TBARS) for a) light mu scle b) dark muscle of fresh Atlantic salmon after irradiation treatment followed by storage for 6 days at 4C. Values are means standard deviations (n=6). Diffe rent letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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113 (a) a a a ab b a b b b a b b b a b b b a b a 0 20 40 60 80 100 120 140 0246umol lipid hydroperoxide/ kg muscleTime (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy (b) a a a a a a a a a a a a a a a a a a a a 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0246umol lipid hydroperoxide/ kg muscleTime (day) 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy Figure 5-8. Changes in lipid oxida tion as measured by the formation of peroxide value (PV) for a) light muscle b) dark muscle of fresh Atlantic salmon after irradiation treatment followed by storage for 6 days at 4C. Values are means standard deviations (n=6). Different letters within each day indicate si gnificant differences at p < 0.05 separated by Tukeys HSD.

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114 a a a a b b a b b b c c b c c 1 2 3 4 5 6 7 8 9 Color Odor Overall AcceptablityHedonics Score 0 kGy 1 kGy 1.5 kGy 2 kGy 3 kGy Figure 5-9. Effect of different irradiation doses on sensory evalua tion of color, odor and overall acceptability of fresh Atlantic salmon fillets Acceptability or hedonic scale: 1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely. ** Mean of hedonic ratings (n = 75) for fresh Atlantic salmon fillets were analyzed by analysis of variance and Tukeys HSD test (P<0.05) for mean separations using Compusense software (Version 5.2 Canada) *** Different letters within each attribute indicate significant differences (P < 0.05)

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115 CHAPTER 6 EFFECT OF IRRADIATION TREATMENT ON THE QUALITY OF FROZ EN ATLANTIC SALMON MUSCLE Introduction Cardiovascular health benefits associated with om ega-3 fatty acids have been well documented. Seafood is recognized as an excellent source of omega-3 fatt y acids in the diet. Consumers prize and purchase s eafood as either fresh or fresh frozen. The consumption of seafood in the U.S. has increased 16.5 pounds per person in 2006 (NOAA, 2007). Seafoods are highly perishable with a 14 day shelf-life for a fresh or thawed product. Usually beyond 7 days of cold storage the product is considered being of a lower grade and fre quently sold at reduced cost or discarded. Moreover, seafoods are more su sceptible to post-mortem texture deterioration than meats from land animals (Ashie and others 1996). Processing techniqu es that can extend the shelf-life of seafood past 14 days can drama tically change the sensory attributes and characteristics of the product beyond the fresh quality demanded by cons umers. Irradiation is a nonthermal treatment due to only a few degrees te mperature rise in foods from the radiation energy absorbed, even at a sterilization dose. Therefore, radiation treatment causes minimal changes in appearance and provides good nutrien t retention. It does not leave any chemical residue, and thus, can substitute for chemical fumi gation; thereby reducing the need for chemical substances and allowing for the treatment of pro ducts with a wide range of sizes and shapes (Nawar, 1995). The value and acceptance of most fish species depend on the color and appearance of products. Color of meat and muscle products plays an important role in consumer perception of meat quality (Jeremiah and others 1972) Studies on seafood products have shown that consumers associate color with the freshness of a product having better flavor and higher quality

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116 (Gormley, 1992). One problem with irradiation is that it can affect the color of seafood depending on the dose amount. Astaxanthin is one of the main caratonoi d pigments found in seafood including salmon, trout, shrimp, lobster and fish egg (Torrissen, 1 989). The intensity of red hue in the muscle of shrimp, salmon, rockfish, and snapper is directly li nked to the grading or pr icing of those species (Sacton, 1986). Seafood muscle, mostly consisti ng of polyunsaturated fatty acids (PUFA), especially highly unsaturated fatty acids (HUFA) of the omega-3 and omega-6 family, are susceptible to quick oxidative de terioration. Oxidation of oils generally depends on their degree of unsaturation, as represente d by double-bond index or methylen e bridge index (Senanayake and Shahidi, 2002). It has been stated that asta xanthin has antioxidant ac tivity, 10 times greater than other carotenoids such as zeaxanthin, lutein, canthaxantin, and -carotene; and 100 times more than -tocopherol. Hence, astaxanthin has been dubbed a super vitamin E (Miki, 1991). Astaxanthin acts as an antioxi dant by quenching singlet oxygen a nd free radicals. This powerful antioxidant ability of astaxant hin comes from its unique molecu lar structure of 11 conjugated double bonds and two hydroxyl groups. Lipid oxidation of seafood is well known and seafood has high amounts of lipids, especially polyunsaturated fatty acids compared to other muscle foods. The oxidative quality changes during processing and following storage di rectly affect the quality of seafood products. Irradiation of lipids at high dose in the presence of oxygen can result in the formation of lipid hydroperoxides, which is not dangerous but elicit undesirable odors and fla vors (rancidity). In addition, the unsaturated fatty aci ds are more prone to develop rancidity. Lipid oxidation can be significantly reduced by freezing, and/or by oxygen removable before irradiation treatment (Miller, 2005).

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117 Many researchers have demonstr ated that irradiation proces sing of food products reduce or eliminate spoilage bacteria and pathogens. Microbi al safety is a major problem associated with seafood products. The demand for irradiation treatm ent has increased as a promising method to enhance shelf-life and safety of pr oducts introduced to consumers. The objective of this study was to investigate the effect of different irradiation treatment doses (1, 2, 3 and 5 kGy) on the quality change s (color, lipid oxidation, fatty acids profile, astaxanthin analysis, total plate count and sensory evaluation) for frozen treated Atlantic salmon fillets and during 6 days storage at 4 C. Materials and Methods Twenty whole guttedAtlantic salm on (Salmo salar ), with average length and weight (69.0.5 cm and 4.0.2 kg, respectively) were purchased from local seafood supplier (Saveon-Seafood, St Petersburg, FL) within two days of harvest and the fish was transported to the laboratory in ice. After fille ting, removing skin and vacuum packaged using FoodSaver Vacloc vacuum bags (Jarden Co. Rye, NY), samples were frozen at -20C by inserting thermocouple in the middle of one fillet to monitor internal temperature of samples. Temperature profile of Atlantic salmon during freezing was shown in Figur e 6-10. All equipments were sterilized using bleach and followed by ethanol before and dur ing the entire experiments to minimize contamination. Irradiation Treatment Frozen fillets were placed in a cooler w ith dry ice and transported to the F lorida Accelerator Services and Tec hnology linear accelerator (Gai nesville, FL, U.S.A). During irradiation, samples were placed into an open corr ugated box, located at center of tray in a single layer. The tray was placed on th e linear accelerator conveyor be lt, and irradiated using a 5.2 MeV Linatron (Varian, Palo Alto, CA) linear accelerator at room temperature. The samples were

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118 subjected to 0, 1, 2 3 and 5 kGy dosages of ir radiation. In order to determine the applied irradiation dose, a minimum four dosimeter s (PMMA Amber 3042, Harwell, UK) were placed evenly at the surface of two fillets; one at the top and one at the bottom of each fillet. Samples were exposed to ambient temperature during irradiation for a maximum of 12 min. Control was the non-irradiated samples kept on the dry ice the entire time. U pon completion of all irradiation treatments, samples were placed into a dry ic e cooler. Samples were thawed and removed aseptically from the vacuum bag and placed into oxygen permeable bags and then stored for 6 days at 4C. Dosimeter Calibration It is im portant that the delivered dose from the irradiation source to samples must be determined accurately and also all radiation sources must be calibrated periodically using traceable methodology systems to prevent overdose or low dose treatment of samples. A single beam 5.2 MeV electron beam linear accelerator, locat ed at the Florida Accelerator Services and Technology facility (Gainesville, FL., U.S. A.) was calibrated in cooperation with Food Technology Service Inc.( FTSI, Mulberry, FL, U. S.A). The calibration methods from ASTM E 1276-93 standard (ASTM, 1993) was used to calibrate Harwell Amber type PMMA dosimeters (Harwell, UK) against NIST approved referen ce standard alanine film dosimeters (Kodak BioMax, Bruker biospin, U.S.A.) in the absorbed dose range of 1 kGy to 10 kGy. One dosimeter packet including three alanine reference dosim eters and five Harwell Amber PMMA dosimeters was used for each calibration point. The pack et contents were sandwiched between two polystyrene sheets with a 5 mm thickness during i rradiation process. Spec ific absorbance, k-1 (ratio of the optical absorbance at 603 nm divided to the optical path length or thickness of PMMA dosimeter) for PMMA dosimeter was calcu lated after spectrophotometrically measuring optical density of the Harwell Amber PMMA dos imeter at 603 nm, and recording its thickness.

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119 The reference alanine dosimeters were transpor ted to FTSI facility to measure the actual absorbed dose. The 2nd order polynomial calibrati on curve was obtained fo r absorbed dose from alanine dosimeter versus specific absorbance from PMMA dosimeter. The absorbed doses for frozen treated salmon samples were determined for microbial, lipid oxidation and fatty acids analysis as 1.1, 2.1, 3.5 and 5.0 kGy, for color and astaxanthin analysis as 1.2, 2.0, 3.2, and 5.6 kGy and for sensory evaluation as 1.1, 2.1, 3.2 and 5.1 kGy. Microbial Analysis Total aerobic m icrobial growth before and after irradiation was determined using PetrifilmTM (3M Laboratories, St. Paul MN) acco rding to the AOAC Official Method 990.12 (AOAC., 1995). Total aerobic coun t was performed on 10 g fish muscle mixed with 90 mL sterile pre-filled dilution vial s of 0.3 mM monopotassium phospha te buffer solution at pH 7.2 (Hardy Diagnostic, Santa Maria CA). The solution was then mixed in a stomacher for 1 min and the pH adjusted to 6.6 7.2 with 1N NaOH and then serially diluted (10-1 10-5). For inoculation, 3M Petrifilm TM was placed on a sterile flat surface and 1.0 mL of the sample was placed at the center of the film and spread by a sterile plastic spreader to an area of ~20 cm2. Duplicate inoculations were conducted for each dilution an d no more than 10 plates were stacked at 35.5C for an incubation time of 48 hours. Color Analysis The surface of color for treated and u ntreated Atlantic salmon muscle was measured during storage by the machine vision system, consis ting of a light box and a CCD color camera connected to a computer with a firewire connec tion. A software program developed was used to capture images, and to obtain color results base d on L* (lightness), a* (redness), b* (yellowness) values and E (Luzuriaga and others 1997; Yoruk and others 2004; Yagiz a nd others 2007). Fish fillets were placed in the light box and the digita l camera captured a picture of the fillets for each

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120 analysis time point. Machine vision system was calibrated by a standard using red plate (L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). Average L*, a*, b* values and E of surface dark and light muscle were calc ulated using a color analysis program. The reason for choosing the color machine vi sion system is discussed in Appendix. HPLC Procedure for Quantification of Astaxanthin Salm on muscle tissue (1 g) was blended with 1 vol water using a Waring commercial blender (Waring Products Division, Dynamics Corp. of America, CT) for 1 min in a disposable glass tube. Two mL of the homogenate was mixed w ith 6 mL of an ethanol-water (2:1) mixture. Five mL of hexane was then added and the mixt ure was shaken for 5 min. The hexane phase was collected in a clean disposable glass tube. The hexane was evaporated using nitrogen. One mL mobile phase (Acetonitrile: dichloromethane: meth anol, 7:2:1) was added to the glass tube. The mixture was filtered through a 0.45 m filter into amber glass vial s under nitrogen prior to HPLC injection. Astaxanthin analysis was conducted usi ng a Dionex HPLC system with a PDA-100 photodiode array detector and a 250 mm 4.6 mm Acclaim 120 A C18 reverse-phase column (Dionex, Sunnyvale, CA) with a C18 guard column (2mm x 4 mm). An isocratic solvent delivery of acetonitrile:dichloromethane:methanol (7:2:1, vol/vol) was run at 1.5 mL/min with detection at 445 nm. Sample injection volume was 50 L. Se veral standards including astaxanthin, lutein, zeaxanthin (Fluka, Switzerland) were run to identify sample peaks. Only one peak was obtained in the HPLC chromatogram. After comparison of re tention time with the standards, the peak was identified as astaxanthin. The amount of asta xanthin in salmon muscle was quantified using calibration curves obtained from different concen tration of astaxanthin standard. Three fillets were used for each treatment for each day with duplicate HPLC injections.

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121 Lipid Oxidation Analysis Measurement of thiobarbituric acid reactive substances (TBARS) TBARS were perform ed based on a modificatio n of Lemon (1974) according to Raghavan and Hultin (2005) by measuring secondary products of oxidation in dark and light muscle. After weighing approximately 1 g of the Atlantic salmon muscle in a disposable glass tube, sample was extracted with 3 mL of 7.5% TCA solution by homogenization with a Biohomogenizer at high speed for 1 min. The sample was centrif uged at 2000 rpm in an Eppendorf 5702 centrifuge (Brinkmann Instruments Inc., Westbury, NY) for 10 min. A 2 mL aliquot of the supernatant was mixed with 2 mL of 0.02 M TBA solution and heat ed in a boiling water bath for 40 min. Sample was cooled under cold running water. The color developed was spectrophotometrically measured at 530 nm. A standard plot was prepared using tetraethoxypropane (TEP). As each mole of TEP would yield one mole of malonaldehyde, the results were expressed as micromoles of malonaldehyde (MDA) per kg tissue. All experiments were done in triplicates. Measurement of lipid hydroperoxides Lipid hydro peroxides were measured accordi ng to the method of to Raghavan and Hultin (2005) by measuring primary products of oxidation in Atlantic salmon dark and light muscle. Approximately 1 g of Atlantic salmon muscle was placed in a disposable glass tube, homogenized for 1 min with 10 mL of chlorofo rm/methanol (2:1) usi ng a Biohomogenizer, and mixed with 3 mL of 0.5 % NaCl solution. The mi xture was first vortexed for 30 sec and then centrifuged at 2000 rpm in an Eppendorf centr ifuge (Brinkmann Instruments Inc., Westbury, NY). The chloroform phase was removed, and a 2 mL volume of the chloroform phase was made to 10 mL using chloroform/methanol (2:1). Ammonium thiocyanate and ferrous chloride were prepared as in Shantha and Decker (1994). A 25 L aliquot of each reagent was added and vortexed for 10 s. The samples were incubate d for 10 min at room temperature, and the

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122 absorbance was measured at 500 nm. A sta ndard curve was prepared using cumene hydroperoxide. All experiments were done in triplicates. Lipid Extraction for Fatty Acids Methyl Esters Lipids were extracted from Atlantic salm on li ght and dark muscle using a modified method of Bligh and Dyer (1959). Salmon muscle ti ssue (1 g) was blende d with 4 mL of a chloroform:methanol (1:2) mixtureusing a Waring commercial blender (Waring Products Division, Dynamics Corp. of America, CT) for 1 min in a disposable glass tube, 1.25 mL of chloroform was added and vortexed for 5 min, and then 2.25 mL of 0.5% KCl solution was added with mixing for 1 min before centrifugatio n. Samples were centrifuged at a speed of 6500 rpm for 10 min and the lower phase collected through the protein disk with a Pasteur pipette into weighed glass tubes. Chloroform (2 mL) was ag ain added to the remaining part, vortexed and centrifuged again and the lower phase collected into the previously weighed glass tube. Chloroform was removed under a nitrogen gas st ream using N-EVAP 112 Nitrogen Evaporator (Organomation Associates Inc., Berlin, MA, and U.S.A). The weights were recorded and percent lipids determined gravimetrically. Oils were flushe d with nitrogen and stor ed in amber vials at 80C until analysis. Preparation of Fatty Acid Methyl Esters Fatty acid methyl esters (FAME) m ethod was performed by modifying the AOAC official method 991.39 (AOAC., 2000). Accurately weigh ca 20 mg oil into a 20 ml round bottom screw cap centrifuge tube with a Teflon liner cap containing 100 L (10 mg/mL) both Methyl tricosanoate (C23:0) and Heptadecan oic acid (C17:0) (Fluka,Switzer land) as internal standards. After adding 1 mL 0.5 M methanoic NaOH solu tion, the mixture was flushed with Nitrogen, vortexed and heated for 5 min at 100 C in an Isotemp dry bath (Fisher Scientific, USA). The mixture was cooled, and 2 mL of 12% BF3 in methanol (Fisher Scientific, USA) was added and

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123 then heated at 100C for 3 min. The mixture was cooled, and 1 mL isooctane was added and this mixture heated at 100C for 1 min. Saturated sodi um chloride (NaCl) solution (5 mL)was added to the mixture, and agitated and cooled to room temperature. The isooctane layer was transferred to a clean glass tube; a small amount of anhydrous Na2SO4 was added. Fatty acids methyl ester was transferred into 2 mL screw thread amber GC vials (National Scientific, Rockwood, TN) for chromatographic analysis. Gas Chromatography (GC) Analysis FAME was analyzed on a GC HP 6890, equipped with flam e ionization detector, ATSilar-100 cyano silicone capillary column (30m x 0.25mm x 0.2 m) (Alltech Assoc. Inc, Nicholasville, KY), and split injection (split ratio 60:1). Operation conditions were as follows: Injection port temperature and detector temper ature were 250C; initial oven temperature was 140C for 2 min, gradually heated to 235C at a rate of 4C /min and held for 10 min. The carrier gas was helium (1ml/min). Retention times and peak areas were computed automatically by a Turbochrom Workstation 6.1.1 (Perkin Elmer, MA, and U.S.A). Compounds were tentatively identified by comparison with the retention times of known standards. All standards used in the identification of peaks were purchased from S upelco (Bellefonte, PA). The standards used were: Supelco 37, Marine Oil PUFA#1, and Me nhaden Oil PUFA#3. Methyl tricosanoate (C23:0) and heptadecanoic acid (C17 :0) were used as internal standards. Three fillets were used for each treatment for each day with duplicate GC injections. Sensory Evaluation After irradiation treatm ent of frozen salmon sa mples (0, 1, 2, 3 and 5 kGy), frozen samples were thawed at 4 C one day before the taste panel and then the samples were kept on ice for taste panels. Samples were cut into cubes of 2x2x2 cm dimension before serving in a small clear cup and lid. Each cup had a three digit random coded sticker. All samples were kept on ice until

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124 served to panelists. Sensory evaluation of irradiated Atlantic salmon was conducted at the Food Science and Human Nutritions taste panel facility (University of Florida, Gainesville, FL). The taste panel facility contains 10 private booths equipped with a monitor, mouse and keyboard for data entry. Seventy five untrained panelists evaluated the acceptability of irradiated Atlantic salmon fillets. When ten panelists at a time entered the room, they were directed to an individual booth. They were met by a panelist coordinato r who asked them to sign-in, gave a brief explanation of the evaluation procedure and re quired the panelist to sign the IRB-approved consent form by the University of Florida. Untr ained panelists (75) evaluated the irradiated frozen treated Atlantic salmon fillets based on color, odor and overall acceptability using a 9point hedonic scale, 1 being extremely dislik e and 9 being extremely like. Five fresh Atlantic salmon samples were given to each panelist; control (0 kGy), 1, 2, 3 and 5 kGy treatments. The samples were randomly assigned three digit codes and they were placed in different orders on a white tray. The panelists were asked a few demographic questions, and then asked to rate samples based on color, odor and ov erall acceptability attributes of samples. The panel design was to first rate the color attribute of samples w ithout removing the clear lid from the cup, then removing the lid from the cup and ra te the odor attribute, an d as a final evaluation rating overall acceptability. The panelists mark on the screen the number to indicate intensity ratings for each attribute. The Compusense software (Version 5.2, Compusense Inc., Ontario, CA) was used to design, conduct the test, and coll ect and analyze the data Mean hedonic ratings (n=75) for frozen treated Atlantic salmon fillets were analyzed by analysis of variance and Tukeys HSD test (p<0.05) for mean separations. Statistical Analysis Color data (L*, a*, b* values), m icrobial anal ysis, fatty acids profile lipid oxidation data (TBARS, PV) and astaxanthin analysis were analyzed by analysis of variance (ANOVA) and the

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125 mean separations were performed by LS Means Tukey HSD (P<0.05) using the JMP 5 software (SAS Institute Inc., Cary, NC). Sensory data were analyzed by analysis of variance and Tukeys HSD test (p<0.05) for mean separations usi ng Compusense software (Version 5.2, Canada). Results Microbial Analysis Frozen Atla ntic salmon samples were subject to irradiation treatment at various dose levels including 0 (control), 1, 2, 3 and 5 kGy. After irradiation treatment, all samples were thawed at 4C, then stored for 6 days at 4C and microbial levels presented in Figu re 6-1. The initial total aerobic count for samples was 1.34.5 log cfu/g. The microbial growth of untreated samples gradually increased and reached 5.7.6 log cfu/g by the end of 6 days storage at 4C. Treatment at 1 kGy and higher doses did significantly (p <0.05) reduced microbial load to undetectable levels and no microbial activity for treated sa mples was observed during 6 days storage. There was no significant (p>0.05) difference among irra diation treated Atlantic salmon samples in terms of total aerobic count during 6 days storage at 4C. There was only a small, but not statistically significant (p>0.05), increase in micr obial growth for untreated fillets on day 0 and 2. After day 2, significant (p<0.05) microbial gr owth was observed for the untreated samples (Figure 6-1). Color Analysis Changes in a* value (redness), L* value (lightness), b* value (yellowness) and E for whole surface of frozen Atlantic salmon light an d dark muscle were performed by using a color machine vision system after irradiation treatm ent and followed by storage for 6 days at 4C (Table 6-1 and Table 6-2). As the irradiation dose level increased to 5 kGy, the samples had discoloration compared with untreated samp les (Figure 6-2). Although not statistically significant (p>0.05), an increase in irradiation dose resulted in an increase in L* value of frozen

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126 light muscle. No significant difference was found in b* value between treated and untreated samples. However, irradiation treatment of light muscle brought about a decrease in a* value from 32.7 (0 kGy) to 17.8 (5 kGy). The 3 kGy a nd less dose showed no significant difference in a* value from control while the 5 kGy had a signif icantly lower a* value than untreated samples. Storage time did not show any major difference in L* and b* values among treated and untreated samples, however, the a* value for 5 kGy decreased 25% during the storage time period. The E value was increased as an increase in irradiation dose level (Table 6-1). Irradiation treatment on color parameters of frozen dark muscle are shown in Table 6-2. No significant difference was found in L* and b* values for dark muscle between treated and untreated samples at each day. Although the a* valu es of dark muscle decr eased gradually as an increase in irradiation level, the 3 kGy and lowe r doses did not significantly (p>0.05) differ from untreated samples. The highest dose (5 kGy) showed significant (p<0.05) difference from untreated sample. An increase in storage time resulte d in an increase in L* value, with a decrease in a* value, with an increase in E value and no significant effect on b* value for dark muscle for both treated and untreated samples. The loss of redness for dark muscle at 0, 1, 2, 3 and 5 kGy doses near the end of storage were 63.3%, 60.4%, 63.8%, 66.9% and 75.1%, respectively. On the other hand, an increase in L* value for da rk muscle for 0, 1, 2, 3 and 5 kGy doses near the end of storage were 11.7%, 8.0%, 1 0.1%, 9.5% and 10.8 %, respectively. Astaxanthin Analysis Astaxanthin analysis of Atlant ic salmon was perfor med using HPLC for frozen irradiation treated and untreated light and dark muscle (Fig ure 6-3a, Figure 64ab). The retention time of astaxanthin was found as 2.67 min at 445 nm (Figure 6-3a). When the light muscle was subjected to different irradiation doses, a significant decrease (p<0.05) in amount of astaxanthin was detected, with a maximum value 4.68 mg/kg for 0 kGy and decreasing to 0.48 mg/kg for 5 kGy

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127 on day 0 (Figure 6-4a). The amount of astaxant hin in light muscle for the 0 kGy was not significantly different from 1 and 2 kGy at da y 4, however, the 0 kGy was significantly higher (p<0.05) for astaxanthin than treated sample s at day 0, 2, and 6. There was no significant (p>0.05) difference in the amount of astaxanthin in light muscle between 1 kGy and 2 kGy for the entire storage periods. The highest treatment showed significantly (p<0.05) low amount astaxanthin in light muscle than the treated and untreated sample s Storage time did not have any effect for astaxanthin in the light muscle betw een treated and untreated samples (Figure 6-4a). The amount of astaxanthin in light muscle was more than 3 times higher than that of dark muscle. The effect of irradiation on the amount of astaxanthin in dark muscle showed similar trends to light muscle (Figure 6-4b). Unlike light muscle, the dark muscle was affected by storage time for both treated and untreated samples. An increase in storage resulted in a significant loss in the amount of as taxanthin for all treated and unt reated dark muscle samples. The amount of astaxanthin in dark muscle signi ficantly (p<0.05) decreas ed as the dose level increased, from 1.3 mg/kg for 0 kGy to 0.3 mg/kg for 5 kGy on day 0. There was no significance difference between the 1 kGy and 2 kGy in the am ount of astaxanthin in the dark muscle during entire storage at 4C. Correlation between Amount of Astaxanthin and a* Value Correlation was obtained between average am ount of astaxanthin and average a*-value (redness) for the different irradiation treatments in frozen light and dark muscle of Atlantic salmon for each storage day at 4C (Figures 6-5 to 6-6). An increase in irradiation dose resulted in decreasing both the amount of astaxanthin and a* value of light muscle for each storage day. The irradiation treatment at 5 kGy resulted in lowest a value and amount of astaxanthin for both light and dark muscle samples. The square of correlation coefficient (R2) for correlation

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128 between amount of astaxanthin a nd a* value in light muscle depending of dose level for day 0, 2, 4, and 6 were 0.84, 0.84, 0.89 and 0.84, respectively (Figure 6-5). The effect of dose level on dark muscle s howed correlation between astaxanthin amount and redness similar to light muscle. Increasing do se level resulted in lo sing astaxanthin amount and redness in dark muscle. The range of R2 was 0.68 to 0.957 for storage of 6 days at 4C. It was observed that the correlation gr adually decreased as storage day increase due to an increase in consumption of astaxanthin in dark muscle with an increase in stor age period (Figure 6-6). Lipid Oxidation Analysis Lipid oxidation was perform ed using thiobarb ituric acid reactive substances (TBARS) assay to monitor levels of secondary oxidati on products formed while Peroxide Value (PV) monitors levels of primary oxidation products formed. TBARS for light and dark muscle of salmon fillets during 6 days storage at 4C are pr esented in Figures 6-7a b and Figures 6-8ab). Irradiation on light and dark muscle forming lipid peroxides (PV) was shown in Figure 68ab. The PV value of untreated light muscle wa s significantly (p<0.05) hi gher than irradiation treatment levels at 2, 3, and 5 kGy for day 0, however, no significant (p>0.05) difference was obtained between 0 kGy and 1 kGy on the same day. There was no significant difference between treated and untreated light mu scle after day 0 (Figure 6-8a). The PV value of dark muscle for both treate d and untreated samples showed an increase during storage, reaching a maximum of 3270 mol lipid hydroperoxide/ kg muscle at the end of 6 days storage at 4C. The control and 3 kGy had significantly (p<0.05) lower PV value than 1 and 5 kGy on day 2. However, no significant difference in PV value for dark muscle was observed between treated and untreated sa mples on days 0, 4 and 6 (Figure 6-8b). TBARS method was used as a second chemical indicator of lipid oxidation. Secondary lipid oxidation products for fro zen irradiation treated and untr eated light and dark muscle

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129 increased during the six day storag e period at 4C. The level of lip id oxidation for the treated and untreated light muscle samples increased, starting at a minimum of 1.6 mol MDA/kg muscle on day 0 and reaching a maximum of 22.6 mol MDA/ kg after 6 days storage. Although 0 kGy was significantly (p<0.05) higher in TBARS value than treated light muscle samples for day 0, no significant (p>0.05) difference wa s observed between treated a nd untreated samples following storage. In addition, no significan t (p>0.05) difference was obtained in TBARS values for all irradiation treated light salmon mu scle at each testing point during the entire storage at 4C. The standard deviations of the salmon samples for TBARS were considerably high, indicating there was a greater amount of va riation in the individual fish (Figure 6-7a). Secondary lipid oxidation products for treated and untreated dark muscle samples showed an increasing trend during the en tire storage. Even though TBARS values of untreated dark muscle were significantly (p<0.05) lower than 3 kGy at day 4, no signifi cant (p>0.05) difference was obtained between treated and untreated samp les at days 0, 2, and 6 (Figure 6-7b). Lipid secondary oxidation products in dark muscle we re higher than light muscle during the entire storage at 4C due to hi gher lipid contents. Fatty Acids Analysis Fatty acid com position of frozen Atlantic salmon light and da rk muscle after irradiation treatment followed by storage for 6 days at 4C ar e shown in Tables 6-3 to 6-10. Selected major fatty acids are shown in Figure 6-3b. The major total saturated fatty acids in light muscle were 14:0, 16:0 and 18:0. The control had significantly (p<0.05) highe r amount of 16:0 and lower amount of 18:0 at day 0. Although the control was significantly (p <0.05) higher in total saturated fatty acids composition (g fatty aci ds/100 g total fatty acids) than the 5 kGy at day 0 (Table 6-3), no significant difference was determined between treated and untreated light muscle samples during storage 2, 4 and 6 days (Tables 6-4 to 6-6). All samples had major monoenes, including

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130 16:1n-7, 18:1n-7, 18:1n-9, 20:1n-9 and 22:1n-11. A lthough the control had significantly lower amount of total monoenes including the treated light muscle samples at day 0, no significant difference was found between untreated and trea ted light muscle samples following storage (Tables 6-4 to 6-6). Total n-6 polyunsaturated fa tty acids (PUFA) were mainly composed of 18:2n-6 and 20:2n-6 and 22:4n6 in light muscle. Although no signifi cant (p>0.05) difference was detected in light muscle between treated samples and control at days 2 an d 4 (Tables 6-4 to 6-5), The control showed signifi cantly (p<0.05) lower amounts of 18:2n6 and total n-6 PUFA compared to treated samples at day 0 (Table 6-3). Several n-3 PUFAs we re detected in light muscle samples, including 18:3n-3, 18:4n-3, 20: 5n-3 (EPA), 22:5n-3, 22:6n-3(DHA). The major individual fatty acids making up the total n3 PUFAs were 20:5n-3 (EPA), 22:5n-3 and 22:6n-3 (DHA). The amount of total n-3 including 20:5n-3 (EPA), 22:6n-3 (DHA) significantly decreased in irradiation treated samples compared to untreated samples at day 0. The 5 kGy dose significantly reduced both the amounts of EPA from 9.6 to 8.0 g/100 g total fatty acids, and DHA from 18.7 to 11.1 g/100 g total fatty acids at day 0 (Table 6-3). No significant difference was found in the total n-3 PUFA, DHA and EPA among irradiation treatments for light muscle at days 0, 2 and 4 (Tables 6-3 to 6-5). Fatty acids composition of dark muscle both treated and untreated samples are shown in Tables 6-7 to 10. It was found that no significant (p>0.05) difference in dark muscle samples for total saturated, total n-3 PUFA a nd ratio of n-3/n-6 PUFA regardless of treatment and storage time at 4oC (Tables 6-7 to 6-10). Sensory Evaluation A total of 75 untrained paneli sts ev aluated the irradiated frozen treated Atlantic salmon fillets based on color, odor and overall acceptability using a 9-point hedonic scale, 1 being extremely dislike and 9 being extremely like (Fig ure 6-9). It was observe d that an increase in

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131 irradiation dose resulted in a decrease in hedonic scores for color, odor, and overall acceptability of Atlantic salmon fillets. Fillets treated with 3 and 5 kGy rated significantly lower (p<0.05) than 0 and 1 kGy in color attributes of samples. C ontrols had the highest rated scores for color evaluation, but these were not si gnificantly different than 1 kGy. The 5 kGy had the lowest score of 4 for color attribute. The 0, 1 and 2 kGy fillets stayed above the acceptability level based on an hedonic score of 5. The odor attribute for untreated and 1 kG y samples showed no significant (p>0.05) difference. Significant (p<0.05) difference occurre d in odor scores of 2, 3 and 5 kGy, compared with that of untreated salmon samples. The treatment at 5 kGy had significantly lower odor scores. No significant different was observed between 1 and 2 kGy in terms of odor attribute. The general trend was an increase in overall acceptability as irradiation dose levels decreased. The Control had the highest odor score with 5.5, just slightly above the acceptability line. Overall acceptability score decreased to 3.2 for 5 kGy. No significant (p>0.05) difference in overall acceptability score was observed between c ontrol and 1 kGy, and also they stayed above the acceptability level. Although no significant difference between 2 and 3 kGy was found for overall acceptability score, both treatments had significantly higher scores than the 5 kGy, and significantly lower scores than control and 1 kGy. Panelists found that 2 kGy and higher doses were not acceptable based on ove rall acceptability (Figure 6-9). Discussion Irrad iation processing of food products is a widely studied field of research and is currently being practiced on several commercial food products, worldwide (Hayashi, 2007; Hileman, 2007). Microbial safety is a potential problem associated with seaf ood products. Irradiation treatment will reduce or eliminate the microorgani sms responsible for spoilage and subsequently extend the fresh-storage shelf-life. The low dose irradiation also has the ability to reduce or

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132 eliminate specific pathogenic bacteria co mmonly associated with seafood (Grodner and Andrews, 1991). In the present study, the irradia tion treatment level at 1 kGy and higher doses significantly (p<0.05) eliminated mi crobial growth in frozen treat ed Atlantic salmon during the 6 days storage period at 4 oC (Figure 6-1). After thawing, the co ntrol samples initial microbial growth was determined as 1.4 log cfu/g, which showed that the salmon were highly fresh. Control samples reached 5.7 log cfu/g by the end of 6 days storage, and did not exceed the maximum acceptable microbial limit, by the International Commission of Microbiological Standards for Foods (ICMSF, 1978). Many authors ha ve studied the influence of irradiation on the microbial growth in seafoods, including oyster, cod, crab, lobster, sw eet-lip, red emperor, mackerel, mullet, whiting, barramundi, crabmeat, prawn and sea bream (Chouliara and others 2004; Novak and others 1966; Chen and others 1996; Dagbjart.B and So lberg, 1973; Poole and others 1994; Thibault and Charbonneau, 1991). These authors have also demonstrated reduction in microbial levels similar to those found in this study. Astaxanthin is a major carotenoid in salmon fish muscle that provides salmon muscle with its characteristics orange color. An increase in irradiation dose caused di scoloration of frozen Atlantic salmon light muscle (Figure 6-2). Samp le treated with irradi ation at 5 kGy caused a significant decrease in a* value, with 46% lo ss in redness compared to untreated samples. However, the 3 kGy and less dose showed no signifi cant difference in a* value for light muscle compared to control during the entire storage tim e. The ice particles in frozen samples could minimize detrimental effects of irradiation on color of salmon samples. In addition, no significant difference was found in L* and b* valu es between treated and untreated samples. Storage time did not show any major difference in L* and b* values among treated and untreated light muscle samples (Table 6-1).

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133 In frozen salmon dark muscle, there was no significant (p>0.05) difference in the L*, a* and b* values for untreated and 3 kGy and lower dose treated samples immediately upon irradiation and each storage day. However, upon 6 da ys of storage at 4C there was a significant decrease (p<0.05) in the a* valu e. The a* value of untreated salmon dark muscle decreased from 18.4 to 6.8 while that of 5 kGy irradiated samp le decreased from 14.9 to 3.7. In addition, there was a significant (p<0.05) difference in the a* and b* values of sa lmon light and dark muscle. In general, the a* and b* values of dark muscle were lower than those of light muscle. Similar results were reported in fully cooked salmon and catfish fillets by McKenna and others (McKenna and others 2003). In sa lmon, the color could be attributed to both carotenoids such as astaxanthin, as well as to heme pigments. The am ount of astaxanthin in salmon light muscle was approximately 3 times higher than that of dark muscle (Figure 6-4a and 6-4b) while the amount of heme (arising from the heme proteins in he moglobin and myoglobin) in dark muscle would be far greater than the amount in light muscle (Ric hards and Hultin, 2002), therefore,, in our present study, the color of salmon light muscle could be pr imarily due to astaxant hin while the color of dark muscle would be due to the heme pigments. In our studies, we found a significant loss in the amount of astaxanthin in both salm on light and dark muscle due to irradiation (Figures 6-4a and 6-4b). Irradiation could lead to loss in the co lor and quality of seafood products. Hammad and others (1992) reported a loss in the color of smoked salmon upon irradiation. Hence, the loss of a* value in salmon light muscle could be due to the loss of astaxanthin. Salmon dark muscle contains a significantly higher amount of heme pr oteins and lipids compared to salmon dark muscle (Katikou and others 2001). Irradiation could lead to the oxidation of unsaturated lipids such as those found in the dark muscle of salm on (Katusinrazem and others 1992). Therefore, the loss in the a* value of salmon dark muscle co uld be due to the oxida tion of lipids. In our

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134 research, we evaluated lipid oxidation in both dark and light muscle of salmon using the primary products of oxidation, i.e., lipid hydroperoxides value (PV), as well as using the secondary products of oxidation, i.e., TBARS. In salmon light muscle, we found no significant difference (p>0.05) in both PV and TBARS values (Figures 6-7a and 6-8a) between treated and untreated samples, as well as for days 2, 4 and 6 of storag e at 4C While in salmon dark muscle, there was no significant difference (p>0.05) in the TBARS value among the different treatments, a significant difference (p<0.05) was observed for different storage days (Figure 6-7b). A trend similar to TBARS was also noticed in the a* va lue of treated and untreated salmon dark muscle, which could imply that oxidation may play an impor tant role in the color of salmon dark muscle. Endogenous antioxidants such as astaxanthin (Miki, 1991) would play a vital role in controlling lipid oxidation in seafood such as salmon. Especia lly, the presence of antioxi dants is important in dark muscle, which contains polyunsaturated tr iglycerides, membrane lipids (Hultin, 1992) and catalysts of lipid oxidation such as heme pigm ents (Richards and Hulti n, 2002). In our studies, a decrease in the astaxanthin leve l (Figure 6-4b) of salmon dark muscle was observed due to both irradiation as well as due to storage (in trea ted samples), which could imply a decrease in protection against oxidation. Irradiation of salmon light and dark muscle on the fatty acid composition was also conducted. A significant difference (p <0.05) in the PUFA of light muscle at day 0 was obtained due to irradiation, while no significant (p>0.05) difference was observed in the fatty acid composition of dark muscle due to irradiation or storage at 4C. A lthough numerous authors have reported loss of fatty acids due to ionizing radiation (Che n and others 2007; Formanek and others 2003), authors such as Hammer and Wills (1979) reported little or no change in the fatty acid composition of various fat sources containing high amount of antioxidants. In addition,

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135 Ghadi and Venugopal (1991) investigated the infl uence of gamma irradiation up to 5 kGy on lipid oxidation in skin and flesh fractions of I ndian mackerel, white pomfret, and seer during ice storage. They found increase in TBA values in bo th control and irradiated fish, especially in mackerel and seer meat. Armstrong and others (1994) also did not find any changes in fatty acids composition of two Australian marine fish species at doses up to 6.0 kGy. The authors also stated that variations in fatty acid composition betw een individual samples were greater than any radiation-induced changes. In our studies, we f ound little change in the fatty acid composition of salmon muscle, which could be due to the pres ence of high amount of antioxidant, astaxanthin. During oxidation, antioxidants are us ually consumed or degraded first, before lipid oxidation would occur. The large decrease in the amount of astaxanthin (Figure 6-4a and 6-4b) due to irradiation indicates that antioxi dants are being consumed or sacr ificed to prevent the oxidation of lipids. However, we conducted a 6 days stor age study at 4C. Maybe, a significant change in the fatty acid content may have been observed for longer storage studies. In addition, we also did sensory analysis using taste panels for frozen treated and untreated salmon light muscle based on the evaluation of co lor, odor and overall acceptability. Irradiation treated samples at 2 kGy and higher doses scor ed significantly less on co lor, odor as well as overall acceptability compared to untreated samples (Figure 6-9). The 1 kGy showed no significance difference from control for all at tributes including color, odor and overall acceptability. The 5 kGy had the lowest score fo r color, odor and overa ll acceptability among treated and untreated samples. Among the color values from co lor machine vision systems, we did not observe significant difference (p>0.05) in the L* and b* values, while significant difference (p<0.05) was observed in the a* value between 5 kGy treated and untreated samples. Hence, it is possible that duri ng sensory evaluation, the panelists were primarily evaluating the

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136 redness (a* value) rather than b* and L* values. However, additional research is required to correlate the color of samples with the evalua tion of consumer panelis ts. The odor of salmon fillets would arise both from lipid oxidation and rancidity, as well as due to the breakdown of compounds from irradiation (Batzer and Doty, 1955; Batzer and others 1957) In our studies, we found no significant difference (p>0.05) in th e PV and TBARS values of untreated and irradiation treated samples for days 2, 4 and 6. Hence, the sensory score of the panelists on the odor of salmon fillets might be due to the br eakdown products of irradiation than due to oxidative rancidity. However, we did not determ ine the type of break down products. Hence, future research in this area would shed light on the type of irradiation products in salmon muscle. Conclusion Frozen salmon fillets were treated with various doses of irradiation and were stored at 4C for 6 days. There was a significant decrease in the microbial growth of frozen irradiation treated fillets. Correlation was obtained between average amount of asta xanthin and average a*value (redness) during different irra diation treatments in frozen light and dark muscle of Atlantic salmon for each storage period up to 6 days at 4 C. It was found that redness (a* value) of salmon light muscle was more related to astaxant hin content, while that of salmon dark muscle was related more to the lipid content and the de gree of lipid oxidation. Sensory score of 2 kGy and higher doses was significantly (p<0.05) lowe r in color, odor and overall acceptability compared to untreated samples, and the sensory score decreased with an increase in irradiation level. The 1 kGy treated samples was a successful treatment, with no significant effect on color, sensory as well as providing inactivation of micr obial growth during 6 da ys storage at 4 C compared to untreated samples.

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137 Table 6-1. Changes in L* valu e (lightness), a* value (redne ss), b* value (yellowness) and E for frozen Atlantic salmon light muscle after irradiation treatment, thawing at 4C and following for 6 days at 4C. L* -value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 56.3.9a 60.6a 59.4 3.9a 60.1.3a63.4.5a 2 56.9.5a 62a 61.2. 8a 61.6.9a62.7.8a 4 56.1a 60.5.8a58.9. 6a 59.8a 63.9.4a 6 56.2.5a 60.3.4a59.6 3.9a 60.1.1a64.5.4a a*-value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 32.7.5a 30.2.4a29.5 2.6a 27.8.9a17.8.2b 2 32.3a 29.7a 28.1.9a 26.2a 16.1.7b 4 32.1.5a 28.7.1a28.6 1.9a 25.7.3a14.7.4b 6 30.8.5a 28.7.2a28. 6a 24.9.4a13.3.5b b*-value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 35.2.6a 36.2a 35.9 1.3a 36.8a 33.2.7a 2 34.7a 34.6.8a34.3. 1a 34.2.5a30.4.6a 4 34.6.7a 34.3a 34.7 1.3a 34.3.4a30.1.7a 6 34.1.7a 34.7a 34.5 1.3a 34.1.5a29.8.1a E Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 2.0.3c 2.5.3bc 3.2 0.7bc 6.3.2b 16.0.7a 2 1.7.5c 3.8.6bc 4.1 1.0bc 7.9.4b 17.5.5a 4 2.4.0c 3.2.9bc 3.2 1.1bc 7.2.5b 19.3.2a 6 3.5.0b 3.1.7b 3.61.3b 8.2.1b 20.9.7a Values are means standard deviations (n=3). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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138 Table 6-2. Changes in L* valu e (lightness), a* value (redne ss), b* value (yellowness) and E for frozen Atlantic salmon dark muscle after irradiation treatment, thawing at 4C and following storage for 6 days at 4C. L* -value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 54.8.7a 58.3.4a57. 7a 56.2.8a56.6.3a 2 57.6a 60.3.1a60.5. 5a 56.9a 58.3.7a 4 56.6.6a 61.2.9a60.2 3.8a 58.5.6a59.5.2a 6 61.2.1a 63.3a 62.8 3.6a 61.5.6a62.7.1a a*value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 18.4.4a 17.3.6ab17.2 0.8ab 16.4.3ab14.9.3b 2 14.4a 12.3.4a13.1a 12.3.9a10.3a 4 10.7.9a 8.8.7a 9. 2.3a 8.7.4a 6.3.7a 6 6.8.3a 6.9.1a 6.20.6a 5.4.5ab3.7.3b b*-value Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 18.3.1a 18.7.9a19. 6a 18.2.5a17.5.8a 2 17.4.5a 17.8a 17.9 1a 17.1.7a16.5.2a 4 18.8a 17.8.2a18.1 1a 17.5.9a17.2.7a 6 18.5.8a 18.9.2a18.6 1.6a 18.7.6a18.3.3a E Storage day 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 0 6.0.5ab 7.4.4a 6.80.5ab 5.5.9b 5.1.6b 2 5.8.1a 6.2.4a 5.90.9a 6.4.6a 7.3.5a 4 9.6.8a 9.1.3a 8.71.2a 9.7.5a 11.4.1a 6 13.0.4a 11.4.3a11.8 1.4a 13.5.7a14.8.4a Values are means standard deviations (n=3). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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139 Table 6-3. Fatty acid composition (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon light muscle after irradiation treatm ent, thawing at 4C and following storage for day 0 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 2.6.3a 2.9.4a 2.9.2a 2.9.1a 2.9.3a 16:00 17.1.6a 15.5.4b 14.7.6b 15.1.7b 14.4.8b 18:00 3.9.2b 4.2ab 4.1.1ab 4.3.2a 4.3.2a Total saturated 24.4a 23.2.7ab 22.4.5b 22.9.7ab 22.3b 16:1n 7 2.9.5b 3.5.4ab 3.7.5a 3.6.3ab 3.8.2a 18:1n 9 14.4.4b 16.5.8ab 17.7.4a 17.1.8a 18.2.8a 18:1n 7 2.5.1b 2.7.1ab 2.8a 2.7.2ab 2.8a 20:1n 9 1.9.8b 2.5.1ab 2.6.1a 2.5.3ab 2.6.1a 22:1n 11 0.3.1a 0.3.1a 0.3.1a 0.4.1a 0.4.1a Total monoe nes 23.6.5b 27.3.4a 29.1.1a 28.1.8a 29.8.1a 18:2n 6 14.8.6b 17.4.4a 18.2.9a 17.6.2a 18.8.3a 20:4n 6 0.9.4a 1.2a 1.2a 1.2a 1.2a 22:4n 6 0.4a 0.4.1a 0.5a 0.4a 0.3.2a Total n6 PUFA 16.6.7b 19.4.2a 20.3.8a 19.7.5a 20.8.5a 18:3n 3 1.1b 1.2.1a 1.3.1a 1.3.1a 1.4.1a 18:4n 3 1.1b 1.1.1ab 1.1.1ab 1.1.1ab 1.2.1a 20:5n 3 9.6.8a 8.7.5b 8.4.5b 8.1.3b 8.3b 22:5n 3 4.1.2a 4.1.4a 4.2a 4.1.3a 4.2.1a 22:6n 3 18.7.2a 13.6.3b 12.2.2b 13.3.8b 11.1.5b Total n3 PUFA 35.4.8a 30.1.9b 28.3.7b 29.2.5b 27.1.7b n3/n 6 2.2.5a 1.6.1b 1.4.2b 1.5.4b 1.3b Results are meansSD, n=12, different letters w ithin a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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140 Table 6-4 Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon light muscle after irradiation treatm ent, thawing at 4C and following storage for day 2 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 2.8.2a 2.8.3a 2.9.3a 2.8.3a 3.4a 16:00 15.3a 14.6.9a 14.6.2a14.5.7a 14.7a 18:00 4.2.3a 4.2.1a 4.2.2a 4.4.1a 4.3.2a Total saturated 22.6.5a 22.2a 22.4.8a22.3.9a 22.6.2a 16:1n 7 3.6.3a 3.6.5a 3.7.1a 3.7.2a 3.9.5a 18:1n 9 17.6.4a 17.8.7a 18.6a 18.1a 18.3.3a 18:1n 7 2.7.1a 2.8a 2.7a 2.8.1a 2.7.1a 20:1n 9 2.6.2a 2.6.1a 2.6.1a 2.6.1a 2.6.1a 22:1n 11 0.4.1a 0.3.1a 0.4.1a 0.4.1a 0.4.1a Total monoe nes 28.8.3a 29.4a29.5a 29.3.2a 30.1a 18:2n 6 18.1.5a 18.7.4a 18.5.9a18.4.5a 18.7a 20:4n 6 1.3a 1.3a 1.3a1.2a 0.9.3a 22:4n 6 0.3.2a 0.4.1a 0.2.2a 0.5.1a 0.4.1a Total n6 PUFA 20.3a 20.7.3a 20.4.7a20.6.5a 20.8a 18:3n 3 1.3.1a 1.3.1a 1.3.1a 1.4.1a 1.4.1a 18:4n 3 1.1.1a 1.2a 1.1.1a 1.2.1a 1.1.1a 20:5n 3 8.2.7a 8.3.8a 8.3.5a 8.3a 7.9.8a 22:5n 3 4.4a 4.1.2a 4.3a 4.1.3a 4.1.2a 22:6n 3 12.6.9a 12.3a 11.6.5a11.8.5a 10.8.1a Total n3 PUFA 28.6.4a 28.1.7a 27.7.3a27.7.6a 26.6a n3/n 6 1.4.3a 1.4.3a 1.4.2a 1.3a 1.3.2a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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141 Table 6-5. Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon light muscle after irradiation treatm ent, thawing at 4C and following storage for day 4 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 2.8.2a 3.4a 3.1.9a 2.9.2a 2.8.4a 16:00 14.4.6a 14.7.3a 14.7.7a14.5.2a 14.2.8a 18:00 4.4.2a 4.4.1a 4.3.1a 4.2.1a 4.2.2a Total saturated 22.2.8a 22.7.6a 22.8.5a22.3.3a 21.8.9a 16:1n 7 3.7.1a 3.8.5a 3.7.7a 3.9.2a 3.7.6a 18:1n 9 18.6.6a 18.1.6a 17.9.4a19.1.3a 18.9a 18:1n 7 2.7a 2.8.1a 2.7.1a 2.6.3a 2.8a 20:1n 9 2.7.1a 2.6.1a 2.6.1a 2.7.1a 2.6.2a 22:1n 11 0.4.1a 0.3.1a 0.3.1a 0.3.1a 0.3.1a Total monoe nes 30.1.8a 29.6.3a 29.3.5a30.9.5a 29.5.8a 18:2n 6 19.2.4a 18.6.8a 18.8.1a19.4.1a 18.5.4a 20:4n 6 0.5.4a 0.9.4a 0.9.4a 0.7.4a 0.9.4a 22:4n 6 0.4.1a 0.6.2a 0.5.2a 0.5.1a 0.6.2a Total n6 PUFA 20.8.9a 20.8.7a 21.1a 21.4.1a 20.7.4a 18:3n 3 1.5.1a 1.4.1a 1.4.1a 1.4.1a 1.4.2a 18:4n 3 0.9.5a 0.9.5a 0.9.5a 0.9.4a 0.8.4a 20:5n 3 8.3a 7.9.7a 8.8a 8.2.4a 8.3.8a 22:5n 3 4.1.2a 4.1.4a 3.9.3a 4.1.1a 4.2.2a 22:6n 3 11.1.2a 11.4a 11.5.1a 9.5.9a 12.3.6a Total n3 PUFA 26.9.4a 26.9.4a 26.9.6a 25.4a 28.4a n3/n 6 1.3.1a 1.3.3a 1.3.3a 1.2.1a 1.4.3a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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142 Table 6-6 Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon light muscle after irradiation treatm ent, thawing at 4C and following storage for day 6 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 3.1.7a 3.5a 2.8.7a 2.9.3a 2.9.4a 16:00 14.7.1a 14.2a 15.5a 14.3.8a 14.3a 18:00 3.9.4a 4.3.1a 4.3a 4.3.1a 4.4.2a Total saturated 22.3.8a22.2.5a 23.9a 22.1.2a 22.3.5a 16:1n 7 4.1.9a 4.3a 3.4.8a 3.8.4a 3.8.2a 18:1n 9 17.9.3a18.9.8a 15.9.8a 18.7.7a 18.8.4a 18:1n 7 2.5.4a 2.7.1a 2.6.1a 2.7.1a 2.8.1a 20:1n 9 2.7.2a 2.7.1ab 2.4.3b 2.7.2a 2.7ab 22:1n 11 0.3.1a 0.4.1a 0.3.1a 0.3.1a 0.3.1a Total monoe nes 30.4ab 30.8.9a 26.4.4b 30.4.2ab 30.6.5a 18:2n 6 19.2.2ab 18.7.7ab 16.4.8b 19.5.7a 19.1.9ab 20:4n 6 0.8.5a 0.9.4a 0.9.4a 0.9.4a 0.9.4a 22:4n 6 0.5.1a 0.5.1a 0.5.2a 0.5.2a 0.5.2a Total n6 PUFA 21.3.9a 20.8.9ab 18.5.8b 21.6.7a 21.3.8a 18:3n 3 1.2.5a 1.5.1a 1.2.2a 1.4.1a 1.5a 18:4n 3 0.9.5a 0.9.4a 0.8.4a 0.9.4a 0.9.4a 20:5n 3 8.1.6a 7.9.2a 8.5.1a 8.4a 8.6a 22:5n 3 4.2.2a 4.1.4a 4.3.3a 4.1.2a 4.4a 22:6n 3 10.3.1b 10.5.4ab 16.4.6a 10.3.6b 10.3.4b Total n3 PUFA 26.1a 26.2.1a 32.1.4a 26.1a 25.9.7a n3/n 6 1.2.2a 1.3.1a 1.8.7a 1.2.3a 1.2.2a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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143 Table 6-7 Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon dark muscle after irradiation treatm ent, thawing at 4C and following storage for day 0 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 3.1.6a 2.8.2a 2.9.3a 3.1.7a 3.3a 16:00 13.9.3a 13.3.1a 13.6a 13.7.9a 13.4.7a 18:00 4.4.2a 4.4.2a 4.4.2a 4.5a 4.4.1a Total saturated 21.9.1a 21.5a 17.8.8a 21.8.6a 21.3.8a 16:1n 7 3.2.5a 3.8.1a 3.8.2a 3.9.4a 4.2a 18:1n 9 18.6.6a 18.7.2a 18.4.9a 18.5.2a 18.9.5a 18:1n 7 2.9.2a 2.9.2a 2.9.2a 2.9.1a 2.9.1a 20:1n 9 2.4a 2.7.1a 2.7.1a 2.7.1a 2.7a 22:1n 11 0.4.1a 0.4.2a 0.3.1a 0.4.1a 0.4.2a Total monoe nes 29.5.2a 30.5.4a 25.2.4a30.5.5a 31.7a 18:2n 6 19.4.9a 19.6.3a 19.3.9a 19.2.4a 19.2.9a 20:4n 6 0.8.4a 0.9.3a 0.7.4a 0.8.4a 0.8.4a 22:4n 6 0.6.1a 0.6.1a 0.6.1a 0.6.1a 0.6.1a Total n6 PUFA 21.5.1a 21.9.3a 17.8.7a 21.3.6a 21.3a 18:3n 3 1.4.1a 1.4.1a 1.4.1a 1.4.1a 1.5.1a 18:4n 3 1.1.1a 1.1.1a 1.1.1a 1.2.1a 1.2a 20:5n 3 8.2.2a 8.1.5a 8.6a 7.9.4a 8.6a 22:5n 3 4.4.4a 4.5.6a 4.3.5a 4.4.3a 4.6.6a 22:6n 3 10.9.5a 10.4.8a 11.2.3a 10.5.5a 10.1.5a Total n3 PUFA 27.1a 26.6.5a 22.6.4a26.4.9a 26.3.4a n3/n 6 1.3.2a 1.2.1a 1.3.2a 1.2.1a 1.2.2a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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144 Table 6-8 Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon dark muscle after irradiation treatm ent, thawing at 4C and following storage for day 2 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 2.8.2a 2.7.4a 2.9.2a 3.4a 3.3a 16:00 13.4.8a 13.2.8a 13.5.7a 13.5.9a 13.3.8a 18:00 4.4.1a 4.4.2a 4.4.2a 4.5.1a 4.5.1a Total saturated 21.2.2a 20.8.1a 21.3a 21.5.2a 21.3.2a 16:1n 7 3.8.1a 3.8.4a 3.8.1a 3.9.3a 4.2a 18:1n 9 18.3.4b 18.9.6ab 18.8.2ab 18.7.2ab 19.1.2a 18:1n 7 3.2a 2.9.2a 3.1a 2.9.1a 3.1a 20:1n 9 2.7.1a 2.7a 2.7.1a 2.7a 2.7.1a 22:1n 11 0.4.1a 0.4.2a 0.4.1a 0.4.2a 0.4.2a Total monoe nes 30.2.3b 30.8.9ab 30.8.2ab 30.8.1ab 31.2.4a 18:2n 6 18.9.8a 19.7.7a 19.8.5a 19.3.8a 19.2.6a 20:4n 6 0.8.4a 0.9.3a 0.9.2a 0.9.2a 0.9.2a 22:4n 6 0.6.1a 0.6.1a 0.6.2a 0.6.1a 0.6.2a Total n6 PUFA 21.9a 22.7a 22.4a 21.6.8a 21.5.7a 18:3n 3 1.4.1ab 1.4.1ab 1.6b 1.4.1ab 1.5.1a 18:4n 3 1.1a 1.2.1a 1.2.1a 1.2.1a 1.2.1a 20:5n 3 8.2.6a 8.1.5a 8.3a 7.8.4a 8.2a 22:5n 3 4.6.6a 4.5.4a 4.4.2a 4.4.5a 4.6.2a 22:6n 3 11.3.3a 10.2.8a 10.3.5a 10.2.2a 9.8.3a Total n3 PUFA 27.6.2a 26.4.5a 25.9.5a 26.1.2a 26.8a n3/n 6 1.3.2a 1.2.1a 1.2.1a 1.2.1a 1.2a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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145 Table 6-9. Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon dark muscle after irradiation treatm ent, thawing at 4C and following storage for day 4 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 3.1.3a 3.1.3a 3.3a 3.1.3a 3.1.4a 16:00 13.6.8a 13.4.8a 13.5.6a 13.4.8a 13.6.8a 18:00 4.4.1ab 4.5.1a 4.5a 4.3b 4.5.2ab Total saturated 21.7.1a 21.5.2a 21.5.9a 21.3.1a 21.6.2a 16:1n 7 4.2a 4.3a 3.9.3a 4.1.2a 4.3a 18:1n 9 19.2.2a 19.4a 19.1.3a 19.6.3a 19.1.5a 18:1n 7 2.9.1a 3.2a 2.9a 3.1a 3.1a 20:1n 9 2.7.1ab 2.7ab 2.7ab 2.8a 2.7.1b 22:1n 11 0.4.2a 0.4.2a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 31.5.2a 31.3.7a 31.2.7a 31.9.4a 31.3.8a 18:2n 6 19.6.4ab 19.3.5b 19.5.4ab 20.1.5a19.3.2b 20:4n 6 0.9.3a 0.9.3a 0.9.3a 0.9.3a 0.9.3a 22:4n 6 0.6.2a 0.6.1a 0.6.1a 0.6.1a 0.6.1a Total n6 PUFA 21.8.4ab 21.6.5b 21.8.4ab 22.3.4a21.6.1b 18:3n 3 1.5.1a 1.5.1a 1.5a 1.5.1a 1.5a 18:4n 3 1.2.1a 1.2.1a 1.2.1a 1.2.1a 1.2.1a 20:5n 3 7.8.5a 8.5a 7.8.4a 7.9.4a 7.9.6a 22:5n 3 4.3.4a 4.5.5a 4.3.3a 4.4.4a 4.3.4a 22:6n 3 9.3.9a 9.6.9a 9.7a 8.5.3a 9.6.1a Total n3 PUFA 25.3a 25.7.8a 25.6.6a 24.5.7a 25.5.9a n3/n 6 1.1.1a 1.2.1a 1.2.1a 1.1.1a 1.2.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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146 Table 6-10. Fatty acid compositions (g fatty acids/100 g total fatty acids) of frozen Atlantic salmon dark muscle after irradiation treatm ent, thawing at 4C and following storage for day 6 at 4C. 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy 14:00 3.1.4a 3.2.4a 3.1.4a 3.1.2a 3.1.2a 16:00 13.8.7a 13.6.5a 13.6a 13.5.3a 13.7.9a 18:00 4.6.1a 4.5.1a 4.5.2a 4.4.2a 4.5.2a Total saturated 21.9.1a 21.7.7a 21.7.5a 21.5.4a 21.8.2a 16:1n 7 4.3a 4.1.3a 4.1.4a 4.2a 4.1.2a 18:1n 9 18.9.4a 19.3.6a 19.1.3a 19.6.4a 19.3.1a 18:1n 7 3.2a 3.1.1a 3.1a 3.1a 3.2a 20:1n 9 2.7a 2.7a 2.7.1a 2.8.1a 2.7.1a 22:1n 11 0.4.2a 0.4.2a 0.4.2a 0.4.2a 0.4.2a Total monoe nes 31.1.6a 31.8.9a 31.5.5a 31.9.5a 31.7.2a 18:2n 6 19.6b 19.3.7ab 19.1.6b 20.1.7a 19.7.2ab 20:4n 6 0.9.3a 0.9.3a 0.9.3a 0.9.3a 0.9.3a 22:4n 6 0.6.1a 0.6.1a 0.6.1a 0.6.1a 0.6.1a Total n6 PUFA 21.2.4b 21.5.5ab 21.3.7b 22.3.6a 21.9.1ab 18:3n 3 1.5.1a 1.5.1a 1.5.1a 1.5.1a 1.5.1a 18:4n 3 1.2.1a 1.1.1a 1.2.1a 1.2a 1.2a 20:5n 3 7.8.5a 7.7.4a 8.4a 7.8.2a 7.7.2a 22:5n 3 4.4.5a 4.4.5a 4.5.4a 4.4.2a 4.2.4a 22:6n 3 10.1a 9.3.4a 9.4.8a 8.4.4a 9.7a Total n3 PUFA 25.8.8a 25.9a 25.5.4a 24.3.3a 24.6.1a n3/n 6 1.2.1a 1.2.1a 1.2.1a 1.1.1a 1.1.1a Results are meansSD, three different fillets were used for each treatment and each day, different letters within a row for each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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147 c c b a 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 01235log cfu/gDose (kGy) Day 0 Day 2 Day 4 Day 6 Figure 6-1. Total aerobic plate count for frozen Atla ntic salmon after irradiation treatment, thawing at 4C and following storage at 4C for 6 days. Values are means standard deviations (n=3). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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148 (a) (b) Figure 6-2. Astaxanthin analysis HPLC chromatogram of Atlant ic salmon at 445 nm UV-visible range with a 2.67 min retention time of asta xanthin (a). Fatty acids analysis by GC chromatogram of Atlantic salmon muscle, only selected major fatty acids peak are labeled. Fatty acids eluted in order of carbon chain length, number of double bonds, and position of double bonds on a polar ca pillary column (see Methods). The 17:0 and 23:0 used as internal standards (b).

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149 Figure 6-3. Images for frozen Atlantic salmon light and dark muscle at different doses after irradiation treatment and subsequent storage for 6 days at 4 C.

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150 (a) a a a a b b a b b bc ab b c c b b d d c c 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0246Amount of Astaxanthin (mg/kg)Time (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy (b) a a a a b bc ab b b ab bc bc bc c cd cd c d d d 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0246Amount of Astaxanthin (mg/kg)Time (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy Figure 6-4. Amount of astaxanthin (mg/kg muscle) for (a) frozen light muscle (b) frozen dark muscle of Atlantic salmon fillets before and after irradiation treatment, thawing at 4C and following storage at 4C for 6 da ys. Three fillets were used for each treatment and each day. Different letters within each day indicate significant differences at P < 0.05 separated by Tukey s HSD. Values are means standard deviations (n=6). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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151 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 3.283x + 19.09 R = 0.8380 5 10 15 20 25 30 35 40 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 0 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 3.045x + 17.87 R = 0.8350 10 20 30 40 0.01.02.03.04.05.06.0a* valueAmount of astaxanthin (mg/kg) Day 2 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 3.972x + 15.26 R = 0.8900 5 10 15 20 25 30 35 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 4 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 4.166x + 14.21 R = 0.8370 5 10 15 20 25 30 35 40 0.01.02.03.04.05.0a* valueAmount of astaxanthin (mg/kg) Day 6 Figure 6-5 Correlation between amount of astaxanthin and average a*-value for different irradiation treatments for frozen light muscle stored at 4C for 6 days.

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152 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 3.444x + 14.27 R = 0.9570 5 10 15 20 0.00.51.01.5a* valueAmount of astaxanthin (mg/kg) Day 0 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 4.293x + 9.566 R = 0.9160 5 10 15 20 0.00.20.40.60.81.01.2a* valueAmount of astaxanthin (mg/kg) Day 2 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 9.245x + 5.787 R = 0.8280 2 4 6 8 10 12 0.00.10.20.30.40.50.6a* valueAmount of astaxanthin (mg/kg) Day 4 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy y = 9.452x + 4.037 R = 0.6750 1 2 3 4 5 6 7 8 0.0 0.1 0.2 0.3 0.4a* valueAmount of astaxanthin (mg/kg) Day 6 Figure 6-6. Correlation between amount of astaxanthin and aver age a*-value for different irradiation treatments for frozen dark muscle stored at 4C for 6 days.

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153 (a) a a a a b a a a b a a a b a a a b a a a 0 5 10 15 20 25 30 35 40 0246TBARS (mol MDA/kg)Time (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy (b) a a b a a a ab a a a ab a a a a a a a ab a 0 20 40 60 80 100 120 140 160 180 200 0246TBARS (mol MDA/kg)Time (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy Figure 6-7. Changes in lipid oxidation as measured by the formation of thiobarbituric acid reactive substances (TBARS) for (a) light musc le (b) dark muscle of frozen Atlantic salmon after irradiation treatment, thawing at 4C and following storage for 6 days at 4C. Values are means standard deviations (n=6). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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154 (a) a a a a ab a a a b a a a b a a a b a a a 0 10 20 30 40 50 60 70 80 90 100 0246mol lipid hydroperoxide/ kg muscleTime (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy (b) a b a a a ab a a a a a a a b a a a a a a 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0246mol lipid hydroperoxide/ kg muscleTime (day) 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy Figure 6-8. Changes in lipid oxidation as measured by the formation of peroxide value (PV) for (a) light muscle (b) dark muscle of frozen Atlantic salmon after irradiation treatment, thawing at 4C and following storage for 6 days at 4C. Values are means standard deviations (n=6). Different letters within each day indicate significant differences at p < 0.05 separated by Tukeys HSD.

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155 a a a ab ab a bc bc b c c b d d c 1 2 3 4 5 6 7 8 9 Color Odor Overall AcceptablityHedonic Score 0 kGy 1 kGy 2 kGy 3 kGy 5 kGy Figure 6-9. Effect of different irradiation doses on sensory evalua tion of color, odor and overall acceptability for frozen Atlant ic salmon fillets. Acceptab ility or hedonic scale: 1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely. ** Mean of hedonic ratings (n = 75) for frozen Atlantic salmon fillets were analyzed by analysis of variance and Tukeys HSD test (P<0.05) for mean separations using Compusense software (Version 5.2 Canada) *** Different letters within each attribute indicate significant differences (P < 0.05)

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156 Figure 6-10. Temperature profile of A tlantic salmon during freezing at -20 C.

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157 CHAPTER 7 SUMMARY AND CONCLUSION Two different types of non-therm al processing, namely, high pressure and irradiation, were performed. High pressure processi ng of rainbow trout and mahi mahi and salmon resulted in low microbial load but higher amount of lipid oxidation, measured as thiobarbituric acid reactive substances. The color of high pressure treated rainbow trout and ma hi mahi and salmon decreased with increase in pre ssure and this was reflected in its sensory quality. 300 MPa for rainbow trout and 450 MPa for mahi mahi were the optimum high pressure processing conditions for controlling microbial load, lipid oxidation and color changes. For Atlantic salmon, HPP and cooking significantly reduced microbial growth. The 150 MPa treatment had a lesser effect on the color compared to cooki ng and 300 MPa. While cooking and 150 MPa led to similar oxidation development as untreated control, th e 300 MPa treatment effectively reduced the samples susceptibility to oxidation. Cooking significantly reduced the amount of total PUFA n-6 and PUFA n-3, including EPA and DHA fatty acids however, HPP did not change the level of those fatty acids. Fresh and Frozen salmon fillets were treated with various doses of irradiation and were stored at 4C for 6 days. There was a significant decrease in th e microbial growth of both fresh and frozen irradiation treated fillets at 1 kGy and higher doses Correlation was obtained between average amount of astaxanthin and average a* -value (redness) during different irradiation treatments in fresh and frozen light and dark muscle of Atlantic salmon for each storage period up to 6 days at 4 C. It was found that redness (a* value) of salmon light muscle was more related to astaxanthin content, while that of salmon dark muscle was related more to the lipid content and the degree of lipid oxidation. Irradiation treatments for fresh and frozen light and dark muscle of Atlantic salmon did not change lipid oxidation and the level of fatty acids

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158 compared to untreated samples. Sensory score of 2 kGy and higher doses was significantly lower in color, odor and overall acceptability compared to untreated samples, and the sensory score decreased with an increase in i rradiation level. The 1 kGy treated fresh and frozen samples was a successful treatment, with no significant eff ect on color, sensory as well as providing inactivation of microbial growth during 6 days storage at 4 C compared to untreated samples.

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159 APPENDIX COMPARISON OF MINOLTA AND MACHIN E VISION S YSTEM IN MEASURING COLOR OF IRRADIATED ATLANTIC SALMON Introduction Color of a meat or m uscle product plays an im portant role in the consumer perception of meat quality (Jeremiah and others 1972). St udies on seafood products have shown that consumers would associate color with the freshn ess of a product having better flavor and higher quality (Gormley, 1992; Anderson, 2000). Electronic instruments used for measuring color would define color in terms of a* value (red and green), b* value (yellow and blue) and L* value (lightness). These three elements along with the chroma and hue values could be used for describing the color of a food product. Minolta Chroma Meter, is a common hand-held colorimeter used for measuring the average color of a food sample area by providi ng a controlled illumination, which is either average daylight illuminant C (6774 K color temperature) or average da ylight illuminant D65 (6504 K color temperature). Minolta colorimeter is widely used for measuring the color of seafood products such as Atlantic salmon (Buttle and others 2001), rainbow trout (Choubert and others 1997), Artic charr (Olsen and Mortensen, 1997) and surimi products (Jaczynski and Park, 2003). Although Minolta provides simple and fast color measurements, it has some limitations. The food should have a uniform surface and color, and the sampling location on the food and the number of readings for obtaining an accurate average color are important (Oliveira and Balaban, 2006). Machine vision system started to be used in the early 90s in the food industry, and became popular in both scientific and industrial areas by providing reliable and reproducible data. There are many applications in the food area such as shrimp (Luzuriaga and others 1997), pork (Lu and others 2000), oysters (Diehl and others 1990) bananas (Yoruk and others 2004), sturgeon

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160 (Oliveira and Balaban, 2006), rainbow trout and mahi mahi (Yagiz and others 2007). There are many advantages of using Machine vision system such as its ability to determine L*a*b* values for each pixel of the samples image, to anal yze the entire surface of the food even with nonuniform shapes and colors, to calculate the aver age and standard deviati on of L*a*b* values, to identify and quantify the colors present in a sa mple, and to provide a permanent record when keeping the picture. During the course of our research, we have us ed both Minolta chromo meter as well as Machine vision system for analyzing the color of various seafood samples and processed muscle food products. However, we observed discrepancie s in values obtained using these two methods. Hence, we wanted to do a more detailed comparison between the color values obtained using Minolta and Machine vision system. The objective of this study was to measure the color of irradiated Atlantic salmon fillets using a hand-held Minolta colorimeter and a Machine vision system, and to compare their performance. Materials and Methods Irradiation Treatment Atlantic salmon ( Salmo salar ) was purchased from local seaf ood supplier (Save-onSeafood, St Petersburg, FL) within two days of harvest and the fish was transported to the laboratory in ice. The fish was filleted and skinless fillets were vacuum packaged using FoodSaver Vacloc vacuum bags (Jarden Co. Ry e, NY). All samples were placed on ice and transported to the electron beam irradiation facility (Florida Accelerator Services and Technology, Gainesville, Fl, U.S.A.). Samples were irradiated at 0, 1.0, 1.5, 2.0 and 3.0 kGy. Controls were kept on ice for th e duration of irradiation process. All fillets were removed from trays and placed back into the ice cooler after each treatment.

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161 Color Analysis The color of three Atlantic salmon fillets from each treatment was measured using a handheld Minolta Chroma Meter CR-200 (Minolta Camera Co., Osaka, Japan) and a Machine vision system. Minolta colorimeter was calibrated before each use with a standard white plate (D65 Y-94.4, x-0.3158 and y-0.3334). The L*, a*, b* values were measured under D65 illumination. Machine vision system, consisted of a light box (Luzuriaga and others 1997) and a digital Nikon D200 color camera (Nikon D200 Digital Ca mera, Nikon Corp., Japan) connected to a computer with a firewire connection. Images were captured using a digital Nikon D200 color camera located inside the light box. The Camera settings was used as 36 mm focal length, ISO 100 sensitivity, 1/3 s F/11 shutter speed -1.0 EV exposure compensation, and direct sunlight white balance. A software program developed wa s used to capture images, and to obtain color results based on L* (lightness), a* (redness), and b* (yellowness) values. Fish fillets were placed in the light box and the digital camera captured a picture of the fillets for each treatment. Machine vision system was calibrated by a standard red plate (L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). Average L*, a *, b* values of the whole fillet surface were calculated using a color analysis program (Luzuriaga and others 1997; Yoruk and others 2004; Yagiz and others 2007). Both Minolta and Machine vision system used the average daylight illuminant D65 mode with a color temperature of 6504K. Result and Discussion Muscle food s are generally subjected to ionizing radiations to kill pathogenic microorganisms and to improve food safety (Thayer, 1995). However, irradiation-induced chemical changes could lead to undesirable odor and color changes in m eat (Thayer and others 1993). As color of meat is an important parameter for consumer acceptability, proper

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162 measurement of color is an important tool in muscle food research. Table 1 represents the L*a*b* values of fillets at different irradiation doses obtaine d by Minolta and Machine vision system, and those of the standard red pl ate (L*=51.13, a*=50.00, b*=24.03) from Labsphere (North Sutton, NH). When the color of irradiated salmon fillets were measured using Minolta and Machine vision system, the L* value increa sed, and a* and b* value decreased with an increase in the irradiation dose. However, Ma chine vision system showed significantly (p<0.05) higher readings for L*a*b* values compared to Minolta (Table 1). The actual colors from the results of the two instrument systems and a representative picture of the salmon fillet for each treatment are shown in Table 2. Although Minolta and Machine vision system showed close or similar readings for the standard red plate, th e two instruments showed significantly (p<0.05) different readings for the color of irradiated At lantic salmon fillets. The color represented by the Minolta readings was purplish, while those measur ed using the Machine vi sion were much closer to the average real color of Atlantic salmon fillets. Our studies were similar to the results of Oliveira and Balaban (2006) who repo rted a significant color difference ( E) when comparing Minolta and Machine vision system during a storage study of sturge on fillets. They stated that the difference could be different average daylight illuminants for Minolta mode D65 with a color temperature of 6504K and their Machine vision system mode D50 with a color temperature 5000K. However, in this study, we used the same illuminant, D65 with a color temperature of 6504K for both instruments. The L*a*b* values of a standard red plate measured using Minolta were similar to the values measured using Machine vision system. He nce, we recommend usi ng caution in reporting color values reported by any measurement system, even when the referen ce tiles are correctly measured. The surface roughness and texture, the am ount of surface shine, the geometry of the

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163 measuring instrument, and other factors may affect the color readings. Checking the actual color represented by the L* a* b* values be fore reporting them is recommended.

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164 Table A-1. Average L*a*b* values from Mach ine Vision and Minolta system at different treatment doses of Atlantic salmon, a nd that of a standard red plate. Machine Vision Minolta Treatment Dose (kGy) Number of Replicates L* a* b* L* a* b* 1 64 28 32 47 14 12 2 59 35 35 43 13 10 0 3 62 31 33 45 14 12 1 59 30 34 46 12 11 2 63 27 35 47 13 13 1 3 63 26 33 48 13 11 1 64 22 31 48 11 10 2 62 26 33 48 11 11 1.5 3 65 21 32 50 9 10 1 66 19 30 52 11 13 2 65 18 30 49 8 8 2 3 62 19 29 46 6 7 1 64 15 28 46 6 9 2 68 13 26 49 5 8 3 3 66 13 27 48 5 7 1 51 46 25 50 55 24 2 51 46 25 50 55 24 Standard Red Plate 3 51 46 25 50 55 24

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165 Table A-2. Color representations of the Minolta and Machine vi sion reading results, and actual pictures of differently treated salmon fillets and standard red plate. Treatment dose (kGy) Minolta Machine Vision Picture (a) 0 (b) 1 (c ) 1.5 (d) 2 (e) 3 (f) Standard Red Plate

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175 Venugopal V, Doke SN, Thomas P. 1999. Radiation pr ocessing to improve the quality of fishery products. Critical Reviews in Food Sc ience and Nutriti on 39(5):391-440. Wetterskog D, Undeland I. 2004. Loss of redness (a*) as a tool to follow hemoglobin-mediated lipid oxidation in washed cod mince. J. Agric. Food Chem. 52(24):7214-7221. Woyewoda AD, Shaw SL, Ke PJ, Bums BG. 1986. Recommended laboratory methods for assessment of fish quality. Can. Tech. Rep. Fish. Aquat. Sci./Rapp. Tech. Can. Sci. Halieut. Aquat. 1448:28-31. Yagiz Y, Kristinsson HG, Balaban MO, Marshall MR. 2005. Effect of hi gh pressure treatment on Atlantic salmon ( Salmo salar ) muscle. IFT Annual Mee ting and Food Expo, Aquatic Food Products New Orleans, LA. p. 89A-17. Yagiz Y, Kristinsson HG, Balaban MO, Marshall MR. 2007. Effect of hi gh pressure treatment on the quality of rainbow trout ( Oncorhynchus mykiss ) and mahi mahi ( Coryphaena hippurus ). J. Food Sci. 72(9):C509-C515. Yoruk R, Yoruk S, Balaban MO, Marshall MR 2004. Machine vision analysis of antibrowning potency for oxalic acid: A comp arative investigation on banana and apple. J. Food Sci. 69(6):E281-E289. Yoshioka K, Yamamoto T. 1998. Changes of ultr astructure and the physical properties of carp muscle by high pressurizati on. Fish. Sci. 64(1):89-94. Young AJ, Lowe GM. 2001. Antioxidant and prooxi dant properties of carotenoids. Arch. Biochem. Biophys. 385(1):20-27.

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176 BIOGRAPHICAL SKETCH Yavuz Yagiz received his Bachelor of Science degree in Chem istry at Orta Dogu Teknik Universitesi in Ankara, Turkey. He went on to pursue his Ph.D. degree with a minor in Packaging Science under the guidance of Dr. Maur ice R. Marshall and Dr. Hordur G. Kristinsson in the Department of Food Scien ce and Human Nutrition at the Univ ersity of Florida. During his graduate career he excelled in his academics a nd was recognized by Department of Food Science and Human Nutrition with the awarding of Check ers scholarship. He received 3rd place of the IFT Aquatic Food Products Divisi on Paper Competition in 2006.