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Euthanasia of Tilapia using Carbon Monoxide for Color Fixation and Color Stabilization


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EUTHANASIA OF TILAPIA USING CARBON MONOXIDE FOR COLOR FIXATION AND COLOR STABILIZATION By DAVID MANTILLA TORRES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by David Mantilla Torres

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This document is dedicated to my loving parents and my sister.

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ACKNOWLEDGMENTS I sincerely want to express my deep gratitude towards my major advisor, Dr. Hordur G. Kristinsson, who with his advice, guidance and support helped me to complete this project. I would also like to thank my committee members, Dr. Murat Balaban, Dr. Steve Otwell and Dr. Frank Chapman, for all their suggestions and help in the completion of this research. It has been a privilege to work with such talented people. I would like to thank to Mr. Gene Evans. His generosity and help providing this research with the amount of live tilapia needed are greatly appreciated. I would like to thank my family for always believing and supporting me, making it possible to reach my goals. Finally, I would like to thank all my friends and my lab mates who became my family and supported me all these years at the University of Florida. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Tilapia and Aquaculture...............................................................................................1 Quality of Seafood........................................................................................................2 2 LITERATURE REVIEW.............................................................................................4 Tilapia Taxonomy.........................................................................................................4 Quality and Shelf Life of Seafood................................................................................5 Heme Proteins and Seafood Quality.............................................................................5 Water Holding Capacity and Muscle pH......................................................................7 Effects of Carbon Monoxide on Fish Muscle.............................................................10 Carbon Monoxide and Euthanasia..............................................................................12 Research Objectives....................................................................................................13 3 MATERIALS AND METHODS...............................................................................14 Euthanasia of Tilapia with Carbon Monoxide (CO)...................................................14 100% CO Post-Mortem Gas Fillets Treatment of Tilapia..........................................16 Color Analysis............................................................................................................16 Quantification of CO in Fish Muscle..........................................................................16 Heme Protein Extraction and Spectroscopic Analysis...............................................17 Muscle pH...................................................................................................................18 Drip Loss....................................................................................................................18 Statistical Analysis......................................................................................................18 v

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4 RESULTS AND DISCUSSION.................................................................................21 Study 1: Frozen Fillets................................................................................................21 Effect of Carbon Monoxide on Color..................................................................21 Heme Spectroscopic Analysis.............................................................................26 Carbon Monoxide Quantification........................................................................30 Muscle pH and Drip Loss....................................................................................34 Study 2: Whole Frozen Tilapia...................................................................................37 Effect of Carbon Monoxide Euthanasia on Color...............................................37 Heme Spectroscopic Analysis.............................................................................41 Carbon Monoxide Quantification........................................................................44 5 SUMMARY AND CONCLUSIONS.........................................................................48 APPENDIX A FIRST STUDY DATA...............................................................................................51 B CO CONCENTRATION IN THE WATER...............................................................53 C PICTURES OF THE TILAPIA FILLETS.................................................................55 LIST OF REFERENCES...................................................................................................57 BIOGRAPHICAL SKETCH.............................................................................................62 vi

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LIST OF TABLES Table page B-1 Concentration of CO in the water used to euthanize tilapias...................................53 B-2 Time and amount of CO needed to euthanize tilapia in the tank.............................54 vii

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LIST OF FIGURES Figure page 3-1 Recirculating water-CO system for the euthanasia of tilapia...................................15 3-2 Experimental design for the first study where tilapia CO treated or untreated and then filleted, frozen (30 days), defrosted and stored at 4 o C for 18 days..................19 3-3 Experimental design for the second study where whole tilapia was either euthanized or left untreated, and then frozen for up to 4 months.............................20 4-1 Red muscle side of 100% CO euthanized, control and 100% CO gassed tilapia fillets.........................................................................................................................21 4-2 White muscle side of 100% CO euthanized, control and 100% CO gassed tilapia fillets..............................................................................................................22 4-3 Effect of CO treatments and no treatment (control) on the a*-values (redness) of tilapia fillet red muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days.........................................................................................................24 4-4 Effect of CO treatments and no treatment (control) on the L*-values (ligthness) of tilapia fillet red muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days.....................................................................................................25 4-5 Effect of CO treatments and no treatment (control) on the a*-values (redness) of tilapia fillet white muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days.....................................................................................................26 4-6 Representative spectra for met-hemoglobin (408 nm), oxy-hemoglobin (414 nm) and carboxy-hemoglobin (418 nm)..........................................................................27 4-7 Maximum heme peak values for red muscle extracts from euthanized and 100% CO gassed tilapia fillets and untreated tilapia stored at 4 o C for 18 days.................27 4-8 Maximum heme peak values for white muscle extracts from euthanized tilapia, 100% CO gassed tilapia fillets and untreated tilapia stored at 4 o C for 18 days.......30 4-9 Concentration of CO (ppb) in tilapia red muscle after 30 minutes exposure to 100% CO, euthanasia with 100% CO or no treatment.............................................31 viii

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4-10 Concentration of CO (ppb) in tilapia white muscle after 30 minutes exposure to 100% CO, euthanasia with 100% CO or no treatment.............................................34 4-11 Change in pH of fillets gassed with 100% CO, fillets from fish euthanized with 100% CO and untreated tilapia fillets stored at 4 o C for 18 days..............................35 4-12 Thaw loss of gassed fillets (100% CO for 30 min), fillets from euthanized fish (100% CO) and untreated fillets after 1 month of freezing. Results obtained are based on the change on weight of the fillets............................................................36 4-13 Change in drip loss of gassed fillets (100% CO for 30 min), fillets from euthanized fish (100% CO) and untreated fillets during 18 days of storage at 4 o C after thawing.............................................................................................................36 4-14 Effects of euthanasia with 100% CO and no treatment on a*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months...................37 4-15 Effect of euthanasia with 100% CO and no treatment on a*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months...................39 4-16 Effect of euthanasia with 100% CO and no treatment on b*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months...................40 4-17 Effect of euthanasia with 100% CO and no treatment on L*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months...................40 4-18 Effect of euthanasia with 100% CO and no treatment on b*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months...................41 4-19 Effect of euthanasia with 100% CO and no treatment on L*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months...................41 4-20 Maximum heme peak values for red muscle of euthanized (100% CO) and untreated fresh and frozen whole tilapia..................................................................42 4-21 Maximum heme peak values for white muscle of euthanized (100% CO) and untreated fresh and frozen whole tilapia..................................................................44 4-22 Concentration of CO (ppb) in the red muscle of fresh and frozen untreated and euthanized (100% CO) tilapia..................................................................................45 4-23 Concentration of CO (ppb) in the white muscle of fresh and frozen untreated and euthanized (100% CO) tilapia..................................................................................46 A-1 Effect of CO treatments on the b*-values of the red muscle of tilapia....................51 A-2 Effect of CO treatments on the b*-values of the white muscle of tilapia................51 A-3 Effect of CO treatments on the L*-values of the white muscle of tilapia................52 ix

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B-1 Change of CO concentration in the water used for the euthanasia process.............54 C-1 Picture of the red muscle side of a tilapia fillet taken by the CMVS...................55 C-2 Picture of the white muscle side of a tilapia fillet taken by the CMVS................56 x

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EUTHANASIA OF TILAPIA USING CARBON MONOXIDE FOR COLOR FIXATION AND COLOR STABILIZATION By David Mantilla Torres December 2005 Chair: Hordur G. Kristinsson Major Department: Food Science and Human Nutrition Tilapia is predominately a white muscle fish that has a small amount of dark lateral muscle. This muscle is an important indicator of fillet freshness, as it changes from a red color to brown on storage. In order to extend the color of the red muscle many tilapia processors are treating their fish with gasses containing carbon monoxide (CO). The most common treatment method is a post-mortem CO gas treatment of fillets. Currently the application of CO into fish muscle via euthanasia is being performed by a few tilapia processors. There is a lack of information on how the euthanasia of the fish with CO will affect fish quality compared to more conventional applications. The objective of this study was to investigate the color retention and quality of fillets from euthanized tilapia, compared to a 100% CO post-mortem gas treatment. Live tilapia was placed in a sealed water tank and was euthanized with 100% CO flushed into a circulatory water system. Two studies were performed. In the first study, tilapia was immediately filleted after euthanasia, vacuum packed and frozen (-20 o C) for xi

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one month, then thawed and kept exposed to air for 18 days at 4 o C. Fillets from non-CO treated fish were subjected to a 30 min treatment with 100% CO. Untreated fillets were used as control. In the second study, CO-euthanized tilapia was gutted, vacuum packed and frozen whole for up to 4 months. For this study, a set of normally slaughtered tilapia was used as control. Before each set of analysis (at 0, 2 and 4 months) the fish was thawed for 24 hours at 4 o C. The change in muscle color was analyzed with a digital Color Machine Vision (CMVS) and L*, a* and b* values were recorded. The uptake and stability of CO in the fish muscle and its binding to heme proteins were analyzed with gas chromatography (FID) and spectrophotometry, respectively. The effect of the different treatments on muscle pH and muscle drip loss was also analyzed. Euthanasia with CO and direct CO treatments on fillets led to a significant (p<0.05) increase in the red color (a*-values) of the muscle, especially the dark muscle. This distinctive cherry red color was maintained for a long period of time for both treatments while a brown color developed for the controls. The color characteristics of the fillets from euthanized fish were more natural than those of the 100% CO treated fillets. The UV-Vis spectra and the concentration of CO in the muscle confirmed the uptake of CO by heme proteins and also demonstrated an increase in heme protein stability. CO uptake was significantly higher in dark muscle compared to white muscle. No significant differences were found in pH or drip loss among the treatments. These results suggest that both CO treatments have a positive effect on color and heme stability, while euthanasia appears to give a more natural looking product. In addition, this new method of processing (i.e., euthanasia) has several advantages such as shorter processing time and less product handling. xii

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CHAPTER 1 INTRODUCTION The variety and the high nutritional content of aquatic foods have created great interest and a high demand for these products. Data provided by the Food and Agriculture Organization (FAO) in 2002[1] shows that the total fish production has reached its highest level ever of 94.8 millions tons in 2000. The high demand for these products is also reflected in an increase of people directly engaged in fisheries and the aquaculture industry. These industries employed an estimated of 35 million people this decade, 7 million more compared to last decade[1]. The high demand for seafood however has depleted many fish stock due to overfishing. World population has been growing faster than the total food fish supply, leading to a decrease in the fish supply per capita[1]. About one billion people rely on fish as their main source of protein [2].The global crisis in capture fisheries and the increasing need for seafood has stimulated the rapid expansion of aquaculture. Aquaculture offers a predictable and consistent supply of high quality seafood [3]. According to the FAO, aquaculture production represented 3.9 % of total fish supply in 1970 and this percentage increased to 27.3% in 2000 [1]. Tilapia and Aquaculture Tilapia is one of many species that is been aquacultured with great success. Tilapia has a broad tolerance to harsh environmental conditions. They are more tolerant to high salinity, high water temperature, low dissolved oxygen and high ammonia concentrations than most other farmed freshwater fish [4, 5]. Illustrations from Egyptians tombs suggest that tilapia was one of the first fish species cultured, more than 3000 years ago [5]. 1

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2 Tilapia is also known as Saint Peters fish and it is believed that tilapia was fed to the multitudes by Jesus Christ [5]. From 1950 to mid 1970s tilapia species moved from their native waters in sub-Saharan Africa to Asia and the rest of the world [2, 6]. They were introduced to different parts of the world for various reasons; for instance, they were used as bait for tuna which is how they were introduced to Hawaii [7]. Apparently the introduction of tilapia in the Caribbean, Central and South America was made in order to reduce mosquitoes through aquatic vegetation control [7]. In 2000 farmed tilapia production surpassed 800 thousand metric tons, second only to carp [3, 5]. Tilapia has a high acceptance in the United States and is one of the fastest growing seafood imports into the U.S. along with salmon. According to Knapp [3] in 2002 close to 70 thousand metric tons of fresh and frozen tilapia were imported to the U. S. Quality of Seafood Seafood is a highly perishable commodity since it is very susceptible to microbial and chemical spoilage which results in economic losses [8]. Its quality declines soon after harvest and continues once the fish has been processed. The value of the product is highly influenced by its appearance, in particular its color. Tilapia is predominately a white muscle fish that has a small amount of dark lateral muscle. This muscle can however be an important indicator of fillet freshness, as it changes from a red color to brown during storage. Maintaining the color of the dark muscle during processing, transport, storage and display is essential and has an influence on the consumer perception of the product. In order to extend the color of the red muscle and reportedly its shelf life, many tilapia processors are treating their fish with carbon monoxide (CO) and filtered wood smokes (FS) containing CO [9]. Several different treatment methods exist, the most common

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3 being exposure of the fillets briefly (<30 min) to CO gas immediately after harvest and processing while the muscle is still respiring (personal communication, B. Olson, Clearsmoke Technologies). Euthanasia of fish using CO dissolved in water has been proposed by Kowalski [10] and is currently performed by some tilapia processors (personal communication, B. Olson, Clearsmoke Technologies). This new method incorporates carbon monoxide to the edible muscle of the fish through the respiratory and circulatory system of the animal. Fillets from fish euthanized with 100% CO have a distinctive and stable cherry red color characteristic of a CO exposure/treatment.

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CHAPTER 2 LITERATURE REVIEW Tilapia Taxonomy Tilapia is a generic term used to designate a group of commercially important food fish that belongs to the Cichlidae family [4]. Tilapias have been classified into three genera based on the type of care the parents provided to their young [2, 4]. The genera Oreochromis and Sarotherodon are mouthbrooders. The eggs are fertilized in the nest but parents immediately pick them up in their mouths and protect them and incubate the young for several days after hatching [2, 4, 5]. A difference between these two genuses is that in the Oreochromis genera only the female parents practice the mouthbrooding. This genus of female mouthbrooding is the most important in aquaculture and it includes the Nile tilapia (O. niloticus), Mozambique tilapia (O. mossambicus) and blue tilapia (O. aureus) [2]. The third genus is called Tilapia. These species are nest builders. Eggs are fertilized, incubated and protected by a brood parent in a pond bottom built-in nest [4, 5]. In the United States, tilapia is grown for commercial purposes manly in Arizona, California and Florida. In 2000 U.S. production reached its peak of 20 million pounds, with a value of 30 million dollars. The production decreased to 17.6 million pounds in 2001 but the total value remained the same [11]. However, domestic production of tilapia is minimal compared to U.S. imports. In 2004 U. S. tilapia imports reached 249 million pounds 25% more from 2003 and 68% higher than in 2002 [12]. 4

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5 Quality and Shelf Life of Seafood Immediately after slaughter, the quality of seafood begins to decline. The initial quality and the type of processing that the fresh product undergoes has a great influence on the shelf life, rate of spoilage and quality of the final product. Some of the factors that influence the spoilage rate of fish are muscle pH, temperature, microbial load, microbial type, amount and type of heme proteins, fat content and fatty acid profile. Refrigeration, frozen storage and modified atmosphere packaging are some of the most common and effective methods to extend the shelf life of seafood. However, these methods normally do not prevent color changes or extend fresh color. In addition, the muscle texture might be affected by some of these methods. Heme Proteins and Seafood Quality Myoglobin and hemoglobin are heme proteins whose main function is the retention and transport of oxygen for enzymatic reactions [13]. Myoglobin is a globular protein consisting of a single polypeptide chain. It is found mainly in muscle tissue where it serves as an intracellular storage site for oxygen [13]. Hemoglobin consists of four myoglobins like subunits linked together as a tetramer. It is found in the red blood cells and forms reversible complexes with oxygen in the lung (or gills in the case of fish), where it transports the bound oxygen through the body to be used in aerobic metabolism pathways [13]. Heme proteins consist of a globin part and a heme part. The heme portion of the molecule is responsible for the color of dark muscle. At the center of the heme is an iron (Fe) atom which possesses six coordination sites. Four of them are occupied by nitrogen atoms. The fifth coordination site is bound to nitrogen from a histidine, leaving the sixth site available to complex with electronegative atoms donated by various ligands [13].

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6 Color depends on the oxidation states of the iron atom (Fe 2+ Fe 3+ and Fe 4+ ) in the protein heme group, and the type of ligands (O 2 CO, NO etc. ) bound to the iron atom. Hemoglobin is the most predominant heme protein found in fish white muscle [14, 15]. The presence of blood, thus hemoglobin, in the muscle leads to changes in color and significant lipid oxidation problems [15, 16]. Soon after death, the heme iron is in the ferrous (Fe 2+ ) valence state [16]. On the surface of fresh muscle oxygen is bound to the ferrous iron yielding oxyhemoglobin/myoglobin which gives the muscle a bright red color. In the interior of the muscle the iron binding site is vacant thus yielding deoxyhemoglobin/myoglobin which gives the muscle a dark purple color. Heme proteins are very sensitive to autoxidation, which is enhanced with temperature increase and pH decrease [17]. Over time, the hemoglobin will oxidize to form methemoglobin (Fe 3+ ). This occurs when oxygen is released from oxyhemoglobin to form ferric (Fe 3+ ) heme iron and the superoxide anion (O 2) [16]. The formation of methemoglobin gives rise to an undesirable brown color. To maintain the red color, the formation of methemoglobin needs to be prevented. This can be achieved by keeping fish at very low temperatures (-50 to -70C) which is highly impractical and expensive for most species. A more practical an inexpensive means to achieve color stability is by exposing the muscle to CO or filtered smoke which contains CO. The CO molecule will combine with the heme group in hemoglobin and myoglobin to form carboxyhemoglobin/myoglobin and give the muscle a bright cherry red color. The CO molecule binds very strongly to the heme group in hemoglobin and myoglobin, over 200 times stronger than O 2 [18] and will thus displace any oxygen present in the heme. This binding leads to a conformational change

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7 in hemoglobin and myoglobin which makes it very resistant to autoxidation and discoloration [19, 20]. Autoxidation of the heme protein to the met form is also a critical step in lipid oxidation. Met-Hb/Mb reacts with peroxides and stimulates formation of chemical compounds capable of initiating and propagating lipid oxidation [14, 21]. Lipid oxidation is a major cause of quality deterioration of seafoods. It often contributes to the formation of off odors and flavors, and the deterioration of color and texture. Toxic compounds can also arise from lipid oxidation [16]. Fish are particularly sensitive and affected by lipid oxidation due to their highly polyunsaturated fatty acid content [14]. Transition metals such as iron and copper can also catalyze lipid oxidation in fish muscle [14, 22]. Iron is the principal transition metal in seafood and a large portion of iron in fish muscle is found in heme proteins. The amount of iron varies greatly among species. White-muscled fish have lower concentrations of iron than dark muscled fish [14]. In tilapia, most of the iron will come from hemoglobin in the white muscle and myoglobin in the dark muscle. Since CO is expected to retard autoxidation of hemoglobin and myoglobin to the met form it is possible that this treatment may retard lipid oxidation, and thus extend the shelf life of tilapia fillets. Water Holding Capacity and Muscle pH Water holding capacity (WHC) of foods can be defined as the ability to hold their own and added water during the application of force, pressing, centrifugation, or heating [23]. Water holding has a great influence on the quality of the final product primarily because of the reduced weight loss during cutting and storage and its ability to retain water during processing [24]. Many factors influence WHC. For example WHC is exponentially related to the protein content of the muscle, as the protein content

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8 increases, WHC increases. The addition of salts also influences the water binding by proteins because of their effects on electrostatic interactions. A change in pH affects as well the conformation of proteins resulting in exposure or burial of the water binding sites [23], as well as increased osmotic pressure within the muscle when is sufficient electrostatic repulsion between proteins [20]. WHC reaches its minimum near the isoelectric points of the major muscle proteins especially myosin (pI~5.4) [25] and rises on either side of this point. After the death of the animal, the anaerobic glycolytic system becomes predominant and ATP is gradually depleted and lactic acid is accumulated leading to a decrease in pH. When the pH is low enough certain critical enzymes are inhibited and glycolysis ceases [13]. The decrease of pH comes from the hydrolysis of ATP [13]. A fast decrease in postmortem pH will cause the denaturation of muscle proteins; the meat produced will be pale soft and exudative (PSE), a condition that is especially troublesome in pork [13, 26]. This phenomenon also occurs in fish; low pH weakens the collagen fibers, they break and gaping takes place. Meat quality and WHC is also influenced by behavioral and physiological status of the animals before slaughter. Stress will exacerbate the drop of pH due to the rising adrenaline levels [26]. An animal is considered in a state of stress if it is required to make abnormal or extreme adjustments in its physiology or behavior in order to cope with adverse aspects of its environment and management [27]. When an animal is under stress, oxygen is not available in sufficient amounts, and the anaerobic pathway becomes predominant and glycogen is depleted. This depletion of glycogen results in an onset of rigor much sooner and in a faster decrease of pH. For example, the time from death to the onset of rigor in unstressed blue tilapia is 6 hours; on the other hand, the time from death

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9 to the onset of rigor is reduced to only 1 hour in stressed tilapias [28]. In one study, CO 2 and live chilling were used on salmon in order to minimize stress and prolong the onset of rigor [29]. The advantage of having a longer onset of rigor is that processing of the raw material can start immediately after slaughter; otherwise the process cannot start until rigor has been resolved. A common practice in Atlantic salmon is to process once rigor mortis has resolved, which takes 3 to 5 days on ice storage [30]. However, it has been shown that there is no major difference between processing pre-rigor salmon and post-rigor salmon [29, 30]. The extension in the onset of rigor is of particular importance to tilapia producers since tilapia is processed pre-rigor. The tilapia industry uses many ways to slaughter the animal. The most common is to transfer tilapia from the pond to a small tank where they are taken one by one and their branchial artery is severed. They are then transferred back to the small tank until they bleed to death. It has been suggested that bleeding the fish has no effect on the quality of the final product since most of the blood in fish is located on the venous side of the cardiovascular system meaning that by gutting most of the blood will be removed along with intestines [31]. The transferring of the tilapia from the pond to the small receiving tank and then the bleeding may thus be an unnecessary and overly stressing slaughtering method. The euthanasia of tilapia can reduce this stress. As mentioned above, salmon industry uses CO 2 as anesthesia in order to avoid stress and to comply with the concept of humane slaughter [29]. Carbon monoxide dissolved in the water where the fish is can also act as an anesthesia for fish. In addition of the benefit of having lower stressed fish, CO will enhance the color of the filleted fish and it can help preserve better the final product.

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10 Effects of Carbon Monoxide on Fish Muscle The bright red color of fish is one of the main attributes that indicates its freshness and quality. The red color arises primarily from the oxygenated and reduced forms of heme proteins which have been discussed in previous sections. The oxidation of these proteins yields a highly undesired brown color. Appearance plays a very important role in consumer buying decisions. The main objective of the use of carbon monoxide is to maintain the attractiveness of the red color characteristic of fresh seafood. As written previously, carbon monoxide binds to heme proteins and forms a very stable complex. Kristinsson and coworkers [20] reported that carboxyhemoglobin was very stable to oxidation even at extreme pH values and temperatures. These results suggest that muscle treated with carbon monoxide may retain its red color even under abusive conditions. The same authors also demonstrated that the Hb-CO complex had decreased pro-oxidative activity in a model system and may thus extend product shelf life with respect to rancidity. Due to its high price and its high content of red muscle, tuna steaks have been the focus of study with respect to the use of carbon monoxide for color preservation. Different studies using different concentration of carbon monoxide and different exposure times have revealed that there is a significant increase in red color as well as color stability when CO is used. For example, tuna steaks treated with 99.5% CO gas for 4 hr showed a significant increase in a*-value (redness) compared to untreated tuna [32]. There were no significant differences between L*(lightness) and b* (yellowness) values between the gas treated tuna and control [32]. Balaban and coworkers [33] reported that exposure to 4% CO increased a*-value and preserved color stability for up to 12 days in refrigerated storage. Danyali [34] compared CO with filtered smoke (FS) treatments and

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11 found little difference between the two with respect to color, heme protein oxidation, lipid oxidation, water holding, and texture. It was however reported that 100% CO led to a reduced microbial growth, and thus could possibly extend shelf life [34]. Studies also show that a higher level of CO leads to more color increase and better color stability [34, 35]. Few studies have been published on applying CO or FS treatment on tilapia and investigating the effect on quality. Ishiwata and coworkers [36] conducted a survey of the concentration of CO in flesh from a variety of fish sold in a local market and also exposed tilapia to CO for 60 minutes at room temperature and measured CO concentration. They reported that the blood colored parts of tilapia exposed to CO were bright red contrasting with the dark brown color exhibited by the untreated tilapia. Kristinsson and coworkers [37] reported that tilapia treated with 100% CO did develop less lipid oxidation products compared to untreated tilapia. Kristinsson and coworkers [19] later found that isolated tilapia carboxy-hemoglobin had dramatically increased stability to autoxidation (i.e., browning) under different environmental conditions compared to oxyhemoglobin, and also had less pro-oxidative activity in a model linoleic acid emulsion system. Leydon and coworkers [38] recently reported that commercially obtained previously frozen tilapia fillets treated with filtered wood smoke had increased color stability, less lipid oxidation and microbial growth than fresh commercially obtained tilapia fillets. The filtered smoke treated fillets were however rejected by a trained sensory panel of 3 people [38]. This study did however use commercially obtained samples, one previously frozen and one fresh, from two different sources, which makes it difficult to interpret the data.

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12 Carbon Monoxide and Euthanasia All studies reported on CO and FS application of seafood employ gas treatments post-mortem on fish steaks or fillets. The practice of euthanizing fish with CO to incorporate CO into muscle is being performed by the industry on tilapia (personal communication, B. Olson, Clearsmoke Technologies). Kowalski [10] issued a patent application in which he described the incorporation of tasteless smoke or carbon monoxide by means of euthanasia; however no supporting data was found. No other research studies have been performed to the best of the authors knowledge. Carbon monoxide is a colorless, odorless and tasteless gas that has about the same density as air, but sustained inhalation of CO has caused many fatalities due to its competitive binding to hemoglobin [39]. At levels of 5% CO-Hb fetuses can be affected and individuals experience many effects. Levels above 10% are life threatening for heart and lung patients, whereas above 30% CO-Hb healthy individuals are at risk and death can rapidly occur at levels above 50% [40]. Carbon monoxide poisoning will severely alter the oxygen transport characteristics of the circulatory system since about 90% of the oxygen consumed is carried to the tissues by hemoglobin [41]. Carbon monoxide attaches to hemoglobin similarly to oxygen but with a binding constant that is 210270 fold stronger [40]. Consequently, CO displaces oxygen from hemoglobin. Due to its great binding affinity, even at low levels of exposure to carbon monoxide, carboxy-hemoglobin will accumulate. Carbon monoxide reduces both the oxygen-carrying capacity of circulating blood by direct displacement and the release of the hemoglobin-bound oxygen to the tissues by shifting the oxygen-hemoglobin dissociation curve [40]. One limitation of killing fish with CO is that it has a relatively low solubility in water. It was reported by Daniels and Lide [42, 43] that CO has a solubility of 1.774x10 -5

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13 mole fraction solubility in water at 25 o C and 101.325 kPa. This solubility however is very similar to the solubility of oxygen in water (2.293x10-5 mole fraction solubility at 101.325 kPa)[43] This suggests that O 2 and CO dissolved volumes are approximately equal. The volume of O 2 or CO dissolved in water is dependent on the partial pressure of the gas and the temperature. The solubility increases as the temperature decreases. Research Objectives The overall objective of this study was to investigate the effect of euthanizing tilapia by dissolving carbon monoxide directly into the water and comparing to 100% CO post-mortem gas treatment of fillets and no gas treatment. The effect on color, color stability, CO uptake and stability, muscle pH and water holding capacity were investigated for both products stored fresh, as well as frozen and defrosted products.

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CHAPTER 3 MATERIALS AND METHODS Euthanasia of Tilapia with Carbon Monoxide (CO) Live tilapia were obtained from Evans Farm in Pierson, FL. The facility produces hybrids from a genetic cross of predominantly aurea tilapia, Oreochromis niloticus and Oreochromis mossambicus. All the production is destined for local consumption and local restaurants. The tilapia were transferred live to the laboratory and kept in holding tanks prior to euthanasia. A tank was constructed from transparent Plexiglas (36x 16x 12) where tilapias were euthanized with CO saturated water (Figure 3-1). 100% CO was flushed into a circulatory system which allows the water to saturate with the gas (Figure B-1). CO was introduced to the animal muscle tissue by its respiratory and circulatory systems. All the experiments were performed at ambient temperature (21 o C). Tilapias were maintained in the euthanizing tank as much time as needed until they all were confirmed dead by visual inspection. On average, 31 minutes were needed for the completion of the euthanasia process (Table B-2). During every trial, thirteen tilapias were euthanized. To flush the remaining CO out of the tank, air was flushed in and the CO converted to CO 2 by passing it through a Hopcalite catalyst tube (Figure 3-1). After euthanasia, two studies were performed. In the first study these tilapia was immediately filleted, vacuum packed in high density polyethylene (HDPE) bags and frozen at -20 o C. After 1 month fillets were thawed at 4 o C for 24 hours and stored aerobically at 4 o C for 18 days. In order to compare the effectiveness of euthanasia with 14

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15 100% CO flushed directly into the water, 100% CO post-mortem gas treated and control fillets were stored under the same conditions for the same amount of time (Figure 3-2). In the second study, tilapia were only gutted, placed in HDPE bags, vacuum packed and stored at -20 o C for up to 4 months. A set of normally slaughtered (no CO) fish was also gutted, vacuum packed and stored at -20 o C. The latter was used as control. Three sampling points were chosen; 0, 2 and 4 months. The whole fish was thawed for 24 hours at 4 o C, then filleted and analyzed (Figure 3-3) Figure 3-1. Recirculating water-CO system for the euthanasia of tilapia. Hopcalite catalyst ( CO CO 2 ) Exhaust Venturi Simple bypass with valve to help regulate pressure in the venturi. Pump Water Tank 100%CO Mixer

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16 100% CO Post-Mortem Gas Fillets Treatment of Tilapia Live tilapia were killed with ice and by bleeding. The 100% CO post-mortem gas treatment can be applied only to the first study since the later study used whole fish. After death, the fish was filleted immediately before rigor and split in two groups: one subjected to CO gas treatment and the other (control) subjected to no treatment. Fillets were placed in a gas tight stainless steel drum on thinly netted stainless steel shelves and 100% CO applied for 30 min at 4 o C. After gas treatment the CO was converted to CO 2 by passing it through a Hopcalite catalyst tube (same one as in Figure 3-1). Untreated, CO-treated and euthanized fillets were then placed in HPDE bags, vacuum packed and frozen for one month. Fillets were then defrosted at 4 o C and kept at 4 o C for 18 days and analyzed every 3 days. Color Analysis A digital Color Machine Vision System (CMVS) was used following the procedures outlined by Balaban and coworkers [33] for detailed color analysis of RGB and L*-(lightness), a*-(redness), and b*-(yellowness) values along with hue values and identifying important color blocks for each treatment. The L*, a* and b*-values were reported. The color analysis was done separately for the white and the dark lateral muscle. The front side of the fillets contains the dark muscle which was used for the analysis of the red muscle. The reverse side which contains practically no red muscle was used for the analysis of the white muscle. Quantification of CO in Fish Muscle The method from Miyazaki and coworkers [44] was used to determine the concentration of CO in white and dark muscle separately. Briefly, 6 g of muscle were minced and introduced into a 60 ml head space bottle. 3 drops of 1-octanol (antifoaming

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17 agent) and 12 ml of 10% sulfuric acid were added. The sulfuric acid denatures heme proteins which causes them to release CO. The mixture was shaken for 10 sec, and then incubated for 5 minutes at 40 o C. After incubation, the tubes were shaken at room temperature for 15 minutes and 100 l of the head space gas was injected into an Agilent gas chromatography system equipped with a stainless steel Poropak Q column (3.17 mm i.d. x 1.82 m; 80-100 mesh) a methanizer (to convert CO to CH 4 ) and a FID detector. Helium was used as the carrier gas with a flow rate of 29.7 mL/min. The injection port, column, methanizer and detector temperatures were maintained at 100 o C, 35 o C, 320 o C and 250 o C respectively. The reducing gas was hydrogen with a flow rate of 40.0 L/min. The retention time and area of the CH 4 peak were compared to those obtained with a calibration CO gas. CO levels were then calculated based on a standard curve constructed by injecting different known levels of 100% CO. For the first study, three fillets were retrieved from the cold room (4 o C) and analyzed on different days after thawing (day 0, 2, 4, 6, 10, 14, 18). These days were chosen due to the rapid decrease of CO concentration in the muscle during the first days of exposure to normal atmospheric conditions [36]. Heme Protein Extraction and Spectroscopic Analysis Spectroscopic analysis can reveal how CO is taken up into the muscle and how stable it is bound to heme proteins on storage. The heme extraction method of Huo and Kristinsson [45] was used. A 10 g sample of tilapia white and red muscle (separately) were mixed with 100 ml of 20 mM Na 2 HPO 4 buffer (pH 8), followed by homogenization with a Ultra-Turrax T19 homogenizer at lowest speed. The sample was filtered through Whatman #1 filter paper at 4C followed by centrifugation at 3000x g. All these steps were done in the cold room to avoid protein denaturation and loss of CO from the heme.

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18 The UV-visible absorbance spectra of the collected supernatant were then read from 350-700 nm. The max wavelength of the heme peak of CO-Hb/Mb is 419 nm, 414 nm for oxyHb/Mb and 408 and below for metHb/Mb. Muscle pH The stress and its influence on the animal were followed by measuring the pH of the muscle. A sample of 5 g red and white muscle was mixed with 45 mL of deionized water at 4 o C and the mixture homogenized with an Ultra-Turrax T19 homogenizer, on ice. The pH was then measured using a Ross Sureflow biological epoxy probe (Thermo Orion, Beverly, MA) attached to a pH meter (Denver Instruments, Fort Collins, CO). The pH meter was calibrated using pH 2, 4, 7, and 10 buffers at 4 o C. Drip Loss Drip loss analysis was performed on fillets from the first study only. Six fillets from each experimental group (euthanized, gassed and control) were analyzed for drip loss for 18 days. Measurements were acquired every third day for 18 days (day 0, 3, 6, 9, 12, 15 and 18). The fillets were kept in open bags during the 18 days of measurements to allow for air to access the fillets. Each fillet was very delicately blotted for any loose liquid on its surface before its weight was recorded. The bag was meticulously cleaned and dried before the fillet was replaced in the bag. Thaw loss and drip loss were calculated based on the weight difference between the different storage days. Statistical Analysis Each analysis was conducted in a minimum of triplicate samples. Analysis of variance (ANOVA) and t-test were used to determine significant differences between treatments and among treatments. The Statistical Analysis Software (SAS) and Microsoft Excel were used for the treatment of the data.

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19 Live tilapia Treatment 1: Euthanized tilapia Euthanized tilapia in water saturated with CO Killed with ice water and bleeding Filleted pre-rigor Treatment 3: Control No gas treatment Treatment 2: Gassed fillets Gassed fillets with 100% CO for 30 min Filleted pre-rigor Vacuum Pack and Freeze fillets for 30 da y s @ -20 o C 1. Color analysis 1.1. Machine vision system (calibrated using Minolta Colorimeter) 2. Quantification of CO in muscle 2.1. Red and white muscle 3. Heme protein extraction and spectroscopic analysis 3.1. Red and white muscle 4. Drip loss 5. Muscle p H Fillets thawed @ 4 o C maintained for 18 days in high air permeability bags. *Analysis every 3 days Figure 3-2. Experimental design for the first study where tilapia CO treated or untreated and then filleted, frozen (30 days), defrosted and stored at 4 o C for 18 days.

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20 Treatment 1: Euthanized tilapia Euthanized tilapia in water saturated with CO Killed with ice water and b leedin g Freeze Whole Fish (g utted ) ( vacuum p acked ) 1. Color analysis 1.1. Machine Vision system (calibrated using Minolta Colorimeter) 2. Quantification of CO in muscle 2.1. Red and white muscle 3. Heme protein extraction and spectroscopic analysis 3.1. Red and white muscle 4. Muscle pH Control Freeze Whole Fish (gutted) (vacuum packed) Analysis at time 0, 2 and 4 months. 3 sampling pts. Live tilapia Figure 3-3. Experimental design for the second study where whole tilapia was either euthanized or left untreated, and then frozen for up to 4 months.

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CHAPTER 4 RESULTS AND DISCUSSION Study 1: Frozen Fillets Effect of Carbon Monoxide on Color The main reason behind the introduction of carbon monoxide in fish processing is to preserve and enhance the red color of the muscle. A Color Machine Vision System (CMVS) was used to analyze the color and color change during the study. The CMVS was chosen because it analyzes every pixel of the sample compared to other color methods that read only a small part of the sample. Other methods such as the Minolta color meter may be good for fillets that are uniform in color. However, tilapia has a red lateral muscle that gives the fillets an uneven color. Euthanized 100% COControlGassed 100% CO Euthanized 100% COControlGassed 100% CO Figure 4-1. Red muscle side of 100% CO euthanized, control and 100% CO gassed tilapia fillets. 21

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22 Euthanized 100%COControlGassed 100% CO Euthanized 100%COControlGassed 100% CO Figure 4-2. White muscle side of 100% CO euthanized, control and 100% CO gassed tilapia fillets Fillets from fish euthanized with 100% CO have a distinctive and stable cherry red color characteristic of a CO exposure/treatment. Figures 4-1 and 4-2 show sample fillets of each treatment for the red muscle side and the white muscle side of a tilapia fillet. Red muscle was defined as the side of the fillets that includes the red muscle (Figure 4-1). The other side was considered as white muscle (Figure 4-2). The degree of redness (a*-values) is the most important indicator of quality and freshness in species rich in red muscle such as tilapia, Spanish mackerel, mahi-mahi, tuna and swordfish [33, 34, 46]. The effect of the two treatments was very noticeable compared to the control fillets (Figures 4-1 and 4-3). Figure 4-3 shows the increase of a*-values in the red muscle of the euthanized and the post-mortem gas treated fillets and the control. The 100% CO post-mortem gassing method of fillets significantly increased (p<0.05) a*-values from 18.36 to 23.63. The a*-values from the euthanized fillets also increased significantly (p<0.05) from 17.24 to 27.48. The a*-values of the fillets from the

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23 euthanized fish remained significantly higher (p<0.05) until day 9 compared to both treatments. From day 12 to day 15 euthanized a*-values were still significantly higher than control a*-values (p<0.05) but no significant differences were found among euthanized and gassed post-mortem fillets. At day 18 there was no significant difference among any of the three treatments (p<0.05). The post-mortem gassing of the fillets maintained significantly higher a*-values (p<0.05) compared to the control samples until day 6. The frozen storage negatively affected the a*values of the control and the post-mortem gassed fillets (p<0.05). Freezing did not affect a*-values of the euthanized samples. Control samples were affected the most by the freezing and thawing as there was a significant decrease (p<0.05) in a*-values from 17.245 to 9.50. Gassed fillets were also affected by the freezing, but to a lesser extent. Euthanized fillets had significantly better stability (p<0.05) during the freezing period, being 8.46 and 17.89 points over CO treated fillets and untreated fillets respectively. The a*-values for the 100% CO post-mortem gas treatment were also significantly increased (p<0.05) compared to the control values. After 6 days at 4 o C there was a significant drop (p<0.05) in a*-values for all treatments. The decrease in a*-values at day 6 corresponded to a decrease in the heme peak wavelength and also in the CO concentration (see Figures 4-6 and 4-8 later) suggesting that the CO is escaping from the hemoglobin and myoglobin (discussed below). These results are consistent with results on CO-treated tuna, mahi mahi and Spanish mackerel, which all show an increase in a*-value on treatment, but a gradual decline after defrosting [33-35, 46].

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24 05101520253035BeforeTreatmentAfterTreament0369121518Daysa* values Control 100% CO Euthanized 100 % CO Gassed Freezing Figure 4-3. Effect of CO treatments and no treatment (control) on the a*-values (redness) of tilapia fillet red muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days. Control and gassed fillets L* (ligthness) values decreased significantly (p<0.05) during the frozen storage. On the other hand, L* values from the euthanized fillets were not significantly (p<0.05) affected until day 6 (Figure 4-4). These results suggest that the euthanasia with 100% CO yielded more natural fresh looking fillets than the post-mortem gassing. Both of the treatments did not have a significant effect on b*(yellowness) values until day 18 where control values increased significantly compare to the euthanized samples (Figure A-1). This slight increase can be attributed to the oxidation of heme proteins and possibly also to lipid oxidation[47]. The reduction in the a*-values for the control corresponds to met-hemoglobin [18], which can produce a brown-yellowish appearance to red muscle, which would explain the increase in b*-value.

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25 01020304050607080BeforeTreatmentAfterTreament0369121518DaysL values Control Euthanized Gassed Figure 4-4. Effect of CO treatments and no treatment (control) on the L*-values (ligthness) of tilapia fillet red muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days. White muscle is the predominant muscle type in tilapia. White muscle has significantly lower amounts of heme proteins than red muscle [48]. Although levels of heme protein are less in the white muscle, its red color was still significantly (p<0.05) influenced by both of the CO treatments (Figure 4-5). Fillets from tilapia euthanized with 100% CO had a pinkish tone to the white muscle which was reflected in a significant (p<0.05) increase in a*-values for the entire study (Figure 4-5). 100% CO post-mortem treatment produced significantly (p< 0.05) higher a*-values to the white muscle until day 6. From day 9 to the end of the study no significant differences were found among post-mortem gas treatment and control fillets. After both CO treatments, euthanized fillets had a*-values significantly (p<0.05) higher than a*-values from post-mortem gassed fillets. This difference was still significant (p<0.05) after the frozen storage and thawing process (at day 0). At days 3 and 6 and after day 15 no significant (p<0.05) differences were found. During day 9 and 12 significantly (p<0.05) higher a*-values were again found. These results suggest higher

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26 stability of a*-values for the euthanized process compared to the 100% CO post-mortem treatment. The white muscle side of the tilapia fillet contains a small central line of red muscle (Figure 4-2) that increased the overall a*-values of the white muscle and gave a higher standard deviations. In addition, the lack of bleeding of the euthanized fillets may also have influenced the results since more blood would leave more hemoglobin in the tissue and thus more CO binding and a larger effect on red color. Overall, the results seen for a*-values in the white muscle followed similar trends as those seen for the dark muscle. 051015202530BeforeTreatmentAfterTreament0369121518Daysa* values Control 100% CO Euthanized 100% CO Gassed Freezing Figure 4-5. Effect of CO treatments and no treatment (control) on the a*-values (redness) of tilapia fillet white muscle before freezing, after freezing and subsequent storage at 4 o C for 18 days. Heme Spectroscopic Analysis Both of the treatments influenced the red color of the fillets according to the CMVS. This increase in color and color stability comes from the binding of CO to heme proteins. The complex carboxy-hemoglobin has been proven by Kristinsson and coworkers [19, 20] to be more stable against oxidation compared to oxy-hemoglobin. The oxidation of heme proteins is the main reason why the red color decreases over time and changes to brown [13]. Figure 4-6 shows a representative spectrum of all three oxidation

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27 states at which hemoglobin/myoglobin is found during these experiments (i.e., Met, Oxy and Carboxy). 00.20.40.60.811.2385405425445465Wavelength (nm)Absorbance (AU) Carboxy-Hemoglobin Oxy-Hemoglobin Met-Hemoglobin 408 nm Met 414 nm Oxy 418 nm Carboxy Figure 4-6. Representative spectra for met-hemoglobin (408 nm), oxy-hemoglobin (414 nm) and carboxy-hemoglobin (418 nm) 395400405410415420BeforeTreatmentAfterTreatment0369121518DaysWavelength (nm) 100% CO Gassed Control 100% CO Euthanized Freezing Figure 4-7. Maximum heme peak values for red muscle extracts from euthanized and 100% CO gassed tilapia fillets and untreated tilapia stored at 4 o C for 18 days From Figures 4-7 it can be seen that the gas treatment as well as the euthanasia process led to a substantial increase in the heme peak wavelength, which indicates that CO is being bound to the heme proteins. A wavelength of 418 nm suggests maximum

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28 binding. Before treatment heme peaks wavelength of ~415 nm were found for all the treatments. This reading corresponded to mostly oxy-hemoglobin. After the gas treatment the heme peak wavelengths reached 416.5 nm on average while a reading of 417.3 nm was observed from the heme extracted from the euthanized tilapia. These UV-Vis spectra of the heme proteins extracted from the muscle of the gassed and euthanized tilapia demonstrated that there was a mixture of oxy-hemoglobin and carboxy-hemoglobin present in the extracts. Similar results were found by Danyali [34] and Garner [46] where muscle from tuna and Spanish mackerel, respectively, were exposed to different concentrations of CO. These high heme peak wavelengths were maintained through the frozen storage proving that the CO was still bound to the heme proteins what explains the high a*-values reported in Figure 4-3. On the other hand, the heme peak for the control decreased after freezing to about 408 nm which shows that it was already oxidized. This explains the decreased in a*-values obtained after freezing in the control samples (Figure 4-3). The significantly higher wavelength peaks seen for both CO treatments after 1 month of frozen storage compared to the control shows how the heme proteins are significantly stabilized when they are bound to CO. A period of 30 minutes of direct exposure of fillets to CO or euthanasia of fish with CO was therefore enough to significantly stabilize the heme proteins during freezing as well as after thawing. There was however a difference in the CO binding between the two CO treatments. The gassed fillets appeared to have more bound CO since wavelength remained mostly constant during the cold storage, while the heme peak wavelength decreased on storage at 4C for the euthanized samples. This decrease suggested that the euthanized samples were loosing CO. This fact is reflected in the a*-values, where a sudden decrease was noticed

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29 at day 6 (Figures 4-3 and 4-5). The heme peak wavelength did however increase after this decrease, suggesting that CO was still present in the muscle and was rebinding to the heme. It is interesting to note that euthanized fish had higher a*-values than the gassed fillets, but show less binding of CO to the heme according to the UV-vis analysis. This was unexpected as the redness of fish muscle is dictated by the level of CO binding to the heme proteins, and thus an opposite result would have been expected. It is possible that during extraction some of the CO may have been lost (e.g. during homogenization which may have denatured the heme proteins) thus giving lower values. However, all samples were extracted identically. Another possibility is that the euthanized fish had lower overall CO levels in the muscle compared to the gassed fish, and therefore during homogenization, some CO could have been lost and since it did not have the excess CO present in the gassed fillets, the CO lost did not get replenished. This is supported by the data on muscle CO levels presented below (Figures 4-9 and 4-10). Similar results were found for the white muscle (Figure 4-8). Heme proteins extracted from control fillets had heme peak wavelengths below 408 nm implying that heme proteins were already oxidized and met-hemoglobin was formed. For some samples heme proteins were below detection limits. This is because the level of heme proteins in white muscle is very low. Higher heme peaks were observed for the treated samples compared to the control. Some of the wavelength values for the euthanized fish suggest that the heme proteins were in part oxidized at days 3, 6, and 9. This is interesting, as the a*-values for the euthanized fish white muscle were higher than those for the gassed fillets, and thus one would have expected higher wavelength for the euthanized fish. The same contradiction was seen for the red muscle. The wavelength then rose again,

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30 suggesting rebinding to CO, similar to what was observed with the red muscle. However, due to the high a*-values maintained by the treated fillets compared with the control fillets and the lack of browning, it can be concluded that these wavelengths may come from some free blood that was present at the surface of the fillets. The free surface blood is expected to oxidize faster than blood in the fillets. 400405410415420BeforeTreatmentAfterTreatment0369121518DaysWavelength (nm) 100% CO Gassed Control 100% CO Euthanized Freezing Figure 4-8. Maximum heme peak values for white muscle extracts from euthanized tilapia, 100% CO gassed tilapia fillets and untreated tilapia stored at 4 o C for 18 days. The heme spectroscopic analysis revealed that treated products with CO can be differentiated from the untreated ones just by analyzing its UV-Vis spectra. Untreated product should not have a heme peak wavelength higher than 414 nm, while treated products will have heme peak wavelengths higher than 414 nm revealing the carboxy-hemoglobin/myoglobin complex. Carbon Monoxide Quantification The concentration of CO in the muscle was quantified using a GC equipped with a flame ionization detector (GC-FID). Few methods are available to measure CO in fish muscle. The Japanese health authority uses a method called the A method [36]. This

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31 method however requires large amounts of muscle for analysis and results have demonstrated that it lacks sensitivity[45]. Due to the small amount of red muscle present in tilapia fillets a different method was chosen [44] which recovered more CO from the muscle than the A method. 500300055008000BeforeTreatmentAfterTreatment0246101418DaysCO ppb Control 100% CO Gassed 100 % CO Euthanized Freezing Figure 4-9. Concentration of CO (ppb) in tilapia red muscle after 30 minutes exposure to 100% CO, euthanasia with 100% CO or no treatment. In Figure 4-9 it can be clearly seen that there was a difference in CO concentration between the treatments after the freezing and thawing process. Control samples had low levels of CO (1408 ppb). It was expected to find some CO in the muscle since endogenous CO is produced during the metabolism of protoheme [36, 49]. This small concentration was maintained for about 10 days, but then increased significantly (p<0.05) in day 14. An increase in CO concentration on extended storage has been reported previously, and is one of the indicators used by the Japanese health authorities that fish has not been treated [36]. If fish has been treated with CO, the level is expected to decline which was the case for the CO treated tilapia in this study.

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32 After treatment a significant (p<0.05) increase in CO concentration was noted for both euthanized and gassed samples (Figure 4-9). CO concentration increased to 6237 ppb and 7020 ppb for the euthanized and the gassed treated fillets respectively. After thawing, initial concentration of CO in the post-mortem gassed fillets remained considerably higher (~6380 ppb) than the level for the euthanized fillets (4712 ppb), but then dropped suddenly after day 4 of storage. The additional amount of CO present in the gassed samples is likely CO trapped in the muscle and thus was not bound to the heme proteins. This is very possible, considering that the initial heme peak wavelengths were similar for both treatments. Thus, the data suggest similar CO saturation of heme proteins, which means the higher level of CO in the CO gassed fillets is due to additional CO trapped in the extracellular matrix. The gas treatment was applied post-mortem by exposing the fillets to 100% CO for 30 min. The exposure is based on surface contact between the muscle and the CO and therefore it is very possible that CO can be trapped in the extracellular muscle matrix. Davenport and coworkers [39] subjected tuna to 100% CO treatment and found that much of the CO is trapped in the extracellular matrix of the muscle, which supports the results seen here with tilapia. Figure 4-9 shows that this excess CO over the CO for the euthanized fish remained in the muscle for four days and then most of it left the muscle since euthanized fish and gassed fillets had similar values at days 6 and beyond (p< 0.05). As discussed before, this additional CO in the muscle may be the reason the CO treated fillets had higher heme peak absorbance (i.e., suggesting more CO saturation) throughout the experiment, since extracellular CO would have replenished CO lost from the heme proteins during extraction. This however would

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33 also suggest that the CO gassed fillets should have had higher a*-values, which was not the case. The euthanized fillets had higher CO concentration than the control but as discussed above, lower levels than the gassed fillets. Since the CO was transferred to the edible muscle tissue through the respiratory and circulatory system it can be inferred that all the CO present in the muscle was bound to the heme proteins. The amount of heme proteins present in the muscle dictates the amount of CO that will be bound. At day 6 both CO treatments had declined to similar CO levels. However, even at the end of the 18 days storage period, both treatments still had significantly (p < 0.05) higher levels than the control. Figure 4-10 represents the amount of CO found in white muscle. The difference between red and white muscle of the two CO treatments is ~2-2.8 fold. Previous work by Miyazaki and coworkers [44] has also shown that CO levels are higher in red muscle compared to white muscle. This suggests that the heme proteins are the main source of bound CO in the muscle since white muscle contains significantly less heme proteins than red muscle. The white muscle of untreated control has similar levels of CO red muscle (943 ppb). Analysis of the CO levels in white muscle of the treated fish confirms that there is entrapment of CO in the extracellular matrix of the muscle when it was treated post-mortem with 100% CO. The starting values of CO for the gassed fillets were significantly higher than the values for the euthanized samples. The difference between the two is close to the difference seen in the red muscle. This difference is therefore very likely explained by additional CO trapped in the muscle, and not bound to heme proteins. The concentration of CO dropped more suddenly in the gassed white muscle than it did in

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34 the red muscle. The higher heme content in the red muscle may have aided in the stabilization of the CO in the muscle during the first days of storage, while the lower level in the white muscle caused CO to be released sooner from the muscle. After 6 days of storage the values stabilized and were similar to those of the euthanized fish. 50025004500BeforeTreatmentAfterTreatment0246101418DaysCO ppb Control 100% CO Euthanized 100% CO Gassed Freezing Figure 4-10. Concentration of CO (ppb) in tilapia white muscle after 30 minutes exposure to 100% CO, euthanasia with 100% CO or no treatment. Muscle pH and Drip Loss The amount of stress at which the animal is put under before being slaughter can have a great influence in its final pH and thus in its water holding capacity [26]. As the muscle pH decreased post-mortem, the number of negative charges decreases on the muscle proteins and they are moved closer to their isoelectric point. Muscle has its lowest water-holding capacity at the isoelectric point of the myofibrillar proteins [50]. It was expected that the euthanasia of tilapia would be less traumatic for the animal and thus a lower drop in pH and higher water holding capacity would be obtained. The pH data does not indicate that there was any significant (p< 0.05) difference between the different

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35 treatments. However, Figure 4-11 shows that fillets from control and euthanized tilapias had a smaller change in pH compared to the gassed fillets. Nevertheless it cannot be inferred that the CO gassing of fillets represented a more stressed process, since the fish used for the gassed fillets were slaughtered in the same way as the control and the pH should therefore have been similar. It can be assumed the level of stress at which the tilapia experienced from every treatment was different from the beginning due to the handling of the live animal. Tilapia were transported live in small coolers from a tilapia farm located 2 hours away from the laboratory. According to Terlouw [26] an animal is under stress when it is confronted with a potentially threatening situation. It is possible that the tilapia used in the CO fillet gassing experiments were under more stress than the other treatments before slaughtering, thus explaining the lower pH values obtained. -0.300.30.60.91.21.5BeforeTreatmentAfterTreatment0369121518DaysChange in pH pH euthanized pH gassed pH control Freezing Figure 4-11. Change in pH of fillets gassed with 100% CO, fillets from fish euthanized with 100% CO and untreated tilapia fillets stored at 4 o C for 18 days. Water lost on thawing (thaw loss) was also recorded for the fillets from the three treatments. Even though statistical analysis showed no significant difference among the three treatments, Figure 4-12 shows that the euthanized samples had the highest thaw loss after 1 month of frozen storage. Thawed fillets lost 2.8% of their original weight. The

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36 gassed samples had the lowest thaw loss with a change of 2% from their weight before freezing. These results are contradictory to the pH results, i.e., fillets from the control and euthanized fish had more stable and higher pH, but still higher thaw loss than gassed fillets which had a lower pH, which is contrary to what was expected. 00.511.522.533.5% Change in Weigth Control 100% CO Euthanized 100% CO Gassed Figure 4-12. Thaw loss of gassed fillets (100% CO for 30 min), fillets from euthanized fish (100% CO) and untreated fillets after 1 month of freezing. Results obtained are based on the change on weight of the fillets. -20246810Day 0Day 3Day 6Day 9Day 12Day 15Day 18Change in Weight (%) Control 100% CO Euthanized 100% CO Gassed Figure 4-13. Change in drip loss of gassed fillets (100% CO for 30 min), fillets from euthanized fish (100% CO) and untreated fillets during 18 days of storage at 4 o C after thawing.

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37 There were no significant differences in the amount drip loss among the three treatments during the 18 day storage at 4C after thawing (Figures 4-12). Apparently the differences in pH did not affect the drip loss of the fillets. Study 2: Whole Frozen Tilapia Effect of Carbon Monoxide Euthanasia on Color Color was analyzed for fillets obtained from fresh control and euthanized tilapia, and also after defrosting the frozen whole fish after 2 and 4 months (Figure 4-14). The fresh data was obtained to compare the color of the muscle at the fresh state to the color of the muscle after being subjected to frozen storage. The effect of euthanizing the fish on the a*-values can be appreciated as the a*-values of the fillets from the euthanized fish were significant higher (p<0.05) than the a*-values of the control. 05101520253035Fresh2 Months4 Monthsa* Value Control 100 % CO Euthanized Figure 4-14. Effects of euthanasia with 100% CO and no treatment on a*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months. The highest a*-values for the control samples were obtained for the fresh fish. At the fresh state, the fillets were exposed to air after filleting which leads to oxygen binding to hemoglobin which gives the bright red color characteristic of oxy-hemoglobin. This is

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38 confirmed by the heme peaks wavelengths obtained for the control samples (see next section). The control a*-values obtained at this point are representative and can be used as reference for a*-values of a fresh tilapia fillet. In addition, the difference between the euthanized samples and the control are less significant at this point than at the other samples points. The euthanized a*-values were however significantly higher than the a*-values of the control. The increase in redness is explained by the formation of the carboxy-hemoglobin complex, as discussed before. The control a*-values progressively decreased after freezing. However there was no significant (p>0.05) difference between the fresh and after 2 months a*-values. Meaning that storing the fish whole in a frozen state preserved the color of the fillets. There was a significant decrease (p<0.05) in a*-values after 2 months of frozen storage. This decrease comes from the oxidation of the heme proteins where the characteristic bright red color of the red muscle of the fresh fillets gradually turns into a more brownish color. The a*-values of the fillets from the euthanized fish were maintained significantly (p<0.05) higher throughout the study compared to the control. It can be seen from Figure 4-13 that the euthanized a*-values were significantly (p<0.05) higher at all times than the control values. After 4 months of frozen storage, the euthanized a*-values were not significantly (p>0.05) different from the initial control values, underlining how stable the a*-values became after the euthanasia with 100% CO. This stability came from the strong binding of CO to the heme proteins. Interestingly an increase in a*-values was observed after 2 months of frozen storage. This agrees with the data shown in the previous chapter on the fillets. Danyali [34] also noted an increase in a*-values when CO treated yellowfin tuna steaks were subjected to 30 days of storage at -25C. The increase in red color of the

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39 tilapia muscle corresponded to an increase in the concentration of CO in the muscle (see later). These results thus suggest that the more CO is bound to the muscle the higher a*-values would be obtained. Similar results were found for the white muscle where fillets from the euthanized fish also show significantly (p<0.05) higher a*-values than control (Figure 4-15). Furthermore, a*-values for the euthanized fish were maintained significantly (p<0.05) higher at all times compared to the control. After four months of frozen storage, a*-values of the white muscle from the euthanized fish were similar (no significant difference (p<0.05)) to the initial a*-values, thus mirroring that seen for the red muscle. The control a*-values on the other hand decreased significantly after 4 months of frozen storage. 051015202530Fresh2 Months4 Monthsa* values Control 100% CO Euthanized Figure 4-15. Effect of euthanasia with 100% CO and no treatment on a*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months. The euthanasia process did not have an influence on b* or L*-values. No significant differences (p>0.05) were found for the euthanized fillets when compared to the control fillets (Figures 4-16 to 4-19).

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40 0246810121416Fresh2 Months4 Monthsb* Value Control 100% CO Euthanized Figure 4-16. Effect of euthanasia with 100% CO and no treatment on b*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months. 010203040506070Fresh2 Months4 MonthsL* value Control 100% CO Euthanized Figure 4-17. Effect of euthanasia with 100% CO and no treatment on L*-values of the red muscle of fresh tilapia and tilapia stored frozen for up to four months.

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41 02468101214161820Fresh2 Months4 Monthsb* Value Control 100% CO Euthanized Figure 4-18. Effect of euthanasia with 100% CO and no treatment on b*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months. 020406080Fresh2 Months4 MonthsL* values Control 100% CO Euthanized Figure 4-19. Effect of euthanasia with 100% CO and no treatment on L*-values of the white muscle of fresh tilapia and tilapia stored frozen for up to four months Heme Spectroscopic Analysis The red color stability throughout the study is caused by the stability of the heme proteins. If the formation of the met-form of the heme proteins is avoided, the red color will be maintained [19]. As shown in the first study on fresh and frozen fillets, the red muscle is where most of the CO is bound due to its high content of heme proteins. Figure 4-18 shows the heme peaks wavelengths obtained of red muscle extracts for the three

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42 samples points. The difference in heme protein ligand binding and stability is clearly different between the euthanized and untreated (control) fish. 405407409411413415417419421Fresh2 months4 monthsWavelength (nm) Control 100% CO Euthanized Figure 4-20. Maximum heme peak values for red muscle of euthanized (100% CO) and untreated fresh and frozen whole tilapia A heme peak wavelength of 415 nm was seen for the fresh control signifying the presence of oxy-hemoglobin/myoglobin (Figure 4-20). This peak was expected since the fillets were exposed to air during the filleting and skinning process (which was part of the sample preparation) which would have allowed oxygen from the air to bind to hemoglobin/hemoglobin. The coupling of oxygen with hemoglobin/myoglobin gives a bright red color, explaining the high a*values obtained for the control in the fresh state. The heme peak wavelengths decreased on freezing, suggesting oxidation of the heme proteins as well as loss of oxygen, which agrees well with red color results (Figures 4-20 and 4-14). At the end of the study, a heme peak wavelength of 409 nm was found for the control samples implying that the heme proteins were significantly oxidized and thus met-hemoglobin/myoglobin had formed. The presence of the metform was also evident in the fillets. As presented and discussed in the section before, a brown color had replaced the bright red color of the fresh fillets.

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43 The euthanized fillets had higher heme peak wavelengths (~418 nm after euthanasia) than control, indicating the presence of carboxy-hemoglobin. These wavelengths demonstrated the intake of CO by the fish during the euthanasia process. The heme proteins in the euthanized fish were also significantly (p<0.05) more stable during the study as they maintained high heme peak wavelengths. After four months of frozen storage, it can be seen that CO was still bound to hemoglobin showing a great stability of the carboxy-hemoglobin complex. This stability is also responsible for the high and stable a*-values discussed earlier. A substantially higher stability of tilapia carboxy-hemoglobin compared to tilapia oxy-hemoglobin during extended frozen storage has been reported [19], which would explain this high heme protein and color stability of the CO euthanized samples during the 4 month frozen storage. The same analyses were done on the white muscle (Figure 4-21). As reported in the first study on white muscle, the heme peak wavelengths were higher for the euthanized samples than the control, however not as high as those for the red muscle. The higher wavelengths were also maintained higher for the euthanized samples for the duration of the study. The heme peak wavelengths found in the white muscle of the euthanized fish correspond to wavelength one would expect for oxy-hemoglobin/myoglobin and not carboxy-hemoglobin/myoglobin. The fact that the heme peak wavelengths of the euthanized fish white muscle is higher than that of the control, does however suggest that CO binding took place. The increase in a*-value for the white muscle of the euthanized fish also supports this (Figure 4-15). These lower wavelengths for the euthanized white muscle compared to the red muscle, suggest that the values represent an average of all three different heme protein derivatives, i.e., met, oxy and carboxy, while the control

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44 value suggest a mixture of met and oxy forms[45]. The heme peak wavelength increase, increased heme protein stability and increased red color of the euthanized white muscle during the four months study therefore must come from partial CO binding to the heme proteins in the white muscle. 406407408409410411412413414415Fresh2 months4 monthsWavelength (nm) Control 100% CO Euthanized Figure 4-21. Maximum heme peak values for white muscle of euthanized (100% CO) and untreated fresh and frozen whole tilapia. It is interesting that the heme peaks wavelength for both red and white muscle did not decrease as much as in the second study compared to that seen in the first study. The fact that the fish were frozen whole appeared to preserve the heme proteins better compared to freezing fillets. Carbon Monoxide Quantification The amount of carbon monoxide that is found in the muscle is important. It reveals the uptake of CO in the muscle and can also be used to differentiate treated products from untreated ones. The heme spectroscopic analyses and the increased redness (a*-values) revealed the intake of CO by the live animal, but does not tell us how much CO is in the muscle. Figure 4-22 shows the difference in CO concentration in the red muscle between a 100% CO euthanized and untreated fish. CO concentration in the untreated red muscle

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45 is low compared to the concentration in the euthanized fish. Furthermore, it can be observed that the CO concentration of the control is very stable and does not change significantly during the four months of study. 500250045006500850010500Fresh2 Months4 MonthsCO ppb Control 100% COEuthanized Figure 4-22. Concentration of CO (ppb) in the red muscle of fresh and frozen untreated and euthanized (100% CO) tilapia. The amount of CO found in the euthanized fillets was considerably higher than the CO concentration in the control fillets and was more variable. Many natural conditions inherent to tilapia may have accentuated these differences. For instance, CO is bound to the heme proteins, explaining the high concentration found in the red muscle compared to the white muscle. Every fillet has different amounts of red muscle influencing the final amount of CO recovered from the muscle. This could explain the higher values found after 2 months of frozen storage. Another possible explanation for the lower values for the fresh fish is that fillets were cut from the euthanized fish very shortly after it was euthanized, which could have led to some CO loss from the muscle. On the other hand, the samples at 2 and 4 months were taken from whole frozen fish filleted after thawing, thus giving the muscle more time to trap CO than the muscle of the freshly killed and filleted fish.

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46 The concentration of CO found in the white muscle follows the same trends as that seen for the red muscle (Figure 4-23). The difference between the euthanized and the control muscle were not as large in the white muscle compared to the red muscle. This relates to the lower amount of heme proteins present in the white compare to the red muscle. 5001000150020002500Fresh2 Months4 MonthsCO ppb Control 100% CO Euthanized Figure 4-23. Concentration of CO (ppb) in the white muscle of fresh and frozen untreated and euthanized (100% CO) tilapia. From Figures 4-22 and 4-23 it can be observed that the amount of CO was maintained high during the whole study and that it did not decrease significantly (p<0.05) over time. Since the tilapia was kept frozen whole and vacuum packed it may have aided in keeping the CO levels high at all times. This can be also observed in the high a*-values obtained throughout the study and in the high heme peak wavelengths discussed earlier. As discussed previously, an increase in a*-values after 2 months of frozen storage was seen in the red muscle, thus corresponding to the increase in CO levels, revealing a close relation between the amount of CO present in the muscle and a*-values (Figures 4-12 and 4-20). However, as seen in Figure 4-13, a*-values from the white muscle did not

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47 follow the same trend as in red muscle. A higher amount of CO after 2 months of frozen storage was found in the white muscle of the fish (Figure 4-23), similar to that seen for the red muscle, however a*-values in the white muscle did not increase. This can possibly be explained to the amount of heme proteins present in each muscle. The small amount of heme proteins present in the white muscle could explain these results. Since there are more heme proteins present in the red muscle, more binding could take place and more CO is needed to stabilize all the heme proteins. Meanwhile, in the white muscle the limit of CO intake is much lower and maybe the excess of CO present is trapped in the muscle are not necessarily bound to the heme proteins and thus has no effect on color.

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CHAPTER 5 SUMMARY AND CONCLUSIONS The practice of treating fish with CO in order to avoid the oxidation of the heme proteins and maintain an appealing and very attractive fresh red color is now widespread and increasingly more common. The red color is stabilized by the formation of the carboxy-hemoglobin/myoglobin complex. In the tilapia industry, a common practice is to expose the fillets pre-rigor, while the muscle is still respiring, to CO for a brief period of time. Another less common practice is to incorporate CO into fish tanks while the animal is still alive. The intake of CO to the muscle by the euthanasia with 100% CO process was confirmed by the heme peaks wavelengths obtained and by the difference in the concentration of CO found in the treated product compared to the untreated ones. The incorporation of CO in the muscle of tilapia by either treatment had a positive effect in the red color of the fillets. After the treatments, the red color was enhanced and maintained for a longer period of time. In addition, the CO treatment affected only the redness (a*-values); b* and L*-values were not significantly affected. Comparing the euthanasia process against the gassing of fillets it was found that both processes had good results preserving the color of the red muscle. The euthanasia with 100% CO had a few advantages compared to the post-mortem 100% CO treatment. A larger increase in a*-values was seen when the fillets came from a euthanized fish. Also, a better stability through the freezing process was seen for the euthanized samples. Finally, the euthanized method was found to preserve better L values giving the fillets a more natural look. 48

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49 Due to the inherent stress of the whole process, muscle pH and water holding capacity were not significant different among treatments. Nevertheless, it is believed that the euthanasia process will be less stressful to the fish if they were initially without the added stress of transportation and laboratory handling. In addition, the euthanasia with 100% CO represents less product handling since there will be no need of post-mortem CO gassing. This advantage may positively influence the shelf life of the final product since there will be less product handling, implying less probability of contamination. Furthermore since the product is already treated via the gas euthanasia process, less labor required is another advantage over a post-mortem gassing process. The different level of acceptance and varying regulations between different countries on the use of CO in seafood processing has created a need to test products for CO content. In this study it has been shown that a simple heme analysis could differentiate a treated from a non treated product. In addition, the method used to measure the amount of CO in the muscle is very important and should be considered and specified when CO regulation and CO limits are put in place. It is worth mentioning that the use of CO in seafood processing has to be very closely studied and regulated. Without the proper control, the use of CO could mask potential health hazards or it could make a product look better than it actually is. In this research study, 100% CO was used to euthanize the fish. It was observed that tilapias remained calm before dying, revealing that the process was not stressful. It was also observed that the use of CO had an anesthetic effect on the animal since they stopped moving and remained calm until euthanasia was completed. This is an important observation since animal welfare has been giving increasingly importance, and more

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50 regulation regarding humane slaughter practices are being required. The euthanasia with 100% CO of fish would be in agreement with the animal welfare act, in my opinion.

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APPENDIX A FIRST STUDY DATA 02468101214161820BeforeTreatmentAfterTreament0369121518Daysb* values Control 100% CO Euthanized 100% CO Gassed Freezing Figure A-1. Effect of CO treatments on the b*-values of the red muscle of tilapia. 0510152025BeforeTreatmentAfterTreament0369121518Daysb* values Control Euthanized Gassed Freezing Figure A-2. Effect of CO treatments on the b*-values of the white muscle of tilapia. 51

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52 01020304050607080BeforeTreatmentAfterTreament0369121518DaysL values Control Euthanized Gassed Figure A-3. Effect of CO treatments on the L*-values of the white muscle of tilapia.

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APPENDIX B CO CONCENTRATION IN THE WATER Table B-1. Concentration of CO in the water used to euthanize tilapias Time Ret. Time Peak Area [CO] ppb Time Ret. Time Peak Area [CO] ppb 0 min 0.490 5.400 2166.504 20 min 0.453 1833.290 27082.385 0 min 0.459 5.490 2167.730 20 min 0.453 1821.400 26920.313 0 min 0.456 5.460 2167.321 20 min 0.457 1369.140 20755.579 0 min N/A N/A N/A 20 min 0.460 1348.140 20469.329 Ave 0.468 5.450 2167.185 Ave 0.456 1592.993 23806.901 Stand Dev 0.019 0.046 0.625 Stand Dev 0.003 270.786 3691.074 5 min 0.465 872.940 13991.900 25 min 0.453 1846.280 27259.451 5 min 0.456 844.440 13603.418 25 min 0.455 1440.570 21729.238 5 min 0.459 633.640 10730.013 25 min 0.456 1547.700 23189.521 5 min 0.455 612.740 10445.126 25 min 0.458 1515.120 22745.425 Ave 0.459 740.940 12192.614 Ave 0.456 1587.418 23730.909 Stand Dev 0.004 136.729 1863.752 Stand Dev 0.002 178.305 2430.472 10 min 0.459 820.970 13283.499 30 min 0.454 1832.360 27069.708 10 min 0.459 802.520 13032.008 30 min 0.460 1822.440 26934.489 10 min 0.462 1109.130 17211.395 30 min 0.454 1334.740 20286.674 10 min 0.464 1104.260 17145.012 30 min 0.458 1318.220 20061.490 Ave 0.461 959.220 15167.979 Ave 0.457 1576.940 23588.090 Stand Dev 0.002 170.468 2323.635 Stand Dev 0.003 289.313 3943.615 15 min 0.466 1676.640 24947.096 35 min 0.453 1814.700 26828.986 15 min 0.452 1665.550 24795.929 35 min 0.453 1796.570 26581.857 15 min 0.457 1583.100 23672.057 35 min 0.452 1518.100 22786.045 15 min 0.454 1559.580 23351.457 35 min 0.450 1511.960 22702.351 Ave 0.457 1621.218 24191.635 Ave 0.452 1660.333 24724.810 Stand Dev 0.006 58.564 798.280 Stand Dev 0.001 167.963 2289.492 53

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54 05000100001500020000250003000005101520253035Minutes[CO] ppb [CO] in water Figure B-1. Change of CO concentration in the water used for the euthanasia process. Table B-2. Time and amount of CO needed to euthanize tilapia in the tank Date Time min CO used (L) 1/19/2005 39 182 1/19/2005 52 110.4 CO did not flow correctly 1/19/2005 32 121.6 2/16/2005 27 500 Pump not working properly 3/14/2005 28 197.3 3/14/2005 26 258 Average 34 228.216 Std Dev 10.01998 143.591 Average of Correct Trials 31.25 189.725 Std Dev of Correct Trials 5.737305 56.034

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APPENDIX C PICTURES OF THE TILAPIA FILLETS Representative pictures of the red muscle side of a tilapia fillet, and a white muscle side of a tilapia fillet are shown below. All the images can be found in the CD D:/Thesismantilla/tilapiaimages (Kept in Dr. Hordur G. Kristinssons office) Figure C-1. Picture of a red muscle side of a tilapia fillet taken by the CMVS 55

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56 Figure C-2. Picture of the white muscle side of a tilapia fillet taken by the CMVS

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LIST OF REFERENCES 1. Food and Agriculture Organization of the United Nations. Fisheries Dept., The state of world fisheries and aquaculture. 2002, Food and Agriculture Organization of the United Nations: Rome. 2. Costa-Pierce, B.A. and J. Rakocy, Tilapia aquaculture in the Americas. 1997, Baton Rouge, LA: World Aquaculture Society; American Tilapia Association. v. 1. 3. Knapp, G., Economic considerations in thinking about United States marine aquaculture. 2004, NOAA Marine Fisheries Advisory Committee: Anchorage, Ak. 4. Chapman, F.A. Culture of hybrid tilapia reference profile. [HTML version PDF version] 2000 [accessed March, 2005]; Available from: http//purl.fcla.edu/UF/lib/CultureOfHybridTilapia.htm http//purl.fcla.edu/UF/lib/CultureOfHybridTilapia.pdf. 5. Popma, T. and M. Masser, Tilapia life history and biology. Aqua KE Government Documents, 2003. 6. Food and Agriculture Organization of the United Nations. Fishery Resources Division., FAO fisheries technical paper 2004, Food and Agriculture Organization of the United Nations.: Rome. 7. Simberloff, D., D.C. Schmitz, and T.C. Brown, Strangers in paradise : impact and management of nonindigenous species in Florida. 1997, Washington, D.C.: Island Press. 8. Reddy, N., C. Schreiber, K. Buzard, G. Skinner, and D. Armstrong, Shelf-life of fresh tilapia fillets packaged in high barrier film with modified atmospheres. Journal of Food Science, 1994. 59(2): p. 260-264. 9. Otwell, W., M. Balaban, and H. Kristinsson. Use of carbon monoxide for color retention in fish. in First Joint Trans-Atlantic Fisheries Technology Conference. 2003. 11-14 June, ReykjavikIceland. 10. Kowalski, W.R., Process to treat fish with tasteless smoke or carbon monoxide through the respiratory and circulatory systems, in US Patent & Trademark Office. 2003: United States. 57

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58 11. National Oceanic and Atmospheric Administration, O.C.S., US Commercial Landings. 2002, NOAA Fisheries. Silver Spring, Maryland. 12. Harvey, D., U.S. aquaculture production higher in 2005, U.S.D. Agriculture, Editor. 2005, World Agricultural Outlook Board. 13. Fennema, O.R., Food chemistry. 3rd ed. 1996, New York: M. Dekker. 14. Shahidi, F. and J.R. Botta, Seafoods: chemistry, processing technology and quality., ed. B.A.a. Professional. 1994, Glasgow, UK. 15. Kristinsson, H. and H. Hultin, Changes in trout hemoglobin conformations and solubility after exposure to acid and alkali pH. Journal of Agricultural and Food Chemistry, 2004. 52(11): p. 3633-3643. 16. Richards, M., H. Ostdal, and H. Andersen, Deoxyhemoglobin-mediated lipid oxidation in washed fish muscle. Journal of Agricultural and Food Chemistry, 2002. 50(5): p. 1278-1283. 17. Cashon, R., M. Vayda, and B. Sidell, Kinetic characterization of myoglobins from vertebrates with vastly different body temperatures. Comparative Biochemistry and Physiology, B: Comparative Biochemistry, 1997. 117(4): p. 613-620. 18. Stryer, L., Biochemistry. 4th ed. 1995, New York: W.H. Freeman. 19. Kristinsson, H., S. Mony, and H. Petty, Properties of tilapia carboxyand oxyhemoglobin at postmortem pH. Journal of Agricultural and Food Chemistry, 2005. 53(9): p. 3643-3649. 20. Kristinsson, H. and H. Hultin, Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding. Journal of Agricultural and Food Chemistry, 2003. 51(24): p. 7187-7196. 21. Everse, J. and N. Hsia, The toxicities of native and modified hemoglobins. Free Radical Biology Medecine, 1997. 22(6): p. 1075-1099. 22. Kanner, J., Oxidative processes in meat and mear-productsquality implications. Meat Science, 1994. 36(1-2): p. 169-189. 23. Zayas, J.F., Functionality of proteins in foods. 1996, New York: Springer. 24. Micklander, E., H. Bertram, H. Marno, L. Bak, H. Andersen, S. Engelsen, and L. Norgaard, Early post-mortem discrimination of water-holding capacity in pig longissimus muscle using new ultrasound method. LWT-Food Science and Technology, 2005. 38(5): p. 437-445.

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59 25. Huff-Lonergan, E. and S. Lonergan, Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes. Meat Science. In press. 26. Terlouw, C., Stress reactions at slaughter and meat quality in pigs: genetic background and prior experience A brief review of recent. Livestock Production Science, 2005. 94(1-2): p. 125-135. 27. Fraser, D., J. Ritchie, and A. Fraser, Term stress in a veterinary context. British Veterinary Journal, 1975. 131(6): p. 653-662. 28. Huss, H.H., Quality and quality changes in fresh fish. 1995, Rome: Food and Agriculture Organization of the United Nations. 29. Erickson, U., L. Hultmann, and J. Steen, Live chilling of Atlantic salmon (Salmo salar) Combined with mild Carbon Dioxide anaesthesia. I. Establishing a method for large-scale processing of farmed fish. Aquaculture, 2005. In press. 30. Einen, O., T. Guerin, S. Fjaera, and P. Skjervold, Freezing of pre-rigor fillets of Atlantic salmon. Aquaculture, 2002. 212(1-4): p. 129-140. 31. Roth, B., O. Torrisen, and E. Slinde, The effects of slaughtering procedures on blood spotting in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Aquaculture. In press. 32. Chow, C., P. Hsieh, M. Tsai, and Y. Chu, Quality changes during iced and frozen storage of tuna flesh treated with carbon monoxide gas. Journal of Food and Drug Analysis, 1998. 6(3): p. 615-623. 33. Demir, N., H. Kristinsson, and M. Balaban. Quality changes in mahi mahi (Coryphaena hippurus) fillets treated by different carbon monoxide concentrations and filleted smoke as assessed by color machine vision and lipid oxidation. 2004. IFT annual meeting, Las Vegas, Nv, Abstract # 63-8. 34. Danyali, N., The effect of carbon monoxide and filtered smoke on quality and safety of yellowfin tuna, in Food Science anf Human Nutrition. 2004, University of Florida: Gainesville. p. v, 112 leaves. 35. Ross, M.P., The influence of exposure to carbon monoxide on the quality attributes for yellowfin tuna muscle, in Food Science and Human Nutrition. 2000, University of Florida: Gainesville. p. v, 72 leaves. 36. Ishiwata, H., Y. Takeda, Y. Kawasaki, R. Yoshida, T. Sugita, S. Sakamoto, and T. Yamada, Concentration of carbon monoxide in commercial fish flesh and in fish flesh exposed to carbon monoxide gas for color fixing. Journal of the Food Hygienic Society of Japan, 1996. 37(2): p. 83-90.

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60 37. Kristinsson, H., Acid-induced unfolding of flounder hemoglobin: Evidence for a molten globular state with enhanced pro-oxidative activity. Journal of Agricultural and Food Chemistry, 2002. 50(26): p. 7669-7676. 38. Leydon, N., S. Suman, P. Ellis, C. Palmer, L. Faustman, and L. Pivarnik, Quality and safety assessment of time/temperature storage conditions for filtered smoked tilapia fillets produced for retail distribution. 2005: IFT annual meeting, New Orleans, LA, Abstract# 89A-16. 39. Davenport, M., Human consumption of CO-exposed tuna sushi. 2002, Greenville College: Greenville. 40. Kalin, B., Diagnosis of carbon monoxide poisoning 1996, Current Approaches in Forensic Toxicology. 41. Holeton, G., Oxygen uptake and tranport by rainbow trout during exposure to carbon monoxide. Journal of Experimental Biology, 1971. 54(1): p. 239-243. 42. Daniels, F. and F.H. Getman, Outlines of physical chemistry. 1st ed. 1948, New York,: J. Wiley. 43. Lide, D., CRC Handbook of chemistry and physics. 86th ed. 2005, Boca Raton: Taylor & Francis Group, LLC. 44. Miyazaki, H., M. Abe, M. Asanoma, Y. Nagai, M. Nakajima, and M. Miyabe, Simple determination of carbon monoxide in fish meat by GC. Journal of the Food Hygienic Society of Japan, 1997. 38(4): p. 233-239. 45. Huo, L. and H. Kristinsson, Rapid detection of carbon monoxide treated seafood products based on spectral properties of heme proteins. 2005: IFT annual meeting, New Orleans, LA, Abstract # 89A-35. 46. Garner, S.K., Effects of carbon monoxide on muscle quality of spanish mackerel, in Food Science and Human Nutrition. 2004, University of Florida: Gainesville. p. v. 96 leaves. 47. Kristinsson, H. and N. Demir, Functional protein isolates from underutilized tropical and subtropical fish species and byproducts, in Advances in Seafood Byproducts, U.A. Alaska Sea Grant College Program, Editor. 2003, P. Bechtel: Anchorage, AK. p. 277-298. 48. Richards, M., S. Kelleher, and H. Hultin, Effect of washing with or without antioxidants on quality retention of mackerel fillets during refrigerated and frozen storage. Journal of Agricultural and Food Chemistry, 1998. 46(10): p. 4363-4371. 49. Wilks, A. and P. Demontellano, Rat-liver heme oxygenase high level expression of a truncated soluble form and nature of the meso-hydroxylanting species. Journal of Biological Chemistry, 1993. 268(30): p. 22357-22362.

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61 50. Foegeding, E., T. Lanier, and H. Hultin, Characteristics of edible muscle tissue, in Food Chemistry, M. Dekked, Editor. 1996: New York. p. 879-942.

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BIOGRAPHICAL SKETCH David Mantilla was born on February 3, 1979, in Quito, Ecuador. He attended a French high school and graduated from it in July 1997. He came to the U.S. in Fall 1999 to the English Language Institute (ELI) at the University of Florida. He attended Santa Fe Community College and graduated in December 2000. David graduated in December 2002 from the University of Florida with a Bachelor of Science degree in food science and human nutrition. He did two internships as an undergrad, one with Tyson Foods and the other with Darden Restaurants. He started his masters degree in August 2003 under Dr. Hordur G. Kristinsson. While at the University of Florida he was invited to join Phi Tau Sigma, the honorary society of food scientists. 62


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EUTHANASIA OF TILAPIA USING CARBON MONOXIDE FOR COLOR
FIXATION AND COLOR STABILIZATION















By

DAVID MANTILLA TORRES


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

UNIVERSITY OF FLORIDA


2005





























Copyright 2005

by

David Mantilla Torres

































This document is dedicated to my loving parents and my sister.















ACKNOWLEDGMENTS

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

Hordur G. Kristinsson, who with his advice, guidance and support helped me to complete

this project. I would also like to thank my committee members, Dr. Murat Balaban, Dr.

Steve Otwell and Dr. Frank Chapman, for all their suggestions and help in the completion

of this research. It has been a privilege to work with such talented people.

I would like to thank to Mr. Gene Evans. His generosity and help providing this

research with the amount of live tilapia needed are greatly appreciated.

I would like to thank my family for always believing and supporting me, making it

possible to reach my goals.

Finally, I would like to thank all my friends and my lab mates who became my

family and supported me all these years at the University of Florida.
















TABLE OF CONTENTS

Page

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

LIST OF TABLES .............. ................. ........... ............... ............ vii

LIST OF FIGURES ..................................................... .......... ............... .. viii

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

CHAPTER

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

T ilapia and A quaculture ................................................. .. .. .. ... ...............1
Quality of Seafood ............... ................. ................................ .... .2

2 LITER A TU R E REV IEW ............................................................. ....................... 4

Tilapia Taxonom y ................... ................................................... .... 4
Q quality and Shelf L ife of Seafood .................................................................... ..... 5
H em e Proteins and Seafood Quality.................................... .......................... ......... 5
W after Holding Capacity and M uscle pH ........................................... ............... 7
Effects of Carbon Monoxide on Fish Muscle..........................................................10
Carbon M monoxide and Euthanasia.................................... ........................... ......... 12
R research O bjectiv es.......... ................................................................. ......... ....... 13

3 M ATERIALS AND M ETHOD S ........................................ ......................... 14

Euthanasia of Tilapia with Carbon Monoxide (CO)........................ ............... 14
100% CO Post-Mortem Gas Fillets Treatment of Tilapia............... ..... ...... ....16
C o lo r A n aly sis ..................................................................... ................ 16
Quantification of CO in Fish M uscle...................................... ........................ 16
Heme Protein Extraction and Spectroscopic Analysis ............................................17
M u scle p H ............................................................................18
D rip L oss ............................................. 18
Statistical A nalysis................................................... 18






v









4 RESULTS AND DISCU SSION ........................................... .......................... 21

Study 1: Frozen Fillets.......................................................... .....21
Effect of Carbon M monoxide on Color....................................... ............... 21
Heme Spectroscopic Analysis ....... ..................... ..................26
Carbon Monoxide Quantification..................... .... .......................... 30
M u scle pH and D rip L oss........................................................................ .. .... 34
Study 2: W hole Frozen Tilapia............... ............................ ............... ...37
Effect of Carbon Monoxide Euthanasia on Color .............................................37
Heme Spectroscopic Analysis ....... ..................... ..................41
Carbon Monoxide Quantification..................... .... .......................... 44

5 SUMMARY AND CONCLUSIONS.....................................................................48

APPENDIX

A F IR ST ST U D Y D A T A .................................................................... .....................5 1

B CO CONCENTRATION IN THE WATER.............................................................53

C PICTURES OF THE TILAPIA FILLETS ............................... ............... 55

LIST OF REFEREN CES ............................................................ .................... 57

B IO G R A PH IC A L SK E TCH ..................................................................... ..................62
















LIST OF TABLES

Table

B-1 Concentration of CO in the water used to euthanize tilapias .................................53

B-2 Time and amount of CO needed to euthanize tilapia in the tank .............................54















LIST OF FIGURES


Figurege

3-1 Recirculating water-CO system for the euthanasia of tilapia.............................. 15

3-2 Experimental design for the first study where tilapia CO treated or untreated and
then filleted, frozen (30 days), defrosted and stored at 40C for 18 days ................19

3-3 Experimental design for the second study where whole tilapia was either
euthanized or left untreated, and then frozen for up to 4 months.............................20

4-1 "Red" muscle side of 100% CO euthanized, control and 100% CO gassed tilapia
fille ts ......................................................................... 2 1

4-2 "White" muscle side of 100% CO euthanized, control and 100% CO gassed
tilap ia fillets ...................................... ............................... ................ 2 2

4-3 Effect of CO treatments and no treatment (control) on the a*-values (redness) of
tilapia fillet red muscle before freezing, after freezing and subsequent storage at
4C for 18 days .........................................................................24

4-4 Effect of CO treatments and no treatment (control) on the L*-values (ligthness)
of tilapia fillet red muscle before freezing, after freezing and subsequent storage
at 4C for 18 days. ......................... .......... .. ....... ... .............. 25

4-5 Effect of CO treatments and no treatment (control) on the a*-values (redness) of
tilapia fillet white muscle before freezing, after freezing and subsequent storage
at 4C for 18 days. ......................... .......... .. ....... ... .............. 26

4-6 Representative spectra for met-hemoglobin (408 nm), oxy-hemoglobin (414 nm)
and carboxy-hem oglobin (418 nm ) ............................................... ............... 27

4-7 Maximum heme peak values for red muscle extracts from euthanized and 100%
CO gassed tilapia fillets and untreated tilapia stored at 40C for 18 days .................27

4-8 Maximum heme peak values for white muscle extracts from euthanized tilapia,
100% CO gassed tilapia fillets and untreated tilapia stored at 40C for 18 days.......30

4-9 Concentration of CO (ppb) in tilapia red muscle after 30 minutes exposure to
100% CO, euthanasia with 100% CO or no treatment................. ........... ......31









4-10 Concentration of CO (ppb) in tilapia white muscle after 30 minutes exposure to
100% CO, euthanasia with 100% CO or no treatment................. ........... ......34

4-11 Change in pH of fillets gassed with 100% CO, fillets from fish euthanized with
100% CO and untreated tilapia fillets stored at 40C for 18 days ............................35

4-12 Thaw loss of gassed fillets (100% CO for 30 min), fillets from euthanized fish
(100% CO) and untreated fillets after 1 month of freezing. Results obtained are
based on the change on weight of the fillets. ........... .............................................36

4-13 Change in drip loss of gassed fillets (100% CO for 30 min), fillets from
euthanized fish (100% CO) and untreated fillets during 18 days of storage at 4C
after thaw ing.............................................................................................. 36

4-14 Effects of euthanasia with 100% CO and no treatment on a*-values of the red
muscle of fresh tilapia and tilapia stored frozen for up to four months. .................37

4-15 Effect of euthanasia with 100% CO and no treatment on a*-values of the white
muscle of fresh tilapia and tilapia stored frozen for up to four months. .................39

4-16 Effect of euthanasia with 100% CO and no treatment on b*-values of the red
muscle of fresh tilapia and tilapia stored frozen for up to four months. .................40

4-17 Effect of euthanasia with 100% CO and no treatment on L*-values of the red
muscle of fresh tilapia and tilapia stored frozen for up to four months. ...............40

4-18 Effect of euthanasia with 100% CO and no treatment on b*-values of the white
muscle of fresh tilapia and tilapia stored frozen for up to four months. ...............41

4-19 Effect of euthanasia with 100% CO and no treatment on L*-values of the white
muscle of fresh tilapia and tilapia stored frozen for up to four months ...................41

4-20 Maximum heme peak values for red muscle of euthanized (100% CO) and
untreated fresh and frozen whole tilapia ...................................... ............... 42

4-21 Maximum heme peak values for white muscle of euthanized (100% CO) and
untreated fresh and frozen whole tilapia. ...................................... ............... 44

4-22 Concentration of CO (ppb) in the red muscle of fresh and frozen untreated and
euthanized (100% CO) tilapia. ...... ............................................................ 45

4-23 Concentration of CO (ppb) in the white muscle of fresh and frozen untreated and
euthanized (100% CO) tilapia. ...... ............................................................ 46

A-1 Effect of CO treatments on the b*-values of the red muscle of tilapia ..................51

A-2 Effect of CO treatments on the b*-values of the white muscle of tilapia. ...............51

A-3 Effect of CO treatments on the L*-values of the white muscle of tilapia................52









B-l Change of CO concentration in the water used for the euthanasia process ............54

C-1 Picture of the "red" muscle side of a tilapia fillet taken by the CMVS ...............55

C-2 Picture of the "white" muscle side of a tilapia fillet taken by the CMVS................56















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

EUTHANASIA OF TILAPIA USING CARBON MONOXIDE FOR COLOR
FIXATION AND COLOR STABILIZATION

By

David Mantilla Torres

December 2005

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

Tilapia is predominately a white muscle fish that has a small amount of dark lateral

muscle. This muscle is an important indicator of fillet freshness, as it changes from a red

color to brown on storage. In order to extend the color of the red muscle many tilapia

processors are treating their fish with gasses containing carbon monoxide (CO).

The most common treatment method is a post-mortem CO gas treatment of fillets.

Currently the application of CO into fish muscle via euthanasia is being performed by a

few tilapia processors. There is a lack of information on how the euthanasia of the fish

with CO will affect fish quality compared to more conventional applications. The

objective of this study was to investigate the color retention and quality of fillets from

euthanized tilapia, compared to a 100% CO post-mortem gas treatment.

Live tilapia was placed in a sealed water tank and was euthanized with 100% CO

flushed into a circulatory water system. Two studies were performed. In the first study,

tilapia was immediately filleted after euthanasia, vacuum packed and frozen (-20C) for









one month, then thawed and kept exposed to air for 18 days at 40C. Fillets from non-CO

treated fish were subjected to a 30 min treatment with 100% CO. Untreated fillets were

used as control. In the second study, CO-euthanized tilapia was gutted, vacuum packed

and frozen whole for up to 4 months. For this study, a set of normally slaughtered tilapia

was used as control. Before each set of analysis (at 0, 2 and 4 months) the fish was

thawed for 24 hours at 40C. The change in muscle color was analyzed with a digital Color

Machine Vision (CMVS) and L*, a* and b* values were recorded. The uptake and

stability of CO in the fish muscle and its binding to heme proteins were analyzed with gas

chromatography (FID) and spectrophotometry, respectively. The effect of the different

treatments on muscle pH and muscle drip loss was also analyzed.

Euthanasia with CO and direct CO treatments on fillets led to a significant (p<0.05)

increase in the red color (a*-values) of the muscle, especially the dark muscle. This

distinctive cherry red color was maintained for a long period of time for both treatments

while a brown color developed for the controls. The color characteristics of the fillets

from euthanized fish were more "natural" than those of the 100% CO treated fillets. The

UV-Vis spectra and the concentration of CO in the muscle confirmed the uptake of CO

by heme proteins and also demonstrated an increase in heme protein stability. CO uptake

was significantly higher in dark muscle compared to white muscle. No significant

differences were found in pH or drip loss among the treatments.

These results suggest that both CO treatments have a positive effect on color and

heme stability, while euthanasia appears to give a more "natural" looking product. In

addition, this new method of processing (i.e., euthanasia) has several advantages such as

shorter processing time and less product handling.














CHAPTER 1
INTRODUCTION

The variety and the high nutritional content of aquatic foods have created great

interest and a high demand for these products. Data provided by the Food and Agriculture

Organization (FAO) in 2002[1] shows that the total fish production has reached its

highest level ever of 94.8 millions tons in 2000. The high demand for these products is

also reflected in an increase of people directly engaged in fisheries and the aquaculture

industry. These industries employed an estimated of 35 million people this decade, 7

million more compared to last decade[l]. The high demand for seafood however has

depleted many fish stock due to overfishing. World population has been growing faster

than the total food fish supply, leading to a decrease in the fish supply per capitall.

About one billion people rely on fish as their main source of protein [2].The global crisis

in capture fisheries and the increasing need for seafood has stimulated the rapid

expansion of aquaculture. Aquaculture offers a predictable and consistent supply of high

quality seafood [3]. According to the FAO, aquaculture production represented 3.9 % of

total fish supply in 1970 and this percentage increased to 27.3% in 2000 [1].

Tilapia and Aquaculture

Tilapia is one of many species that is been aquacultured with great success. Tilapia

has a broad tolerance to harsh environmental conditions. They are more tolerant to high

salinity, high water temperature, low dissolved oxygen and high ammonia concentrations

than most other farmed freshwater fish [4, 5]. Illustrations from Egyptians tombs suggest

that tilapia was one of the first fish species cultured, more than 3000 years ago [5].









Tilapia is also known as Saint Peter's fish and it is believed that tilapia was fed to the

multitudes by Jesus Christ [5]. From 1950 to mid 1970s tilapia species moved from their

native waters in sub-Saharan Africa to Asia and the rest of the world [2, 6]. They were

introduced to different parts of the world for various reasons; for instance, they were used

as bait for tuna which is how they were introduced to Hawaii [7]. Apparently the

introduction of tilapia in the Caribbean, Central and South America was made in order to

reduce mosquitoes through aquatic vegetation control [7].

In 2000 farmed tilapia production surpassed 800 thousand metric tons, second only

to carp [3, 5]. Tilapia has a high acceptance in the United States and is one of the fastest

growing seafood imports into the U.S. along with salmon. According to Knapp [3] in

2002 close to 70 thousand metric tons of fresh and frozen tilapia were imported to the U.

S.

Quality of Seafood

Seafood is a highly perishable commodity since it is very susceptible to microbial

and chemical spoilage which results in economic losses [8]. Its quality declines soon after

harvest and continues once the fish has been processed. The value of the product is highly

influenced by its appearance, in particular its color. Tilapia is predominately a white

muscle fish that has a small amount of dark lateral muscle. This muscle can however be

an important indicator of fillet freshness, as it changes from a red color to brown during

storage. Maintaining the color of the dark muscle during processing, transport, storage

and display is essential and has an influence on the consumer perception of the product.

In order to extend the color of the red muscle and reportedly its shelf life, many tilapia

processors are treating their fish with carbon monoxide (CO) and filtered wood smokes

(FS) containing CO [9]. Several different treatment methods exist, the most common









being exposure of the fillets briefly (<30 min) to CO gas immediately after harvest and

processing while the muscle is still respiring (personal communication, B. Olson,

Clearsmoke Technologies). Euthanasia of fish using CO dissolved in water has been

proposed by Kowalski [10] and is currently performed by some tilapia processors

(personal communication, B. Olson, Clearsmoke Technologies). This new method

incorporates carbon monoxide to the edible muscle of the fish through the respiratory and

circulatory system of the animal. Fillets from fish euthanized with 100% CO have a

distinctive and stable cherry red color characteristic of a CO exposure/treatment.














CHAPTER 2
LITERATURE REVIEW

Tilapia Taxonomy

Tilapia is a generic term used to designate a group of commercially important food

fish that belongs to the Cichlidae family [4]. Tilapias have been classified into three

genera based on the type of care the parents provided to their young [2, 4]. The genera

Oreochromis and Sarotherodon are mouthbrooders. The eggs are fertilized in the nest but

parents immediately pick them up in their mouths and protect them and incubate the

young for several days after hatching [2, 4, 5]. A difference between these two genuses is

that in the Oreochromis genera only the female parents practice the mouthbrooding. This

genus of female mouthbrooding is the most important in aquaculture and it includes the

Nile tilapia (0. niloticus), Mozambique tilapia (0. mossambicus) and blue tilapia (0.

aureus) [2]. The third genus is called Tilapia. These species are nest builders. Eggs are

fertilized, incubated and protected by a brood parent in a pond bottom built-in nest [4, 5].

In the United States, tilapia is grown for commercial purposes manly in Arizona,

California and Florida. In 2000 U.S. production reached its peak of 20 million pounds,

with a value of 30 million dollars. The production decreased to 17.6 million pounds in

2001 but the total value remained the same [11]. However, domestic production of tilapia

is minimal compared to U.S. imports. In 2004 U. S. tilapia imports reached 249 million

pounds 25% more from 2003 and 68% higher than in 2002 [12].









Quality and Shelf Life of Seafood

Immediately after slaughter, the quality of seafood begins to decline. The initial

quality and the type of processing that the fresh product undergoes has a great influence

on the shelf life, rate of spoilage and quality of the final product. Some of the factors that

influence the spoilage rate of fish are muscle pH, temperature, microbial load, microbial

type, amount and type of heme proteins, fat content and fatty acid profile. Refrigeration,

frozen storage and modified atmosphere packaging are some of the most common and

effective methods to extend the shelf life of seafood. However, these methods normally

do not prevent color changes or extend fresh color. In addition, the muscle texture might

be affected by some of these methods.

Heme Proteins and Seafood Quality

Myoglobin and hemoglobin are heme proteins whose main function is the retention

and transport of oxygen for enzymatic reactions [13]. Myoglobin is a globular protein

consisting of a single polypeptide chain. It is found mainly in muscle tissue where it

serves as an intracellular storage site for oxygen [13]. Hemoglobin consists of four

myoglobins like subunits linked together as a tetramer. It is found in the red blood cells

and forms reversible complexes with oxygen in the lung (or gills in the case of fish),

where it transports the bound oxygen through the body to be used in aerobic metabolism

pathways [13].

Heme proteins consist of a globin part and a heme part. The heme portion of the

molecule is responsible for the color of dark muscle. At the center of the heme is an iron

(Fe) atom which possesses six coordination sites. Four of them are occupied by nitrogen

atoms. The fifth coordination site is bound to nitrogen from a histidine, leaving the sixth

site available to complex with electronegative atoms donated by various ligands [13].









Color depends on the oxidation states of the iron atom (Fe2+, Fe3 and Fe4+) in the protein

heme group, and the type of ligands (02, CO, NO etc. ) bound to the iron atom.

Hemoglobin is the most predominant heme protein found in fish white muscle [14,

15]. The presence of blood, thus hemoglobin, in the muscle leads to changes in color and

significant lipid oxidation problems [15, 16]. Soon after death, the heme iron is in the

ferrous (Fe2+) valence state [16]. On the surface of fresh muscle oxygen is bound to the

ferrous iron yielding oxyhemoglobin/myoglobin which gives the muscle a bright red

color. In the interior of the muscle the iron binding site is vacant thus yielding

deoxyhemoglobin/myoglobin which gives the muscle a dark purple color. Heme proteins

are very sensitive to autoxidation, which is enhanced with temperature increase and pH

decrease [17]. Over time, the hemoglobin will oxidize to form methemoglobin (Fe3+).

This occurs when oxygen is released from oxyhemoglobin to form ferric (Fe3+) heme iron

and the superoxide anion (02-) [16]. The formation of methemoglobin gives rise to an

undesirable brown color. To maintain the red color, the formation of methemoglobin

needs to be prevented. This can be achieved by keeping fish at very low temperatures (-

50 to -70C) which is highly impractical and expensive for most species. A more

practical an inexpensive means to achieve color stability is by exposing the muscle to CO

or filtered smoke which contains CO. The CO molecule will combine with the heme

group in hemoglobin and myoglobin to form carboxyhemoglobin/myoglobin and give the

muscle a bright cherry red color. The CO molecule binds very strongly to the heme group

in hemoglobin and myoglobin, over 200 times stronger than 02 [18] and will thus

displace any oxygen present in the heme. This binding leads to a conformational change









in hemoglobin and myoglobin which makes it very resistant to autoxidation and

discoloration [19, 20].

Autoxidation of the heme protein to the met form is also a critical step in lipid

oxidation. Met-Hb/Mb reacts with peroxides and stimulates formation of chemical

compounds capable of initiating and propagating lipid oxidation [14, 21]. Lipid oxidation

is a major cause of quality deterioration of seafoods. It often contributes to the formation

of off odors and flavors, and the deterioration of color and texture. Toxic compounds can

also arise from lipid oxidation [16]. Fish are particularly sensitive and affected by lipid

oxidation due to their highly polyunsaturated fatty acid content [14]. Transition metals

such as iron and copper can also catalyze lipid oxidation in fish muscle [14, 22]. Iron is

the principal transition metal in seafood and a large portion of iron in fish muscle is found

in heme proteins. The amount of iron varies greatly among species. White-muscled fish

have lower concentrations of iron than dark muscled fish [14]. In tilapia, most of the iron

will come from hemoglobin in the white muscle and myoglobin in the dark muscle. Since

CO is expected to retard autoxidation of hemoglobin and myoglobin to the met form it is

possible that this treatment may retard lipid oxidation, and thus extend the shelf life of

tilapia fillets.

Water Holding Capacity and Muscle pH

Water holding capacity (WHC) of foods can be defined as the ability to hold their

own and added water during the application of force, pressing, centrifugation, or heating

[23]. Water holding has a great influence on the quality of the final product primarily

because of the reduced weight loss during cutting and storage and its ability to retain

water during processing [24]. Many factors influence WHC. For example WHC is

exponentially related to the protein content of the muscle, as the protein content









increases, WHC increases. The addition of salts also influences the water binding by

proteins because of their effects on electrostatic interactions. A change in pH affects as

well the conformation of proteins resulting in exposure or burial of the water binding

sites [23], as well as increased osmotic pressure within the muscle when is sufficient

electrostatic repulsion between proteins [20]. WHC reaches its minimum near the

isoelectric points of the major muscle proteins especially myosin (pl~5.4) [25] and rises

on either side of this point. After the death of the animal, the anaerobic glycolytic system

becomes predominant and ATP is gradually depleted and lactic acid is accumulated

leading to a decrease in pH. When the pH is low enough certain critical enzymes are

inhibited and glycolysis ceases [13]. The decrease of pH comes from the hydrolysis of

ATP [13]. A fast decrease in postmortem pH will cause the denaturation of muscle

proteins; the meat produced will be pale soft and exudative (PSE), a condition that is

especially troublesome in pork [13, 26]. This phenomenon also occurs in fish; low pH

weakens the collagen fibers, they break and "gaping" takes place.

Meat quality and WHC is also influenced by behavioral and physiological status of

the animals before slaughter. Stress will exacerbate the drop of pH due to the rising

adrenaline levels [26]. An animal is considered in a state of stress if it is required to make

abnormal or extreme adjustments in its physiology or behavior in order to cope with

adverse aspects of its environment and management [27]. When an animal is under stress,

oxygen is not available in sufficient amounts, and the anaerobic pathway becomes

predominant and glycogen is depleted. This depletion of glycogen results in an onset of

rigor much sooner and in a faster decrease of pH. For example, the time from death to the

onset of rigor in unstressed blue tilapia is 6 hours; on the other hand, the time from death









to the onset of rigor is reduced to only 1 hour in stressed tilapias [28]. In one study, CO2

and live chilling were used on salmon in order to minimize stress and prolong the onset

of rigor [29]. The advantage of having a longer onset of rigor is that processing of the raw

material can start immediately after slaughter; otherwise the process cannot start until

rigor has been resolved. A common practice in Atlantic salmon is to process once rigor

mortis has resolved, which takes 3 to 5 days on ice storage [30]. However, it has been

shown that there is no major difference between processing pre-rigor salmon and post-

rigor salmon [29, 30]. The extension in the onset of rigor is of particular importance to

tilapia producers since tilapia is processed pre-rigor.

The tilapia industry uses many ways to slaughter the animal. The most common is

to transfer tilapia from the pond to a small tank where they are taken one by one and their

branchial artery is severed. They are then transferred back to the small tank until they

bleed to death. It has been suggested that bleeding the fish has no effect on the quality of

the final product since most of the blood in fish is located on the venous side of the

cardiovascular system meaning that by gutting most of the blood will be removed along

with intestines [31]. The transferring of the tilapia from the pond to the small receiving

tank and then the bleeding may thus be an unnecessary and overly stressing slaughtering

method. The euthanasia of tilapia can reduce this stress.

As mentioned above, salmon industry uses CO2 as anesthesia in order to avoid

stress and to comply with the concept of humane slaughter [29]. Carbon monoxide

dissolved in the water where the fish is can also act as an anesthesia for fish. In addition

of the benefit of having lower stressed fish, CO will enhance the color of the filleted fish

and it can help preserve better the final product.









Effects of Carbon Monoxide on Fish Muscle

The bright red color of fish is one of the main attributes that indicates its freshness

and quality. The red color arises primarily from the oxygenated and reduced forms of

heme proteins which have been discussed in previous sections. The oxidation of these

proteins yields a highly undesired brown color. Appearance plays a very important role in

consumer buying decisions. The main objective of the use of carbon monoxide is to

maintain the attractiveness of the red color characteristic of fresh seafood. As written

previously, carbon monoxide binds to heme proteins and forms a very stable complex.

Kristinsson and coworkers [20] reported that carboxyhemoglobin was very stable to

oxidation even at extreme pH values and temperatures. These results suggest that muscle

treated with carbon monoxide may retain its red color even under abusive conditions. The

same authors also demonstrated that the Hb-CO complex had decreased pro-oxidative

activity in a model system and may thus extend product shelf life with respect to

rancidity.

Due to its high price and its high content of red muscle, tuna steaks have been the

focus of study with respect to the use of carbon monoxide for color preservation.

Different studies using different concentration of carbon monoxide and different

exposure times have revealed that there is a significant increase in red color as well as

color stability when CO is used. For example, tuna steaks treated with 99.5% CO gas for

4 hr showed a significant increase in a*-value (redness) compared to untreated tuna [32].

There were no significant differences between L*(lightness) and b* yellownesss) values

between the gas treated tuna and control [32]. Balaban and coworkers [33] reported that

exposure to 4% CO increased a*-value and preserved color stability for up to 12 days in

refrigerated storage. Danyali [34] compared CO with filtered smoke (FS) treatments and









found little difference between the two with respect to color, heme protein oxidation,

lipid oxidation, water holding, and texture. It was however reported that 100% CO led to

a reduced microbial growth, and thus could possibly extend shelf life [34]. Studies also

show that a higher level of CO leads to more color increase and better color stability [34,

35].

Few studies have been published on applying CO or FS treatment on tilapia and

investigating the effect on quality. Ishiwata and coworkers [36] conducted a survey of the

concentration of CO in flesh from a variety of fish sold in a local market and also

exposed tilapia to CO for 60 minutes at room temperature and measured CO

concentration. They reported that the blood colored parts of tilapia exposed to CO were

bright red contrasting with the dark brown color exhibited by the untreated tilapia.

Kristinsson and coworkers [37] reported that tilapia treated with 100% CO did

develop less lipid oxidation products compared to untreated tilapia. Kristinsson and

coworkers [19] later found that isolated tilapia carboxy-hemoglobin had dramatically

increased stability to autoxidation (i.e., browning) under different environmental

conditions compared to oxyhemoglobin, and also had less pro-oxidative activity in a

model linoleic acid emulsion system. Leydon and coworkers [38] recently reported that

commercially obtained previously frozen tilapia fillets treated with filtered wood smoke

had increased color stability, less lipid oxidation and microbial growth than fresh

commercially obtained tilapia fillets. The filtered smoke treated fillets were however

rejected by a trained sensory panel of 3 people [38]. This study did however use

commercially obtained samples, one previously frozen and one fresh, from two different

sources, which makes it difficult to interpret the data.









Carbon Monoxide and Euthanasia

All studies reported on CO and FS application of seafood employ gas treatments

post-mortem on fish steaks or fillets. The practice of euthanizing fish with CO to

incorporate CO into muscle is being performed by the industry on tilapia (personal

communication, B. Olson, Clearsmoke Technologies). Kowalski [10] issued a patent

application in which he described the incorporation of tasteless smoke or carbon

monoxide by means of euthanasia; however no supporting data was found. No other

research studies have been performed to the best of the author's knowledge.

Carbon monoxide is a colorless, odorless and tasteless gas that has about the same

density as air, but sustained inhalation of CO has caused many fatalities due to its

competitive binding to hemoglobin [39]. At levels of 5% CO-Hb fetuses can be affected

and individuals experience many effects. Levels above 10% are life threatening for heart

and lung patients, whereas above 30% CO-Hb healthy individuals are at risk and death

can rapidly occur at levels above 50% [40]. Carbon monoxide poisoning will severely

alter the oxygen transport characteristics of the circulatory system since about 90% of the

oxygen consumed is carried to the tissues by hemoglobin [41]. Carbon monoxide attaches

to hemoglobin similarly to oxygen but with a binding constant that is 210- 270 fold

stronger [40]. Consequently, CO displaces oxygen from hemoglobin. Due to its great

binding affinity, even at low levels of exposure to carbon monoxide, carboxy-hemoglobin

will accumulate. Carbon monoxide reduces both the oxygen-carrying capacity of

circulating blood by direct displacement and the release of the hemoglobin-bound oxygen

to the tissues by shifting the oxygen-hemoglobin dissociation curve [40].

One limitation of killing fish with CO is that it has a relatively low solubility in

water. It was reported by Daniels and Lide [42, 43] that CO has a solubility of 1.774x105









mole fraction solubility in water at 25 C and 101.325 kPa. This solubility however is

very similar to the solubility of oxygen in water (2.293x10-5 mole fraction solubility at

101.325 kPa)[43] This suggests that 02 and CO dissolved volumes are approximately

equal. The volume of 02 or CO dissolved in water is dependent on the partial pressure of

the gas and the temperature. The solubility increases as the temperature decreases.

Research Objectives

The overall objective of this study was to investigate the effect of euthanizing

tilapia by dissolving carbon monoxide directly into the water and comparing to 100% CO

post-mortem gas treatment of fillets and no gas treatment. The effect on color, color

stability, CO uptake and stability, muscle pH and water holding capacity were

investigated for both products stored fresh, as well as frozen and defrosted products.














CHAPTER 3
MATERIALS AND METHODS

Euthanasia of Tilapia with Carbon Monoxide (CO)

Live tilapia were obtained from Evan's Farm in Pierson, FL. The facility produces

hybrids from a genetic cross of predominantly aurea tilapia, Oreochromis niloticus and

Oreochromis mossambicus. All the production is destined for local consumption and

local restaurants.

The tilapia were transferred live to the laboratory and kept in holding tanks prior to

euthanasia. A tank was constructed from transparent Plexiglas (36"x 16"x 12") where

tilapias were euthanized with CO saturated water (Figure 3-1). 100% CO was flushed

into a circulatory system which allows the water to saturate with the gas (Figure B-l). CO

was introduced to the animal muscle tissue by its respiratory and circulatory systems. All

the experiments were performed at ambient temperature (21C). Tilapias were maintained

in the euthanizing tank as much time as needed until they all were confirmed dead by

visual inspection. On average, 31 minutes were needed for the completion of the

euthanasia process (Table B-2). During every trial, thirteen tilapias were euthanized. To

flush the remaining CO out of the tank, air was flushed in and the CO converted to CO2

by passing it through a Hopcalite catalyst tube (Figure 3-1).

After euthanasia, two studies were performed. In the first study these tilapia was

immediately filleted, vacuum packed in high density polyethylene (HDPE) bags and

frozen at -200C. After 1 month fillets were thawed at 40C for 24 hours and stored

aerobically at 40C for 18 days. In order to compare the effectiveness of euthanasia with









100% CO flushed directly into the water, 100% CO post-mortem gas treated and control

fillets were stored under the same conditions for the same amount of time (Figure 3-2).

In the second study, tilapia were only gutted, placed in HDPE bags, vacuum packed

and stored at -200C for up to 4 months. A set of normally slaughtered (no CO) fish was

also gutted, vacuum packed and stored at -20 C. The latter was used as control. Three

sampling points were chosen; 0, 2 and 4 months. The whole fish was thawed for 24 hours

at 4C, then filleted and analyzed (Figure 3-3)


Figure 3-1. Recirculating water-CO system for the euthanasia of tilapia.









100% CO Post-Mortem Gas Fillets Treatment of Tilapia

Live tilapia were killed with ice and by bleeding. The 100% CO post-mortem gas

treatment can be applied only to the first study since the later study used whole fish. After

death, the fish was filleted immediately before rigor and split in two groups: one

subjected to CO gas treatment and the other (control) subjected to no treatment. Fillets

were placed in a gas tight stainless steel drum on thinly netted stainless steel shelves and

100% CO applied for 30 min at 40C. After gas treatment the CO was converted to CO2

by passing it through a Hopcalite catalyst tube (same one as in Figure 3-1). Untreated,

CO-treated and euthanized fillets were then placed in HPDE bags, vacuum packed and

frozen for one month. Fillets were then defrosted at 40C and kept at 40C for 18 days and

analyzed every 3 days.

Color Analysis

A digital Color Machine Vision System (CMVS) was used following the

procedures outlined by Balaban and coworkers [33] for detailed color analysis of RGB

and L*-(lightness), a*-(redness), and b*-(yellowness) values along with hue values and

identifying important color blocks for each treatment. The L*, a* and b*-values were

reported. The color analysis was done separately for the white and the dark lateral

muscle. The front side of the fillets contains the dark muscle which was used for the

analysis of the red muscle. The reverse side which contains practically no red muscle was

used for the analysis of the white muscle.

Quantification of CO in Fish Muscle

The method from Miyazaki and coworkers [44] was used to determine the

concentration of CO in white and dark muscle separately. Briefly, 6 g of muscle were

minced and introduced into a 60 ml head space bottle. 3 drops of 1-octanol (antifoaming









agent) and 12 ml of 10% sulfuric acid were added. The sulfuric acid denatures heme

proteins which causes them to release CO. The mixture was shaken for 10 sec, and then

incubated for 5 minutes at 400C. After incubation, the tubes were shaken at room

temperature for 15 minutes and 100 [il of the head space gas was injected into an Agilent

gas chromatography system equipped with a stainless steel Poropak Q column (3.17 mm

i.d. x 1.82 m; 80-100 mesh) a methanizer (to convert CO to CH4) and a FID detector.

Helium was used as the carrier gas with a flow rate of 29.7 mL/min. The injection port,

column, methanizer and detector temperatures were maintained at 1000C, 35C, 320C

and 250C respectively. The reducing gas was hydrogen with a flow rate of 40.0 L/min.

The retention time and area of the CH4 peak were compared to those obtained with a

calibration CO gas. CO levels were then calculated based on a standard curve constructed

by injecting different known levels of 100% CO. For the first study, three fillets were

retrieved from the cold room (4C) and analyzed on different days after thawing (day 0,

2, 4, 6, 10, 14, 18). These days were chosen due to the rapid decrease of CO

concentration in the muscle during the first days of exposure to normal atmospheric

conditions [36].

Heme Protein Extraction and Spectroscopic Analysis

Spectroscopic analysis can reveal how CO is taken up into the muscle and how

stable it is bound to heme proteins on storage. The heme extraction method of Huo and

Kristinsson [45] was used. A 10 g sample of tilapia white and red muscle (separately)

were mixed with 100 ml of 20 mM Na2HPO4 buffer (pH 8), followed by homogenization

with a Ultra-Turrax T19 homogenizer at lowest speed. The sample was filtered through

Whatman #1 filter paper at 40C followed by centrifugation at 3000x g. All these steps

were done in the cold room to avoid protein denaturation and loss of CO from the heme.









The UV-visible absorbance spectra of the collected supernatant were then read from 350-

700 nm. The max wavelength of the heme peak of CO-Hb/Mb is 419 nm, 414 nm for

oxyHb/Mb and 408 and below for metHb/Mb.

Muscle pH

The stress and its influence on the animal were followed by measuring the pH of

the muscle. A sample of 5 g red and white muscle was mixed with 45 mL of deionized

water at 40C and the mixture homogenized with an Ultra-Turrax T19 homogenizer, on

ice. The pH was then measured using a Ross Sureflow biological epoxy probe (Thermo

Orion, Beverly, MA) attached to a pH meter (Denver Instruments, Fort Collins, CO).

The pH meter was calibrated using pH 2, 4, 7, and 10 buffers at 40C.

Drip Loss

Drip loss analysis was performed on fillets from the first study only. Six fillets

from each experimental group (euthanized, gassed and control) were analyzed for drip

loss for 18 days. Measurements were acquired every third day for 18 days (day 0, 3, 6, 9,

12, 15 and 18). The fillets were kept in open bags during the 18 days of measurements to

allow for air to access the fillets. Each fillet was very delicately blotted for any loose

liquid on its surface before its weight was recorded. The bag was meticulously cleaned

and dried before the fillet was replaced in the bag. Thaw loss and drip loss were

calculated based on the weight difference between the different storage days.

Statistical Analysis

Each analysis was conducted in a minimum of triplicate samples. Analysis of

variance (ANOVA) and t-test were used to determine significant differences between

treatments and among treatments. The Statistical Analysis Software (SAS) and Microsoft

Excel were used for the treatment of the data.










I Live tilapia


a$--
Treatment 1: Euthanized tilapia
Euthanized tilapia in water saturated
with CO


Killed with ice water and
bleeding


Filleted pre-rigor


Treatment 2: Gassed fillets Treatment 3:
Gassed fillets with 100% CO Control
for 30 min No gas treatment






Vacuum Pack and Freeze fillets for 30
days @ -20C



Fillets thawed @ 4C maintained for 18
days in high air permeability bags.
*Analysis every 3 days


1. Color analysis
1.1. Machine vision system (calibrated using Minolta Colorimeter)
2. Quantification of CO in muscle
2.1. Red and white muscle
3. Heme protein extraction and spectroscopic analysis
3.1. Red and white muscle
4. Drip loss
5. Muscle pH
Figure 3-2. Experimental design for the first study where tilapia CO treated or untreated
and then filleted, frozen (30 days), defrosted and stored at 40C for 18 days.


^^^^^^-^^^^^_^^^^^^-^^^^^_


Fillete
SFlet










I Live tilapia


Treatment 1: Euthanized tilapia
Euthanized tilapia in water saturated with
CO



Freeze Whole Fish
(gutted) (vacuum packed)


Bleeding


Freeze Whole Fish
(gutted) (vacuum packed)


Analysis at time 0, 2 and
4 months. 3 sampling pts.


1. Color analysis
1.1. Machine Vision system (calibrated using Minolta Colorimeter)
2. Quantification of CO in muscle
2.1. Red and white muscle
3. Heme protein extraction and spectroscopic analysis
3.1. Red and white muscle
4. Muscle pH

Figure 3-3. Experimental design for the second study where whole tilapia was either
euthanized or left untreated, and then frozen for up to 4 months.


-L














CHAPTER 4
RESULTS AND DISCUSSION

Study 1: Frozen Fillets

Effect of Carbon Monoxide on Color

The main reason behind the introduction of carbon monoxide in fish processing is

to preserve and enhance the red color of the muscle. A Color Machine Vision System

(CMVS) was used to analyze the color and color change during the study. The CMVS

was chosen because it analyzes every pixel of the sample compared to other color

methods that read only a small part of the sample. Other methods such as the Minolta

color meter may be good for fillets that are uniform in color. However, tilapia has a red

lateral muscle that gives the fillets an uneven color.





















Figure 4-1. "Red" muscle side of 100% CO euthanized, control and 100% CO gassed
tilapia fillets.






























figure 4-2. -wnite" muscle side ot lUU/o CU eutmamzea, control ana iuu/o LU gassea
tilapia fillets

Fillets from fish euthanized with 100% CO have a distinctive and stable cherry red

color characteristic of a CO exposure/treatment. Figures 4-1 and 4-2 show sample fillets

of each treatment for the "red" muscle side and the "white" muscle side of a tilapia fillet.

"Red" muscle was defined as the side of the fillets that includes the red muscle (Figure 4-

1). The other side was considered as "white" muscle (Figure 4-2).

The degree of redness (a*-values) is the most important indicator of quality and

freshness in species rich in red muscle such as tilapia, Spanish mackerel, mahi-mahi, tuna

and swordfish [33, 34, 46]. The effect of the two treatments was very noticeable

compared to the control fillets (Figures 4-1 and 4-3). Figure 4-3 shows the increase of a*-

values in the red muscle of the euthanized and the post-mortem gas treated fillets and the

control. The 100% CO post-mortem gassing method of fillets significantly increased

(p<0.05) a*-values from 18.36 to 23.63. The a*-values from the euthanized fillets also

increased significantly (p<0.05) from 17.24 to 27.48. The a*-values of the fillets from the









euthanized fish remained significantly higher (p<0.05) until day 9 compared to both

treatments. From day 12 to day 15 euthanized a*-values were still significantly higher

than control a*-values (p<0.05) but no significant differences were found among

euthanized and gassed post-mortem fillets. At day 18 there was no significant difference

among any of the three treatments (p<0.05). The post-mortem gassing of the fillets

maintained significantly higher a*-values (p<0.05) compared to the control samples until

day 6.

The frozen storage negatively affected the a*values of the control and the post-

mortem gassed fillets (p<0.05). Freezing did not affect a*-values of the euthanized

samples. Control samples were affected the most by the freezing and thawing as there

was a significant decrease (p<0.05) in a*-values from 17.245 to 9.50. Gassed fillets were

also affected by the freezing, but to a lesser extent. Euthanized fillets had significantly

better stability (p<0.05) during the freezing period, being 8.46 and 17.89 points over CO

treated fillets and untreated fillets respectively. The a*-values for the 100% CO post-

mortem gas treatment were also significantly increased (p<0.05) compared to the control

values. After 6 days at 40C there was a significant drop (p<0.05) in a*-values for all

treatments. The decrease in a*-values at day 6 corresponded to a decrease in the heme

peak wavelength and also in the CO concentration (see Figures 4-6 and 4-8 later)

suggesting that the CO is escaping from the hemoglobin and myoglobin (discussed

below). These results are consistent with results on CO-treated tuna, mahi mahi and

Spanish mackerel, which all show an increase in a*-value on treatment, but a gradual

decline after defrosting [33-35, 46].













30 T

25

20

15


5

Before After 0 3 6 9 12 15 18
Treatment Treament
r Days
Freezing D
SControl 1 100% CO Euthanized 0100 % CO Gassed

Figure 4-3. Effect of CO treatments and no treatment (control) on the a*-values (redness)
of tilapia fillet red muscle before freezing, after freezing and subsequent
storage at 40C for 18 days.

Control and gassed fillets' L* (ligthness) values decreased significantly (p<0.05)

during the frozen storage. On the other hand, L* values from the euthanized fillets were

not significantly (p<0.05) affected until day 6 (Figure 4-4). These results suggest that the

euthanasia with 100% CO yielded more "natural" fresh looking fillets than the post-

mortem gassing.

Both of the treatments did not have a significant effect on b*(yellowness) values

until day 18 where control values increased significantly compare to the euthanized

samples (Figure A-i). This slight increase can be attributed to the oxidation of heme

proteins and possibly also to lipid oxidation[47]. The reduction in the a*-values for the

control corresponds to met-hemoglobin [18], which can produce a brown-yellowish

appearance to red muscle, which would explain the increase in b*-value.
















40





Before After 0 3 6 9 12 15 18
Treatment Treament Days
SControl l Euthanized l Gassed

Figure 4-4. Effect of CO treatments and no treatment (control) on the L*-values
(ligthness) of tilapia fillet red muscle before freezing, after freezing and
subsequent storage at 40C for 18 days.

White muscle is the predominant muscle type in tilapia. White muscle has

significantly lower amounts of heme proteins than red muscle [48]. Although levels of

heme protein are less in the white muscle, its red color was still significantly (p<0.05)

influenced by both of the CO treatments (Figure 4-5). Fillets from tilapia euthanized with

100% CO had a pinkish tone to the white muscle which was reflected in a significant

(p<0.05) increase in a*-values for the entire study (Figure 4-5). 100% CO post-mortem

treatment produced significantly (p< 0.05) higher a*-values to the white muscle until day

6. From day 9 to the end of the study no significant differences were found among post-

mortem gas treatment and control fillets.

After both CO treatments, euthanized fillets had a*-values significantly (p<0.05)

higher than a*-values from post-mortem gassed fillets. This difference was still

significant (p<0.05) after the frozen storage and thawing process (at day 0). At days 3 and

6 and after day 15 no significant (p<0.05) differences were found. During day 9 and 12

significantly (p<0.05) higher a*-values were again found. These results suggest higher











stability of a*-values for the euthanized process compared to the 100% CO post-mortem

treatment.

The white muscle side of the tilapia fillet contains a small central line of red muscle

(Figure 4-2) that increased the overall a*-values of the white muscle and gave a higher

standard deviations. In addition, the lack of bleeding of the euthanized fillets may also

have influenced the results since more blood would leave more hemoglobin in the tissue

and thus more CO binding and a larger effect on red color. Overall, the results seen for

a*-values in the white muscle followed similar trends as those seen for the dark muscle.


30

25

20

"5 15
5


5

0


Before After 0 3 6 9 12 15 18
Treatment Treament I Days
Freezing U Control B 100% CO Euthanized 0 100% CO Gassed

Figure 4-5. Effect of CO treatments and no treatment (control) on the a*-values (redness)
of tilapia fillet white muscle before freezing, after freezing and subsequent
storage at 40C for 18 days.

Heme Spectroscopic Analysis

Both of the treatments influenced the red color of the fillets according to the

CMVS. This increase in color and color stability comes from the binding of CO to heme

proteins. The complex carboxy-hemoglobin has been proven by Kristinsson and

coworkers [19, 20] to be more stable against oxidation compared to oxy-hemoglobin. The

oxidation of heme proteins is the main reason why the red color decreases over time and

changes to brown [13]. Figure 4-6 shows a representative spectrum of all three oxidation


T



TTT T T





7 7











states at which hemoglobin/myoglobin is found during these experiments (i.e., Met, Oxy

and Carboxy).


1.2

1

0.8
0)
0.6

0 0.4
C.Q

0.2

0


385 405 425 445 465
Wavelength (nm)
Carboxy-Hemoglobin -o-Oxy-Hemoglobin --Met-Hemoglobin

Figure 4-6. Representative spectra for met-hemoglobin (408 nm), oxy-hemoglobin (414
nm) and carboxy-hemoglobin (418 nm)



420

415 r

410

405

400

395
Before After 0 3 6 9 12 15 18
Treatment Treatment Days
Freezing -4-100% CO Gassed -- Control -A-100% CO Euthanized


Figure 4-7. Maximum heme peak values for red muscle extracts from euthanized and
100% CO gassed tilapia fillets and untreated tilapia stored at 4C for 18 days

From Figures 4-7 it can be seen that the gas treatment as well as the euthanasia

process led to a substantial increase in the heme peak wavelength, which indicates that

CO is being bound to the heme proteins. A wavelength of 418 nm suggests maximum


414 nm



Sarboxy
408 nm Met |









binding. Before treatment heme peaks wavelength of -415 nm were found for all the

treatments. This reading corresponded to mostly oxy-hemoglobin. After the gas treatment

the heme peak wavelengths reached 416.5 nm on average while a reading of 417.3 nm

was observed from the heme extracted from the euthanized tilapia. These UV-Vis spectra

of the heme proteins extracted from the muscle of the gassed and euthanized tilapia

demonstrated that there was a mixture of oxy-hemoglobin and carboxy-hemoglobin

present in the extracts. Similar results were found by Danyali [34] and Garner [46] where

muscle from tuna and Spanish mackerel, respectively, were exposed to different

concentrations of CO. These high heme peak wavelengths were maintained through the

frozen storage proving that the CO was still bound to the heme proteins what explains the

high a*-values reported in Figure 4-3. On the other hand, the heme peak for the control

decreased after freezing to about 408 nm which shows that it was already oxidized. This

explains the decreased in a*-values obtained after freezing in the control samples (Figure

4-3). The significantly higher wavelength peaks seen for both CO treatments after 1

month of frozen storage compared to the control shows how the heme proteins are

significantly stabilized when they are bound to CO. A period of 30 minutes of direct

exposure of fillets to CO or euthanasia of fish with CO was therefore enough to

significantly stabilize the heme proteins during freezing as well as after thawing. There

was however a difference in the CO binding between the two CO treatments. The gassed

fillets appeared to have more bound CO since wavelength remained mostly constant

during the cold storage, while the heme peak wavelength decreased on storage at 40C for

the euthanized samples. This decrease suggested that the euthanized samples were

loosing CO. This fact is reflected in the a*-values, where a sudden decrease was noticed









at day 6 (Figures 4-3 and 4-5). The heme peak wavelength did however increase after this

decrease, suggesting that CO was still present in the muscle and was rebinding to the

heme. It is interesting to note that euthanized fish had higher a*-values than the gassed

fillets, but show less binding of CO to the heme according to the UV-vis analysis. This

was unexpected as the redness of fish muscle is dictated by the level of CO binding to the

heme proteins, and thus an opposite result would have been expected. It is possible that

during extraction some of the CO may have been lost (e.g. during homogenization which

may have denatured the heme proteins) thus giving lower values. However, all samples

were extracted identically. Another possibility is that the euthanized fish had lower

overall CO levels in the muscle compared to the gassed fish, and therefore during

homogenization, some CO could have been lost and since it did not have the excess CO

present in the gassed fillets, the CO lost did not get replenished. This is supported by the

data on muscle CO levels presented below (Figures 4-9 and 4-10).

Similar results were found for the white muscle (Figure 4-8). Heme proteins

extracted from control fillets had heme peak wavelengths below 408 nm implying that

heme proteins were already oxidized and met-hemoglobin was formed. For some samples

heme proteins were below detection limits. This is because the level of heme proteins in

white muscle is very low. Higher heme peaks were observed for the treated samples

compared to the control. Some of the wavelength values for the euthanized fish suggest

that the heme proteins were in part oxidized at days 3, 6, and 9. This is interesting, as the

a*-values for the euthanized fish white muscle were higher than those for the gassed

fillets, and thus one would have expected higher wavelength for the euthanized fish. The

same contradiction was seen for the red muscle. The wavelength then rose again,










suggesting rebinding to CO, similar to what was observed with the red muscle. However,

due to the high a*-values maintained by the treated fillets compared with the control

fillets and the lack of browning, it can be concluded that these wavelengths may come

from some free blood that was present at the surface of the fillets. The free surface blood

is expected to oxidize faster than blood in the fillets.


420


415


410


405


400
Before After 0 3 Days 6 9 12 15 18
Treatment Treatment
Freezing 0100% CO Gassed 0 Control E 100% CO Euthanized

Figure 4-8. Maximum heme peak values for white muscle extracts from euthanized
tilapia, 100% CO gassed tilapia fillets and untreated tilapia stored at 4C for
18 days.

The heme spectroscopic analysis revealed that treated products with CO can be

differentiated from the untreated ones just by analyzing its UV-Vis spectra. Untreated

product should not have a heme peak wavelength higher than 414 nm, while treated

products will have heme peak wavelengths higher than 414 nm revealing the carboxy-

hemoglobin/myoglobin complex.

Carbon Monoxide Quantification

The concentration of CO in the muscle was quantified using a GC equipped with a

flame ionization detector (GC-FID). Few methods are available to measure CO in fish

muscle. The Japanese health authority uses a method called the "A method" [36]. This










method however requires large amounts of muscle for analysis and results have

demonstrated that it lacks sensitivity[45]. Due to the small amount of red muscle present

in tilapia fillets a different method was chosen [44] which recovered more CO from the

muscle than the "A method".



8000




5500

0

3000




500
Before After 0 2 4 6 10 14 18
Treatment Treatment Days
Freezing Control -- 100% CO Gassed -*- 100 % CO Euthanized

Figure 4-9. Concentration of CO (ppb) in tilapia red muscle after 30 minutes exposure to
100% CO, euthanasia with 100% CO or no treatment.

In Figure 4-9 it can be clearly seen that there was a difference in CO concentration

between the treatments after the freezing and thawing process. Control samples had low

levels of CO (1408 ppb). It was expected to find some CO in the muscle since

endogenous CO is produced during the metabolism of protoheme [36, 49]. This small

concentration was maintained for about 10 days, but then increased significantly (p<0.05)

in day 14. An increase in CO concentration on extended storage has been reported

previously, and is one of the indicators used by the Japanese health authorities that fish

has not been treated [36]. If fish has been treated with CO, the level is expected to decline

which was the case for the CO treated tilapia in this study.









After treatment a significant (p<0.05) increase in CO concentration was noted for

both euthanized and gassed samples (Figure 4-9). CO concentration increased to 6237

ppb and 7020 ppb for the euthanized and the gassed treated fillets respectively. After

thawing, initial concentration of CO in the post-mortem gassed fillets remained

considerably higher (-6380 ppb) than the level for the euthanized fillets (4712 ppb), but

then dropped suddenly after day 4 of storage. The additional amount of CO present in the

gassed samples is likely CO trapped in the muscle and thus was not bound to the heme

proteins. This is very possible, considering that the initial heme peak wavelengths were

similar for both treatments. Thus, the data suggest similar CO saturation of heme

proteins, which means the higher level of CO in the CO gassed fillets is due to additional

CO trapped in the extracellular matrix. The gas treatment was applied post-mortem by

exposing the fillets to 100% CO for 30 min. The exposure is based on surface contact

between the muscle and the CO and therefore it is very possible that CO can be trapped

in the extracellular muscle matrix. Davenport and coworkers [39] subjected tuna to 100%

CO treatment and found that much of the CO is trapped in the extracellular matrix of the

muscle, which supports the results seen here with tilapia. Figure 4-9 shows that this

excess CO over the CO for the euthanized fish remained in the muscle for four days and

then most of it left the muscle since euthanized fish and gassed fillets had similar values

at days 6 and beyond (p< 0.05). As discussed before, this additional CO in the muscle

may be the reason the CO treated fillets had higher heme peak absorbance (i.e.,

suggesting more CO saturation) throughout the experiment, since extracellular CO would

have replenished CO lost from the heme proteins during extraction. This however would









also suggest that the CO gassed fillets should have had higher a*-values, which was not

the case.

The euthanized fillets had higher CO concentration than the control but as

discussed above, lower levels than the gassed fillets. Since the CO was transferred to the

edible muscle tissue through the respiratory and circulatory system it can be inferred that

all the CO present in the muscle was bound to the heme proteins. The amount of heme

proteins present in the muscle dictates the amount of CO that will be bound. At day 6

both CO treatments had declined to similar CO levels. However, even at the end of the 18

days storage period, both treatments still had significantly (p < 0.05) higher levels than

the control.

Figure 4-10 represents the amount of CO found in white muscle. The difference

between red and white muscle of the two CO treatments is -2-2.8 fold. Previous work by

Miyazaki and coworkers [44] has also shown that CO levels are higher in red muscle

compared to white muscle. This suggests that the heme proteins are the main source of

bound CO in the muscle since white muscle contains significantly less heme proteins

than red muscle. The white muscle of untreated control has similar levels of CO red

muscle (943 ppb). Analysis of the CO levels in white muscle of the treated fish confirms

that there is entrapment of CO in the extracellular matrix of the muscle when it was

treated post-mortem with 100% CO. The starting values of CO for the gassed fillets were

significantly higher than the values for the euthanized samples. The difference between

the two is close to the difference seen in the red muscle. This difference is therefore very

likely explained by additional CO trapped in the muscle, and not bound to heme proteins.

The concentration of CO dropped more suddenly in the gassed white muscle than it did in










the red muscle. The higher heme content in the red muscle may have aided in the

stabilization of the CO in the muscle during the first days of storage, while the lower

level in the white muscle caused CO to be released sooner from the muscle. After 6 days

of storage the values stabilized and were similar to those of the euthanized fish.





4500




S2500





500
Before After 0 2 4 6 10 14 18
Treatment Treatment Days
Freezing -- Control -- 100% CO Euthanized -- 100% CO Gassed

Figure 4-10. Concentration of CO (ppb) in tilapia white muscle after 30 minutes exposure
to 100% CO, euthanasia with 100% CO or no treatment.


Muscle pH and Drip Loss

The amount of stress at which the animal is put under before being slaughter can

have a great influence in its final pH and thus in its water holding capacity [26]. As the

muscle pH decreased post-mortem, the number of negative charges decreases on the

muscle proteins and they are moved closer to their isoelectric point. Muscle has its lowest

water-holding capacity at the isoelectric point of the myofibrillar proteins [50]. It was

expected that the euthanasia of tilapia would be less traumatic for the animal and thus a

lower drop in pH and higher water holding capacity would be obtained. The pH data does

not indicate that there was any significant (p< 0.05) difference between the different










treatments. However, Figure 4-11 shows that fillets from control and euthanized tilapias

had a smaller change in pH compared to the gassed fillets. Nevertheless it cannot be

inferred that the CO gassing of fillets represented a more stressed process, since the fish

used for the gassed fillets were slaughtered in the same way as the control and the pH

should therefore have been similar. It can be assumed the level of stress at which the

tilapia experienced from every treatment was different from the beginning due to the

handling of the live animal. Tilapia were transported live in small coolers from a tilapia

farm located 2 hours away from the laboratory. According to Terlouw [26] an animal is

under stress when it is confronted with a potentially threatening situation. It is possible

that the tilapia used in the CO fillet gassing experiments were under more stress than the

other treatments before slaughtering, thus explaining the lower pH values obtained.



1.5

1.2

c0.9

S0.6

L=0.3

0"

-0.3 i
Before After 0 3 6 9 12 15 18
Treatment Treatment Days

Freezing --pH euthanized ---pH gassed -A-pH control

Figure 4-11. Change in pH of fillets gassed with 100% CO, fillets from fish euthanized
with 100% CO and untreated tilapia fillets stored at 40C for 18 days.

Water lost on thawing (thaw loss) was also recorded for the fillets from the three

treatments. Even though statistical analysis showed no significant difference among the

three treatments, Figure 4-12 shows that the euthanized samples had the highest thaw loss

after 1 month of frozen storage. Thawed fillets lost 2.8% of their original weight. The










gassed samples had the lowest thaw loss with a change of 2% from their weight before

freezing. These results are contradictory to the pH results, i.e., fillets from the control and

euthanized fish had more stable and higher pH, but still higher thaw loss than gassed

fillets which had a lower pH, which is contrary to what was expected.


2.5

. 2
a,
=M 1.5

0 1
5M


* Control 0 100% CO Euthanized 2 100% CO Gassed


Figure 4-12. Thaw loss of gassed fillets (100% CO for 30 min), fillets from euthanized
fish (100% CO) and untreated fillets after 1 month of freezing. Results
obtained are based on the change on weight of the fillets.


Day 0


Day 3


Day 6


Day 9


Day 12 Day 15


Day 18


--- Control ---100% CO Euthanized -A-100% CO Gassed


Figure 4-13. Change in drip loss of gassed fillets (100% CO for 30 min), fillets from
euthanized fish (100% CO) and untreated fillets during 18 days of storage at
4C after thawing.


,--,-,-,-,- T










There were no significant differences in the amount drip loss among the three

treatments during the 18 day storage at 40C after thawing (Figures 4-12). Apparently the

differences in pH did not affect the drip loss of the fillets.


Study 2: Whole Frozen Tilapia

Effect of Carbon Monoxide Euthanasia on Color

Color was analyzed for fillets obtained from fresh control and euthanized tilapia,

and also after defrosting the frozen whole fish after 2 and 4 months (Figure 4-14). The

fresh data was obtained to compare the color of the muscle at the fresh state to the color

of the muscle after being subjected to frozen storage. The effect of euthanizing the fish on

the a*-values can be appreciated as the a*-values of the fillets from the euthanized fish

were significant higher (p<0.05) than the a*-values of the control.


35 -
30
25



10 -




Fresh 2 Months 4 Months
SControl I 100 % CO Euthanized

Figure 4-14. Effects of euthanasia with 100% CO and no treatment on a*-values of the
red muscle of fresh tilapia and tilapia stored frozen for up to four months.

The highest a*-values for the control samples were obtained for the fresh fish. At

the fresh state, the fillets were exposed to air after filleting which leads to oxygen binding

to hemoglobin which gives the bright red color characteristic of oxy-hemoglobin. This is









confirmed by the heme peaks wavelengths obtained for the control samples (see next

section). The control a*-values obtained at this point are representative and can be used

as reference for a*-values of a fresh tilapia fillet. In addition, the difference between the

euthanized samples and the control are less significant at this point than at the other

samples points. The euthanized a*-values were however significantly higher than the a*-

values of the control. The increase in redness is explained by the formation of the

carboxy-hemoglobin complex, as discussed before.

The control a*-values progressively decreased after freezing. However there was

no significant (p>0.05) difference between the fresh and after 2 months a*-values.

Meaning that storing the fish whole in a frozen state preserved the color of the fillets.

There was a significant decrease (p<0.05) in a*-values after 2 months of frozen storage.

This decrease comes from the oxidation of the heme proteins where the characteristic

bright red color of the red muscle of the fresh fillets gradually turns into a more brownish

color. The a*-values of the fillets from the euthanized fish were maintained significantly

(p<0.05) higher throughout the study compared to the control. It can be seen from Figure

4-13 that the euthanized a*-values were significantly (p<0.05) higher at all times than the

control values. After 4 months of frozen storage, the euthanized a*-values were not

significantly (p>0.05) different from the initial control values, underlining how stable the

a*-values became after the euthanasia with 100% CO. This stability came from the strong

binding of CO to the heme proteins. Interestingly an increase in a*-values was observed

after 2 months of frozen storage. This agrees with the data shown in the previous chapter

on the fillets. Danyali [34] also noted an increase in a*-values when CO treated yellowfin

tuna steaks were subjected to 30 days of storage at -250C. The increase in red color of the









tilapia muscle corresponded to an increase in the concentration of CO in the muscle (see

later). These results thus suggest that the more CO is bound to the muscle the higher a*-

values would be obtained.

Similar results were found for the white muscle where fillets from the euthanized

fish also show significantly (p<0.05) higher a*-values than control (Figure 4-15).

Furthermore, a*-values for the euthanized fish were maintained significantly (p<0.05)

higher at all times compared to the control. After four months of frozen storage, a*-

values of the white muscle from the euthanized fish were similar (no significant

difference (p<0.05)) to the initial a*-values, thus mirroring that seen for the red muscle.

The control a*-values on the other hand decreased significantly after 4 months of frozen

storage.


30

25

~, 20

S15

S10 -

5

0
Fresh 2 Months 4 Months
SControl 0 100% CO Euthanized

Figure 4-15. Effect of euthanasia with 100% CO and no treatment on a*-values of the
white muscle of fresh tilapia and tilapia stored frozen for up to four months.

The euthanasia process did not have an influence on b* or L*-values. No

significant differences (p>0.05) were found for the euthanized fillets when compared to

the control fillets (Figures 4-16 to 4-19).











Fresh 2 Months 4 Months
Control S 100% CO Euthanized
Figure 4-16. Effect of euthanasia with 100% CO and no treatment on b*-values of the red
muscle of fresh tilapia and tilapia stored frozen for up to four months.


i


"Ii


Fresh 2 Months 4 Months
SControl S 100% CO Euthanized
Figure 4-17. Effect of euthanasia with 100% CO and no treatment on L*-values of the red
muscle of fresh tilapia and tilapia stored frozen for up to four months.


L


L







41



20
18
16 T
14 2 Months 4 Months
12 -





Control 100% CO Euthanized
8 -
6-
4-
2 -
0




80 i---------------------
Fresh 2 Months 4 Months

SControl 100% CO Euthanized


Figure 4-18. Effect of euthanasia with 100% CO and no treatment on b*-values of the
white muscle of fresh tilapia and tilapia stored frozen for up to four months.


80

60

4 4

2O
0

Fresh 2 Months 4 Months
U Control 3 100% CO Euthanized

Figure 4-19. Effect of euthanasia with 100% CO and no treatment on L*-values of the
white muscle of fresh tilapia and tilapia stored frozen for up to four months


Heme Spectroscopic Analysis

The red color stability throughout the study is caused by the stability of the heme

proteins. If the formation of the met-form of the heme proteins is avoided, the red color

will be maintained [19]. As shown in the first study on fresh and frozen fillets, the red

muscle is where most of the CO is bound due to its high content of heme proteins. Figure

4-18 shows the heme peaks wavelengths obtained of red muscle extracts for the three










samples points. The difference in heme protein ligand binding and stability is clearly

different between the euthanized and untreated (control) fish.


421
419
E 417--
415
^ 413
411
cc 409
407
405
Fresh 2 months 4 months

-*-Control --100% CO Euthanized

Figure 4-20. Maximum heme peak values for red muscle of euthanized (100% CO) and
untreated fresh and frozen whole tilapia

A heme peak wavelength of 415 nm was seen for the fresh control signifying the

presence of oxy-hemoglobin/myoglobin (Figure 4-20). This peak was expected since the

fillets were exposed to air during the filleting and skinning process (which was part of the

sample preparation) which would have allowed oxygen from the air to bind to

hemoglobin/hemoglobin. The coupling of oxygen with hemoglobin/myoglobin gives a

bright red color, explaining the high a*- values obtained for the control in the fresh state.

The heme peak wavelengths decreased on freezing, suggesting oxidation of the heme

proteins as well as loss of oxygen, which agrees well with red color results (Figures 4-20

and 4-14). At the end of the study, a heme peak wavelength of 409 nm was found for the

control samples implying that the heme proteins were significantly oxidized and thus

met-hemoglobin/myoglobin had formed. The presence of the met- form was also evident

in the fillets. As presented and discussed in the section before, a brown color had

replaced the bright red color of the fresh fillets.









The euthanized fillets had higher heme peak wavelengths (-418 nm after

euthanasia) than control, indicating the presence of carboxy-hemoglobin. These

wavelengths demonstrated the intake of CO by the fish during the euthanasia process.

The heme proteins in the euthanized fish were also significantly (p<0.05) more stable

during the study as they maintained high heme peak wavelengths. After four months of

frozen storage, it can be seen that CO was still bound to hemoglobin showing a great

stability of the carboxy-hemoglobin complex. This stability is also responsible for the

high and stable a*-values discussed earlier. A substantially higher stability of tilapia

carboxy-hemoglobin compared to tilapia oxy-hemoglobin during extended frozen storage

has been reported [19], which would explain this high heme protein and color stability of

the CO euthanized samples during the 4 month frozen storage.

The same analyses were done on the white muscle (Figure 4-21). As reported in the

first study on white muscle, the heme peak wavelengths were higher for the euthanized

samples than the control, however not as high as those for the red muscle. The higher

wavelengths were also maintained higher for the euthanized samples for the duration of

the study. The heme peak wavelengths found in the white muscle of the euthanized fish

correspond to wavelength one would expect for oxy-hemoglobin/myoglobin and not

carboxy-hemoglobin/myoglobin. The fact that the heme peak wavelengths of the

euthanized fish white muscle is higher than that of the control, does however suggest that

CO binding took place. The increase in a*-value for the white muscle of the euthanized

fish also supports this (Figure 4-15). These lower wavelengths for the euthanized white

muscle compared to the red muscle, suggest that the values represent an average of all

three different heme protein derivatives, i.e., met, oxy and carboxy, while the control










value suggest a mixture of met and oxy forms[45]. The heme peak wavelength increase,

increased heme protein stability and increased red color of the euthanized white muscle

during the four months study therefore must come from partial CO binding to the heme

proteins in the white muscle.


415
414
413
S412
S411
w 410
I 409
3 408
407
406
Fresh 2 months 4 months
-*- Control -m- 100% CO Euthanized

Figure 4-21. Maximum heme peak values for white muscle of euthanized (100% CO) and
untreated fresh and frozen whole tilapia.

It is interesting that the heme peaks wavelength for both red and white muscle did

not decrease as much as in the second study compared to that seen in the first study. The

fact that the fish were frozen whole appeared to preserve the heme proteins better

compared to freezing fillets.

Carbon Monoxide Quantification

The amount of carbon monoxide that is found in the muscle is important. It reveals

the uptake of CO in the muscle and can also be used to differentiate treated products from

untreated ones. The heme spectroscopic analyses and the increased redness (a*-values)

revealed the intake of CO by the live animal, but does not tell us how much CO is in the

muscle. Figure 4-22 shows the difference in CO concentration in the red muscle between

a 100% CO euthanized and untreated fish. CO concentration in the untreated red muscle









is low compared to the concentration in the euthanized fish. Furthermore, it can be

observed that the CO concentration of the control is very stable and does not change

significantly during the four months of study.


10500

8500

6500

o 4500 __
C--


2500

500
Fresh 2 Months 4 Months

Control 19 100% COEuthanized

Figure 4-22. Concentration of CO (ppb) in the red muscle of fresh and frozen untreated
and euthanized (100% CO) tilapia.

The amount of CO found in the euthanized fillets was considerably higher than the

CO concentration in the control fillets and was more variable. Many natural conditions

inherent to tilapia may have accentuated these differences. For instance, CO is bound to

the heme proteins, explaining the high concentration found in the red muscle compared to

the white muscle. Every fillet has different amounts of red muscle influencing the final

amount of CO recovered from the muscle. This could explain the higher values found

after 2 months of frozen storage. Another possible explanation for the lower values for

the fresh fish is that fillets were cut from the euthanized fish very shortly after it was

euthanized, which could have led to some CO loss from the muscle. On the other hand,

the samples at 2 and 4 months were taken from whole frozen fish filleted after thawing,

thus giving the muscle more time to "trap" CO than the muscle of the freshly killed and

filleted fish.










The concentration of CO found in the white muscle follows the same trends as that

seen for the red muscle (Figure 4-23). The difference between the euthanized and the

control muscle were not as large in the white muscle compared to the red muscle. This

relates to the lower amount of heme proteins present in the white compare to the red

muscle.


2500


2000


0. 1500
0

1000


500
Fresh 2 Months 4 Months
SControl 100% CO Euthanized

Figure 4-23. Concentration of CO (ppb) in the white muscle of fresh and frozen untreated
and euthanized (100% CO) tilapia.


From Figures 4-22 and 4-23 it can be observed that the amount of CO was

maintained high during the whole study and that it did not decrease significantly (p<0.05)

over time. Since the tilapia was kept frozen whole and vacuum packed it may have aided

in keeping the CO levels high at all times. This can be also observed in the high a*-values

obtained throughout the study and in the high heme peak wavelengths discussed earlier.

As discussed previously, an increase in a*-values after 2 months of frozen storage

was seen in the red muscle, thus corresponding to the increase in CO levels, revealing a

close relation between the amount of CO present in the muscle and a*-values (Figures 4-

12 and 4-20). However, as seen in Figure 4-13, a*-values from the white muscle did not









follow the same trend as in red muscle. A higher amount of CO after 2 months of frozen

storage was found in the white muscle of the fish (Figure 4-23), similar to that seen for

the red muscle, however a*-values in the white muscle did not increase. This can possibly

be explained to the amount of heme proteins present in each muscle. The small amount of

heme proteins present in the white muscle could explain these results. Since there are

more heme proteins present in the red muscle, more binding could take place and more

CO is needed to stabilize all the heme proteins. Meanwhile, in the white muscle the limit

of CO intake is much lower and maybe the excess of CO present is trapped in the muscle

are not necessarily bound to the heme proteins and thus has no effect on color.














CHAPTER 5
SUMMARY AND CONCLUSIONS

The practice of treating fish with CO in order to avoid the oxidation of the heme

proteins and maintain an appealing and very attractive fresh red color is now widespread

and increasingly more common. The red color is stabilized by the formation of the

carboxy-hemoglobin/myoglobin complex. In the tilapia industry, a common practice is to

expose the fillets pre-rigor, while the muscle is still respiring, to CO for a brief period of

time. Another less common practice is to incorporate CO into fish tanks while the animal

is still alive.

The intake of CO to the muscle by the euthanasia with 100% CO process was

confirmed by the heme peaks wavelengths obtained and by the difference in the

concentration of CO found in the treated product compared to the untreated ones. The

incorporation of CO in the muscle of tilapia by either treatment had a positive effect in

the red color of the fillets. After the treatments, the red color was enhanced and

maintained for a longer period of time. In addition, the CO treatment affected only the

redness (a*-values); b* and L*-values were not significantly affected. Comparing the

euthanasia process against the gassing of fillets it was found that both processes had good

results preserving the color of the red muscle. The euthanasia with 100% CO had a few

advantages compared to the post-mortem 100% CO treatment. A larger increase in a*-

values was seen when the fillets came from a euthanized fish. Also, a better stability

through the freezing process was seen for the euthanized samples. Finally, the euthanized

method was found to preserve better L values giving the fillets a more "natural" look.









Due to the inherent stress of the whole process, muscle pH and water holding capacity

were not significant different among treatments. Nevertheless, it is believed that the

euthanasia process will be less stressful to the fish if they were initially without the added

stress of transportation and laboratory handling. In addition, the euthanasia with 100%

CO represents less product handling since there will be no need of post-mortem CO

gassing. This advantage may positively influence the shelf life of the final product since

there will be less product handling, implying less probability of contamination.

Furthermore since the product is already treated via the gas euthanasia process, less labor

required is another advantage over a post-mortem gassing process.

The different level of acceptance and varying regulations between different

countries on the use of CO in seafood processing has created a need to test products for

CO content. In this study it has been shown that a simple heme analysis could

differentiate a treated from a non treated product. In addition, the method used to measure

the amount of CO in the muscle is very important and should be considered and specified

when CO regulation and CO limits are put in place. It is worth mentioning that the use of

CO in seafood processing has to be very closely studied and regulated. Without the

proper control, the use of CO could mask potential health hazards or it could make a

product look better than it actually is.

In this research study, 100% CO was used to euthanize the fish. It was observed

that tilapias remained calm before dying, revealing that the process was not stressful. It

was also observed that the use of CO had an anesthetic effect on the animal since they

stopped moving and remained calm until euthanasia was completed. This is an important

observation since animal welfare has been giving increasingly importance, and more






50


regulation regarding humane slaughter practices are being required. The euthanasia with

100% CO of fish would be in agreement with the animal welfare act, in my opinion.


















APPENDIX A
FIRST STUDY DATA


20
18
16
14
S12
1 10
8
6
4
2
0


* Control i 100% CO Euthanized o 100% CO Gassed


Figure A-1. Effect of CO treatments on the b*-values of the red muscle of tilapia.




25


20


S15o





5


0-


Freezing U Control 1 Euthanized [ Gassed


Figure A-2. Effect of CO treatments on the b*-values of the white muscle of tilapia.








52




80

70

60

50

40
-J
30

20

10



Before After 0 3 6 9 12 15 18
Treatment Treament Days

U Control U Euthanized 0 Gassed


Figure A-3. Effect of CO treatments on the L*-values of the white muscle of tilapia.















APPENDIX B
CO CONCENTRATION IN THE WATER


Table B-1. Concentration of CO in the water used to euthanize tilapias


Time


Ret.
Time


Peak
Area


rCO1 ppb


0 min 0.490 5.400 2166.504
0min 0.459 5.490 2167.730
0min 0.456 5.460 2167.321
0 min N/A N/A N/A
Ave 0.468 5.450 2167.185
Stand
Dev 0.019 0.046 0.625
5 min 0.465 872.940 13991.900
5 min 0.456 844.440 13603.418
5 min 0.459 633.640 10730.013
5 min 0.455 612.740 10445.126
Ave 0.459 740.940 12192.614
Stand
Dev 0.004 136.729 1863.752
10 min 0.459 820.970 13283.499
10 min 0.459 802.520 13032.008
10min 0.462 1109.130 17211.395
10min 0.464 1104.260 17145.012
Ave 0.461 959.220 15167.979
Stand
Dev 0.002 170.468 2323.635
15min 0.466 1676.640 24947.096
15min 0.452 1665.550 24795.929
15min 0.457 1583.100 23672.057
15min 0.454 1559.580 23351.457


Ave
Stand
Dev


0.457

0.006


1621.218


58.564


24191.635

798.280


Time


Ret.
Time


Peak
Area


rCO1 ppb


20min 0.453 1833.290 27082.385
20min 0.453 1821.400 26920.313
20min 0.457 1369.140 20755.579
20 min 0.460 1348.140 20469.329
Ave 0.456 1592.993 23806.901
Stand
Dev 0.003 270.786 3691.074
25 min 0.453 1846.280 27259.451
25 min 0.455 1440.570 21729.238
25min 0.456 1547.700 23189.521
25min 0.458 1515.120 22745.425
Ave 0.456 1587.418 23730.909
Stand
Dev 0.002 178.305 2430.472
30 min 0.454 1832.360 27069.708
30 min 0.460 1822.440 26934.489
30min 0.454 1334.740 20286.674
30 min 0.458 1318.220 20061.490
Ave 0.457 1576.940 23588.090
Stand
Dev 0.003 289.313 3943.615
35 min 0.453 1814.700 26828.986
35min 0.453 1796.570 26581.857
35min 0.452 1518.100 22786.045
35min 0.450 1511.960 22702.351


Ave
Stand
Dev


0.452

0.001


1660.333

167.963


24724.810

2289.492











30000

25000

S20000
0.
S15000
0
,-' 10000

5000

0


S--[CO] in water


0 5 10 15 20 25 30 35
Minutes


Figure B-1. Change of CO concentration in the water used for the euthanasia process.


Table B-2. Time and amount of CO needed to euthanize tilapia in the tank


Date


1/19/2005 39 182

1/19/2005 52 110.4
1/19/2005 32 121.6

2/16/2005 27 500
3/14/2005 28 197.3
3/14/2005 26 258
Average 34 228.216
Std Dev 10.01998 143.591
Average of
Correct Trials 31.25 189.725
Std Dev of
Correct Trials 5.737305 56.034


CO did not flow
correctly

Pump not working
properly


Time
min


CO used
(L)

















APPENDIX C
PICTURES OF THE TILAPIA FILLETS



Representative pictures of the "red" muscle side of a tilapia fillet, and a 'white" muscle

side of a tilapia fillet are shown below. All the images can be found in the CD

D:/Thesismantilla/tilapiaimages (Kept in Dr. Hordur G. Kristinsson's office)


Figure C-1. Picture of a "red" muscle side of a tilapia fillet taken by the CMVS



































Figure C-2. Picture of the "white" muscle side of a tilapia fillet taken by the CMVS













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BIOGRAPHICAL SKETCH

David Mantilla was born on February 3, 1979, in Quito, Ecuador. He attended a

French high school and graduated from it in July 1997. He came to the U.S. in Fall 1999

to the English Language Institute (ELI) at the University of Florida. He attended Santa Fe

Community College and graduated in December 2000. David graduated in December

2002 from the University of Florida with a Bachelor of Science degree in food science

and human nutrition. He did two internships as an undergrad, one with Tyson Foods and

the other with Darden Restaurants. He started his master's degree in August 2003 under

Dr. Hordur G. Kristinsson. While at the University of Florida he was invited to join Phi

Tau Sigma, the honorary society of food scientists.