1 CARBON MONOXIDE TREATMENT AND APPL ICATION OF CRYOPROTECTANTS ON THE QUALITY OF FROZEN/THAWED SALMON By MAX OCHSENIUS A DISSERTATION 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 2009
2 2009 Max Ochsenius
3 To my loving parents
4 ACKNOWLEDGMENTS I express m y deep gratitude to my major advisors, Dr. Murat Balaban and Dr. Charles Sims, for their advice, guidance and support. I al so thank my committee members: Dr. Hordur G. Kristinsson for his advice, guidance and suggestions and Dr.Allan Wysocki for his support in the completion of this research. It has been a privilege to work with them. I also thank my parents for their love and suppor t. I thank them for always believing in me and supporting me, making it possible for me to reach my goals, with their guidance and understanding. Finally, I thank all my friends and lab mate s especially Wendy Gomez, Alberto Azeredo, Dr.Yavuz Yagiz, Dr.Sivakumar Raghavan, Sara Aldaous, and Stefan Crynen for their support and help during my studies and research work.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........7LIST OF FIGURES.........................................................................................................................8ABSTRACT...................................................................................................................................10 CHAPTER 1 INTRODUCTION..................................................................................................................132 LITERATURE REVIEW.......................................................................................................16Salmon Taxonomy................................................................................................................ ..16Quality of Fresh Salmon........................................................................................................ .17Evaluation of Freshness...................................................................................................22Color of Salmon..............................................................................................................23Salmon Color Measurement............................................................................................ 25Odor Attributes.......................................................................................................................27Lipid oxidation................................................................................................................ ........27Textural Deterioration............................................................................................................32Cryoprotectants.......................................................................................................................34Fish Proteins (hydrolysates)...................................................................................................36Effects of Carbon Monoxide on Fish Muscle......................................................................... 40Electronic Nose................................................................................................................ .......44Machine Vision.......................................................................................................................47Research Objective............................................................................................................. ....493 MATERIALS AND METHODS........................................................................................... 54Fresh salmon................................................................................................................... ........54Determination of Protein by Biuret Reaction......................................................................... 54Preparation of Fish Protein Hydrolyzates............................................................................... 55Protein Injection.............................................................................................................. ........56CO Treatment of Salmon Fillets............................................................................................. 56Color Analysis by Machine Vision System............................................................................ 58Texture Analysis............................................................................................................... ......59Lipid Oxidation.......................................................................................................................59Water Holding Capacity......................................................................................................... 60Quantification of CO in Muscle............................................................................................. 60Aerobic Microbial Growth.....................................................................................................61Freshness (Odor) by Electronic Nose..................................................................................... 61Statistical Analysis........................................................................................................... .......63
6 4 RESULTS AND DISCUSSION............................................................................................. 71Texture....................................................................................................................................74Carbon Monoxide Quantification byGC................................................................................75Water Holding Capacity......................................................................................................... 76Lipid Oxidation.......................................................................................................................78Microbial Analysis..................................................................................................................80E-Nose Results........................................................................................................................815 SUMMARY AND CONCLUSION....................................................................................... 92 APPENDIX A VISUAL ANALYSIS............................................................................................................. 94B PHYSICAL ANALYSE S....................................................................................................... 96C CHEMICAL ANALYSIS.....................................................................................................100D MICROBIAL ANALYSES..................................................................................................106LIST OF REFERENCES.............................................................................................................107BIOGRAPHICAL SKETCH.......................................................................................................118
7 LIST OF TABLES Table page 2-1 Intrinsic characteristics of wild and farmed salmon.......................................................... 502-2 Fresh SalmonPremium quality indicators........................................................................ 503-1 Nikon D200 camera settings.............................................................................................. 644-1 Means separation. L* values for control and treated samples........................................... 844-2 Means separation. a* values for control and treated samples............................................ 844-3 Means separation. b* values for control and treated samples............................................84B-1 Mean Separation. Results for firmness in control, CO treated, and CO+protein salmon samples................................................................................................................. .96B-2 Mean Separation. Results for WHC. Differe nces in water loss between control and treated samples. Water loss from 10 g. of sample............................................................. 96B-3 Squared Mahalanobis dist ances for all treatments............................................................. 97B-4 Discriminant function analysis F-va lues for control and treated samples......................... 97B-5 Discriminant function analysis p-le vels for control and treated samples.......................... 97C-1 ANOVA analysis in lipi d oxidation of salmon fillets untreated, treated with 100%CO, and treated-inj ected with proteins................................................................... 100C-2 ANOVA Analysis. Concentrati on of CO (ppm) in the muscle of control, treated, and treated+protein salmon fillets.......................................................................................... 101C-3 GC data results. Concentration of CO (p pm) in the muscle of control, treated, and treated+protein salmon fillets.......................................................................................... 104C-4 Lipid Oxidation values obtained over 45 days of study for untreated and treated salmon fillets................................................................................................................. ...105D-1 Microbial results CFU per (g) average............................................................................ 106
8 LIST OF FIGURES Figure page 2-1 Relationship between redness (a*) and carotenoid concentration (mg/kg) in Atlantic salmon fillets. ................................................................................................................ .....512-2 Relationship between redness (a*), yello wness (b*), lightness (L*) and carotenoid concentration (mg/kg) of Atlantic salmon fillets (Based on data from Bjerkeng et al., 1997a)................................................................................................................................522-3 The Roche Salm oFan.....................................................................................................533-1 Salmon filleting process................................................................................................... ..643-2 Equal size fillets......................................................................................................... ........643-3 Skin removing process.......................................................................................................653-4 Air pressure protein injector used in the application of hydrolysates................................ 653-5 CO box gas used for the salmon treatments....................................................................... 663-6 Front view of CO box treatment. Tubing system is depicted in this view......................... 663-7 Outline of the determination of protein content in salmon frame by Biuret reaction........ 68 3-8 Outline of enzima tic hydrolisis process of salmon frame mince 69 3-9 Experimental design for the control, CO treated, and CO treated injected samples. ...... 704-1 Lightness (L*values). Statistical analysis of the co lor of salmon samples over 39 days....................................................................................................................................844-2 Redness (a*values). Statistical analysis of the color of salmon samples over 39 days..... 854-3 Yellowness (b*values). Statistical analys is of the color of salmon samples over 39 days....................................................................................................................................854-4 Statistical analysis of salmon firmne ss during 30 days stored at -30C............................ 864-5 Concentation of CO (ppm) in salmon fillets after 48 hours treatment with 100% CO. in refrigeration, then frozen for 30 days. Treatment means with the same letter are not significantly different from each other........................................................................ 874-6 Water Holding capacity based on injected weight of salmon fillets injected with hydrolysates to obtain a 10% injection level..................................................................... 87
9 4-7 Statistical analysis in lipid oxidation of salmon fillets un treated and treated with 100%CO and injected proteins.......................................................................................... 884-8 Aerobic microbial growth (log CFU/g) in untreated and treated salmon fillets................ 894-9 R/R values for each sensor for the contro l, treated, and treated-injected salmon fillets..................................................................................................................................904-10 Scatterplot of root 1 vs. root 2 of unsta ndardized canonical scores for control, treated samples with 100% CO, and CO treated+injected samples............................................... 91 A-1 Salmon f illets pictures analyzed by a machine vision system. (a) Control samples frozen 30 d. (b) Samples treated with CO and frozen for 30 da ys, thawed a week after 94 B-1 Electronic nose data R/R for each sensor in control sam ples.......................................... 98B-2 Electronic nose data R/R for each sensor in CO treated samples....................................98B-3 Electronic nose data R/R for each sensor in CO treated + protein samples.................. 99C-1 Chromatogram of the injection of 100l of the headspace analysis over each sample, at day 2 after treatment.................................................................................................... 102C-2 Chromatogram of the injection of 100l of the headspace analys is over each sample, at day 32 after 30 days at frozen temperature.................................................................. 103
10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CARBON MONOXIDE TREATMENT AND APPL ICATION OF CRYOPROTECTANTS ON THE QUALITY OF FROZEN/THAWED SALMON By Max Ochsenius May 2009 Chair: Charles Sims Co chair: Murat Balaban Major: Food Science and Human Nutrition Seafood is highly susceptible to spoilage and deterioration. The high levels of polyunsaturated lipids in fish lead to lipid oxidation thus to rancidity in fish muscle, which is a major cause of quality deterioration in fish. Unlike most preservation techniques like freezing or refrigeration, Carbon monoxide (CO) can stabil ize the heme proteins of fish muscle by maintaining these in their reduced state, leading to less lipid oxid ation, and retaini ng the color of the fish muscle. The objectives of this research were to qua ntify the effects of CO in Atlantic salmon ( Salmo Salar) and the use of protein injection (sal mon hydrolysates) as cryoprotectants. An enzymatic hydrolysis process was used to extrac t functional proteins from salmon frames using Protamex (a bacterial protease.) Fish byproducts can be converted in to useful functional proteins and injected to fish muscle as cryoprotectan ts. Phosphates, whey and soy protein, sucrose sorbitol mixture, and salts are being used to im prove the water holding cap acity thus texture of muscle foods. One objective was to study the e ffect of hydrolysates, prepared by enzymatic hydrolysis of salmon frames, on the water hol ding capacity, texture and lipid oxidation, by
11 injecting the protein hydrolysates solution to in crease the weight of the fish muscle by up to 10%. Atlantic salmon ( Salmo salar ) from Chile was received from a certified distributor in Miami FL. not more than 3 days after harvest. Th e fish were filleted and divided into 3 groups: control, CO treated, and CO treat ed and injected with hydrolysat es. Each group had 3 replicates. For CO treatment, fresh salmon (fillets) were be placed in a box built with Lexan sheets (polycarbonate material) and flushed with 100% CO. The gas was flushed seven times the volume of the box to assure the desired concentra tion. The box was sealed with a door that had a gasket to avoid any leakage. Befo re CO treatment, fish proteins ( hydrolysates) were injected into the fish muscle as cryoprotectants to stabilize protein against denaturation and their effects were evaluated after the freez ing-thawing cycle. All samples were vacuum packed and frozen for 30 days at minus thirty degrees Celcius. Lipid oxi dation was measured by (TBARS), CO levels in the fish by GC (FID) and spectrophotometer, co lor by Machine Vision and L, a and b values were recorded, texture by Instron TPA prof ile, water holding capaci ty by a Sorvall RC-5B Refrigerated Superspeed Centrifuge, freshness by E-nose, and microbial count by Total Plate Count(TPC). All the analyses were done in tripli cate Results were analyzed statistically using analysis of variance to look for significant differences between the 3 treatments at each storage day. CO significantly (P value higher than 0.05) changed the color (a value) of Atlantic salmon fillets on day 5 after treatment, but the color remained about the same during frozen storage. The study showed that there was mini mal effect of CO on the growth of microorganisms. Before treatment, all samples had le ss than 100 cfu/g. At day 39 af ter thawing, the control had 103 cfu/g, and the treated 104 cfu/g. TBARS values confirmed the effect iveness of CO in reducing lipid oxidation. These values were si gnificantly less (P< 0.05) for the CO and CO+ injected treated
12 salmon compared to the controls during day12 through day 45 of frozen storage. CO uptake was significantly higher at day 2 af ter treatment between samples. Water holding capacity was significantly improved by using cryoprot ectants at 10% injection level.
13 CHAPTER 1 INTRODUCTION The use of carbon monoxide in fish processi ng is a significant recen t development in the seafood industry. This technology has emerged at the same time when the increasing demand for seafood resulted in seeking supplie s from more distant markets. The importation of seafood into United States has grown to satisfy the demand that exceeds the annual domestic supply and there is no sign to suggest this trend will change. Simi lar situations are occurring in Europe, Canada, and Japan. This trend had induced the search for new processing methods to protect and maintain product quality and appeal for long transportation distances. The use of CO offers a method of preservation combined with freezing while mainta ining product appeal. This has prompted fish technologists and the fish trade to pay more atte ntion to aquaculture techniques as a source of fish and other seafood products (FAO 2001). 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, initial microbial load, amount and type of heme proteins, fat content and fatty acid profile (Mantilla,2005). Freshness and spoilage are the pr incipal terms used to descri be quality changes of seafood. Final product quality is affected by factors su ch as handling, gutting, and storage temperatures. Although refrigeration and frozen storage can extend the shelf lif e of seafood products, proliferation of psychotropic bacteria at refriger ated temperatures still contributes to spoilage (Hobbs1983) and deterioration of texture. Atlantic salmon, both wild and farmed species, are known to deteriorat e rapidly after death due to different mechanisms, such as microbiol ogical proliferation, endogenous enzyme activity,
14 lipid oxidation, and browning. During chilled storage of salmon, a strong effect of lipid damage has been detected as a cause of fish quality loss (Hwang 1999, Undeland 1999) that leads to a reduction of commercial value. Research has shown that the use of CO can significantly retard and minimize lipid oxidation in seafood (Huo and others. 2005). A study by Balaban and others (2005) demonstrated that a 4% CO treatment did not have a noticeable eff ect on microbial levels of tuna, but a 100% CO treatment showed a reduction in the bacter ial level. The use of CO can minimize bacterial growth and reduce the amount of microbially produced compounds that cause spoilage and also oxidation of lipids in the flesh during refrigerated and frozen storage as well as stabilize color. Long storage time, especially at temperatures around -18 C or lower, brings significant deterioration of texture of frozen fish descri bed as increased toughness,chewiness,rubberiness,or stringiness (Sikorski and others, 1976). Storage time also, contribu tes to continued growth of ice crystals increasing salt concentration, leading to protein denaturation, then loss of protein functionality and textur e hardening (Shenouda, 1980). One of the major problems with any muscle food product, in cluding sea foods, is the loss in the ability of muscle and muscle proteins to hold water. This loss in water-holding capacity costs millions of dollars to the seafood industry. The ability of fresh meat to retain moisture is one of the most important quality characteristics of raw products as well as their appearance. However, this loss of water rete ntion and deterioration of fish muscle can be prevented using some compounds against freeze-induced deterioration, known as cryoprotectants. Proper cryoprotective ingredients can be used to improve fro zen storability. In general, phosphates, salts, starches, gums and plant based proteins have been used to improve water holding capacity in fish muscle. Phosphates have strict usage restrictions because of potential
15 abuse in the seafood industry. Cryopro tectants such as sucrose or sorbitol are commonly used to prevent ice crystal growth and the migration of water molecules from the protein (Matsumoto and Noguchi, 1992). Additions of salts and phosphates elevate the sodium content of the muscle food which is not very desirable in the food indus try. The use of sorbitol or sucrose mixture also imparts a taste that is too sweet, leading to a sear ch for other cryoprotectants as an alternative to the above ingredients. Every year tons of fish byproducts are discar ded. The recovery and alteration of the fish muscle proteins present in the byproducts for use as functional ingredients in food muscle is an excellent and a natural al ternative to improve waterholding capacity and reduce texture deterioration sinc e weight decrease due to loss of water is also of economic importance. By applying enzymes for protein recovery in fish processing, it is possible to produce hydrolysates and modify functiona l properties of proteins that can be used as cryoprotectants. These proteins can be obtained by enzymatic hydrolysis using pr oteolytic enzymes that improve or modify the physicochemical and functional propert ies of the proteins. These proteins can help to improve the texture of fish muscle and preven t water loss since water is important for muscle texture. This study focused in determining the effects of CO and the use of cryoprotectants (hydrolysates) on salmon fillets, and assessing ch anges that occur in color, texture, WHC, growth or inhibition of aerobic microorganisms, CO concentration in the muscle, lipid oxidation development, and differences in odor between treatments.
16 CHAPTER 2 LITERATURE REVIEW Salmon Taxonomy Worldwide, there are ma ny va rieties or species of salm on. Among these species are King, Coho, Sockeye, Chum, Pink and Steelhead salmon which are native from northern California to Japan. Atlantic salmon are native to the nor thern Atlantic Ocean, from New England to Scandinavia. Atlantic salmon is the most recognizable and farm ed species in Canada and around the world. They are reared in bot h the Atlantic and the Pacific oceans. The share of farm-raised salmon produced worldwide rose to 32 percent in 1992 and exceeded 60 percent in 2002 (Babcock and Weninger, 2004). In 2003, 105, 050 tons of Atlantic salmon were produced in Canada valued at CAD $434 million (Canada FAO 2006). Atlantic salmon is farmed in North America, South America, Australia and Europe. Atlantic salmon ( Salmo salar) is important among the various species cultured worldwide and its contribution to the total aquaculture production was 2.39% in 2001. Norway, Chile and UK (Scotland) are the three major producers of fa rmed Atlantic salmon. Scotlands production of Atlantic salmon was estimated around 158,000 tons in 2001. Salmon farming is an important enterprise in Scotland and the product has achieved a superior qua lity reputation reflected in a premium price. Ninety percent of the farmed salmon consumed in the United States are Atlantics (FAO 2001). Atlantic salmon, or Salmo salar has a long, thin body, a large mouth, fairly large scales, and a fleshy adipose fin on the back just in front of the tail fin. It is a silver-skinned fish with distinct dark blue-gr een, cross-like spots over the body and h ead, and above the lateral line. The underside of the fish is nearly all white and its fl esh color ranges from pink to deep orange. The
17 flesh of farmed Atlantic salmon is firm and has a large moist flake. It has a mild flavor compared to other salmon species (FAO Canada 2006). Salmon are an anadromous species, meaning th ey spend a portion of their life in fresh water and a portion in seawater. The freshwater phase occurs wh en migrating salmon return to their rivers of origin to spawn. After hatching, juvenile salmon remain in the river until they undergo physiological and behavioral change, a process called smoltification, which prepares them for their life at sea. The Salmon life cy cle follows this process: 1) Adults spawn 2) Incubation& emergence 3) Freshwater rearing 4) Ocean entry 5) Ocean residence 6) Upstream migration. Salmon farming is based on the natural salmon life cycle and requires both freshwater and saltwater operations, fish are spawned, eggs inc ubated and juvenile fish reared at freshwater hatcheries, usually located on land. When the fish are ready for seawater, th ey are brought to salt water farms. Here, they are reared to adult size and either harvested or returned to the hatchery to produce the next generation. Since farmed salmon are grown in a controlled environment, they are available for harvest at the discretion of the farmer. In general, growing periods are shorter and harvest weights are usually more uniform for farmed salmon as compared to wild salmon. This keeps the farming process efficient and allows farmed salmon to be an economically viable product (DSM. Service 2005). Quality of Fresh Salmon Quality of s almon is a function of both the innate traits of the salmon species and the extrinsic handling procedures the fish receives at harvest, catching, handling, processing and storage practices that can impact the overall qua lity of the salmon. The innate quality of salmon species varies significantly. Innate quality refe rs to the physical char acteristics or natural condition a fish possesses before it is harvested such as size, age, sex, color of skin and flesh, oil
18 content, flesh texture, and degree of maturity. These characteristics are inherent to a particular fish and will not change much during handling. Inna te quality is evaluated at catch or harvest and can be impacted by species, size, age and sex. Age is one of the major factors influencing innate salmon quality. As the fish matures, biochemical changes occur in the flesh and reduce its overall value. Most significantly, in the final stage of the salmon life cycle, hormonal changes cause proteins, oils, and flesh color to change in ways that decrease quality. Skin color and markings, which also change over time, can be used to determine the maturity of the fish. These markings vary by species. Generally, the skin of premium salmon is brig ht and shiny. Further, when the whole fish is bent slightly, the skin s hould not hold wrinkles. Sk in color is transformed from bright silver to mottled blacks, grays, and greens. Farmed salmon are harvested prior to the onset of discoloration (Bab cock and Weninger, 2004). Location of the salmon catch can also affect innate qual ity. Once salmon enter a fresh water environment they stop eating. Fat is lost and the flesh becomes soft and watery. Season of catch can also impact the fish. In wild salm on, flesh fat content varies depending on the season. Variations in fat content can also occur betw een years due to changi ng weather conditions. For farm-raised salmon, fish harvested in summer mont hs will have a higher fa t content than those from winter harvest (DSM service, 2005). Als o, fat content varies ac ross and within each species. A determinant of the total fat content is the distance that the salmon must travel swimming upstream to reach the spawning beds on which they were born. The fat content of farmed salmon flesh is usually in the range 5% to 17% but may reach 20% or more; the fat content varies from head to tail and from dorsal to ventral. Fat deposition increases as the fish increase in size, but the fat concentration a nd composition of salmon flesh depends heavily on
19 the type and amount of fat in the diet (Austr eng and Kroghdal 1987) which also influences the taste, flavor and texture (Hardy and others 1987) and the degree of gaping. Others important characteristics that vary across salmon species in clude the amount of health-enhancing Omega-3 fatty ac ids contained in their flesh, si ze and coloring of the flesh and skin, and average timing of spawning. Table 2-1 summarizes key characteristics that determine the intrinsic quality of each salmon species. Extrinsic quality refers to the changes in fish flesh during and after ha rvest, shown in Table 2-2. Fish handling, from the time of harvest to the time of consumption, is the primary determinant of extrinsic quality. Natural biologica l deterioration processe s begin at the time of death. The nature of death itself can influence the duration of rigor mortis and the rate at which natural chemical processes begin to break dow n flesh (Babcock and Weninger, 2004). A violent or prolonged death expedites th e deterioration process so a quick, non violent death by stunning and bleeding causes the least dama ge. Poor conditions at harves t can stress the fish. As the salmon is stressed, flesh pH falls rapidly. Also, the initial quality and the t ype of processing that the fresh product undergoes has a great influence on th e shelf life, rate of spoilage and quality of the final product (Hardy, 2003). Some of the factors that influence the spoilage rate of fish are muscle pH, temperature, microbial load, microbial type, amount a nd type of heme proteins, fat content and fatty acid profile (Mantilla,2005). The result is a decrease in shelf life. After harvest, if the salmon is subjected to rough or abusive handling, quality can be significantly impacted. Some of the signs of a busive handling are gaping, bruising, and chemical damage that also reduce extrinsic quality. Gaping is the separation of muscle layers (flakes in the fillet separate from each other) due to weakeni ng of connective tissue, which causes holes or splits to appear between the muscle layers After the death of the animal, the anaerobic glycolytic
20 system becomes predominant and ATP is gradually depleted and lactic acid accumulates, leading to a decrease in pH. When the pH is low enough certain critical enzymes are inhibited and glycolysis ceases (Fennema, 1996). The decrease of pH comes from the hydrolysis of ATP (Fennema, 1996). A fast decrease in postmortem pH will cause the denaturation of muscle proteins; the meat produced will be pale soft an d exudative (PSE), a conditi on that is especially troublesome in pork (Terlouw, 2005). This phenomenon also occurs in fish; low pH weakens the collagen fibers, they break and gaping takes place. Gaping damage can cause the fish to be unsuitable for particular processing uses such as curing and cold smoking. Gaping also affects the quality of fillets and steaks ru ining the appearance of the salmon. The main causes of gaping are: allowing th e fish to go through rigor mortis at high temperatures, bending the fish while it is in rigo r, and lifting and pulling the fish by its tail (Babcock and Weninger, 2004). When an animal is under stress, oxygen is not available in sufficient amounts, and the anaerobic pathway b ecomes predominant and glycogen is depleted. This depletion of glycogen results in an onset of rigor much sooner and in a faster decrease of pH (Huss, 1995). A common practice in Atlantic salmon is to process once rigor mortis has resolved which takes 3 to 5 days on ice storage (Einen and others, 2002). However, it has been shown that there is no major difference between processi ng pre-rigor salmon and post-rigor salmon (Erickson and others, 2005, Einen and others, 2002). Internal bruising creates soft, mushy flesh th at is unsightly and una ppetizing. Bruising can preclude certain processed forms, such as lox and steaks, and is the most serious deterrent to the overall quality of the product. Bruising can occu r during the handling process and can be caused by lifting the salmon by the tail, dr opping it on the tail, bending it by th e tail when the fish is in
21 rigor, breaking the backbone. Handlers should avoid lifting the fish by the tail; avoid throwing, kicking or stepping on fish (Babcock and Weninger, 2004). Others causes of quality decline are spoilage from bacteria, rancidity caused by the oxidation of oils (Aubourg and others, 2005), burn caused by careless freezing and contamination from dirt or others materials during transportation and processing process. Similar quality issues can occur if the harvested fish is subjected to temperature changes. The goal of post-death handling practices is to mi nimize further reductions in intrinsic quality and to control the activities that contribute to extrinsic quality decline. The most effective way to control the natural decaying process is to reduc e the body temperature and slow bacterial growth. The shelf life of fresh salmon is about 12-14 days if the body temperature is maintained at freezing temperature (-25C) immediately followi ng death. The rate of shelf life decline is proportional to the degrees above fr eezing at which the salmon is stored and to the exposure time to higher temperatures. If the fish are not iced or bled until so me time after harvest, significant temperature fluctuations can occur, resulting in lower product quality and reduced shell life. Temperature fluctuations impact salmon in two dis tinct ways. First, there is an increase in the natural release of enzymes. These enzymes act to break down the cell stru cture of the flesh. The result is a loss of flesh firmness (Tironi and others, 2007). Enzymatic activity is also proportionally related to temperature. Enzymatic breakdown may cause a condition referred to as belly burn. This occurs when stomach enzyme s break down and begin to digest the stomach wall, causing discoloration and tissue damage. In less severe cases, the belly wall takes on a deep reed color. Severe belly burn includes exposed rib bones (Babcock and Weninger, 2004). A rise in temperature also promotes bacteria l growth, which decreases shelf life.
22 Optimally, the fish should be bled and chille d with ice immediately upon harvest. Once the entrails are removed, the fish shoul d be stored in ice and under refrigeration at all times. The fish must be chilled to a core temperature of (<1 C) as quickly as possible. Th is temperature must be maintained until the salmon is delivered to the cu stomer. Even slight increases in temperature lead to a significant decrease in the time the salmon can be held. Whether wild or farmed, Atlantic salmon, properly handled, provide for an excellent quality product, with a moist and tender texture and uniform pink to red flesh (DSM. Service 2005). As with fresh salmon, well handled frozen salmon can be of excellent quality. However, freezing can hide significant problems. Frozen fish quality is affected by handling before freezing, the actual freezing proces s and the quality of storag e. Once thawed, well handled frozen product will be similar in quality to fresh salmon. To maintain flesh quality, the freezing process must be as quick as possible and the prod uct should be stored at a constant temperature of (-25 C) or lower (DSM Service 2005). Freezer burn, which can appear as whitish spot s or patches on frozen fish, is caused by dehydration. It causes a distinctive off odor and flavor that is the result of oxidation after the dehydration begins. To protect against dehydration and oxidation, salmon can be glazed or shrink-wrapped. In short, gentle handling, a ttention to temperature, and sanitary and uncontaminated storage facilities are essential fr om harvest to end use to minimize extrinsic handling practices that can negatively impact the fish. Evaluation of Freshness Due to an increasing consumption and im portance of salm on in the U.S.(FAO 2001), quality has become an important factor that should be monitored for a safe and high quality commercial product. Current quality evaluation of most seafood produc t relies on subjective methods that use the senses of vision, smell and touch. These senses indirectly examine the
23 microbiological, chemical and physical changes th at have occurred in the product. The freshness of fish is an important factor when determining wh ether the fish meat is edible, or in considering other possible uses. Traditionally, several factors have been used to determine the freshness in fish. These included visual inspectio n (signs of quality), such as gi ll color, shine, shape, size, color and texture of the skin a nd the color and thickne ss of the slime on its skin. Smell is also used to detect any perceptible sign of off odor s or presence of chemi cals. (Luzuriaga, 1999). Other methods used in the determination of freshness are by chemical and microbiological means. The determination of spoilage of fi sh by chemical means is based on the products resulting from the breakdown of various compon ents present in the fish, both the macrocomponents (fats and proteins) and the microcomponents (nucleotides, non protein nitrogen compounds, enzymes etc.) (Fraser and Sumar, 1998). The breakdown of these compounds generally leads to unfavorable changes in the sensory characteri stics of the fish meat which coincides in most cases with its physical deterioration. These ne w compounds formed in fish are responsible for the changes in odor, flavor and texture of deteriora ting fish meat, and can be used as indices of fish deterioration of freshness. Color of Salmon Salmons flesh color is one of the m ost im portant attributes used during its quality evaluation. Color depends on the species, genetic di fferences, the spawning cycle, diet and other environmental factors. Sockeye salmon has the reddest flesh, while pink salmon has the palest. The color of individual fish also varies greatly from one another (Dore, 1990; Luzuriaga, 1999). The flesh color is a good measure of the freshness of the fish. Consumers equate freshness with the vibrancy (redder) of the fl esh color (Beaudoin, 1997). Fresh salmon has an attractive vivid pink-orange color, which changes to a dull pink or even beige color during storage. In farm raised salmon, flesh color depends on the feed given to the animal. Since farmed salmon do not
24 have access to naturally occurring sources of carotenoids, many salmon farmers use carotenoid compounds in the feed to give the salmon the prope r color that attracts th e consumers attention. The fish farming industry is working to devel op strains of fish whic h have redder flesh and which absorb more easily the pigments from their feed (Luzuriaga, 1999). Salmon flesh color is due to carotenoids. Caro tenoids that are composed entirely of carbon and hydrogen are known as carotenes, while thos e that contain oxygen are termed xanthophylls (Anderson, 2000). In nature, carotenoids are pro duced by certain microorganisms and plants. Wild salmon obtain carotenoids from their f ood, begining with microscopic algae, a marine source of carotenoids. The algae are eaten by crus taceans, which in turn are eaten by small fish. Most salmon consume either crustaceans or small fish. Once the carotenoid is absorbed it is transported in the blood and then it is deposited in the skin and muscles (Bell and others, 2000). Although 20 carotenoids have been identified in salmon, at least 90% of the total carotenoids in the fish is astaxanthin which play s a role as a potent antioxidant that works to protect the fish from the harmful byproducts of biological processe s. Cold water fishes, like salmon, have a high level of polyunsaturated fat in their membranes, and protection of lipid tissue from peroxidation seems to be a metabolic function for astaxant hin (Anderson, 2000). Astaxa nthin has been shown to be one hundred times more effective than vita min E as an antioxidant (Miki, 1991). As is the case with other carotenoids, salmonids cannot en dogenously synthesize astaxa nthin; therefore, it must be supplemented in the fishs ration (Anderson, 2000). Salmon can also convert astaxanthin to vitamin A, an essential nutrient, and astaxanthin has also been shown to support normal growth and immune system functions (Bjerke ng, 2000). Also salmon are a natural source of protein, vitamins, minerals and the polyunsaturated omega-3 fatty acids, EPA and DHA. Studies
25 by the American Heart Association have showed their health benefits and positive impact in human health conditions like heart di sease and Alzheimers disease. Salmon Color Measurement It is ge nerally accepted that the color of sa lmon products is one of the most important quality parameters. Color of food can be asse ssed by various analytical methods: sensory analysis using trained panelists for descriptive or comparative tests, comparison of fish samples with standardized colors, or by electronic instrumental analysis based on light reflectance from fresh samples ( Luzuriaga, 1999). Spectral characteristics have frequently b een used to evaluate the pigmentation of salmonids. Salmon flesh has a uniform color, ma king it easier to be analyzed with current instrumental techniques. Salmon color has been described by the parameters L, a and b (Skrede and Storebakken, 1986). These color parameters are commonly obtaine d from commercially available tristimulus colorimeters. Colorimeters measure the color of a sample by shining a beam of light at a certain angle on the surface of th e object. These measure the reflectance from the surface of the sample. The light reflected from the sample is sensed by a photocell, after passing through filters which converts the light inform ation into its tristimulus values. The most frequently used instrumental color scales are th e CIE L*, a*, b* and the Hunter L, a, b systems, which primary parameters are lightness (L*) valu e, redness (a*) value red/green and yellowness (b*) value-yellow/blue. Other impor tant calculated values are the Hue (H*) (specific color from lighter to darker) and Chroma (C*) (the purity of color, how it differs from gray) of a color (Anderson, 2000; Bjerkeng, 2000). Visual color assessments may be aided by the us e of color charts such as the Roche Color Card for salmonids and the SalmonFan (Hoffm an-La Roche) which is the internationally recognized method for color measurement. A color card for raw flesh of astaxanthin-fed salmon
26 was developed based on instrumentally obtaine d L*, a* b* values (Skrede and others, 1990). These procedures focused only on the red portion of the flesh. Such assessments represent crude measures to characterize and classify pigmentatio n of fish flesh, primarily for the use by fish farmers, fish feed producers, the processing industry and marketing organizations. However, the degree of precision in color determination is re latively low due to influences of illumination, individual abilities to distinguish colors, a nd qualities of the objects surface (Anderson, 2000). Color of salmon flesh depends on the content of certain carotenoids. Muscle coloration and the concentration of carotenoids in Atlantic salmon are si gnificantly related, but when astaxanthin concentration exceeds about 8 mg/kg, the eye becomes saturated; the increase in color perception upon increas e in pigment concentration levels off, and color hues cannot be distinguished accurately. The a* value generally exhibits the best correlation to increasing carotenoids levels (Wathne and others, 1998) There is also a high correlat ion between a*, b* and chroma (Cab*) and the logarithm of fillet carotenoid concentration in Atlantic salmon (Christiansen and others, 1995a). The logarithm of the a* values and th e logarithm of fillet ca rotenoid concentration, and L* values are negatively linear with the fillet carotenoid concentration giving the highest correlation (Bjerkeng and others, 1997a). Color parameters L* a* b* are also correlated to the fat content of the muscle (Einen and Skrede, 1998). As the astaxanthin levels in muscle increases, increasing redness (a* value), increasing of fillet fat content contributes to higher values a* and b*, but a less red and more yellowish hue (hab) (Bjerkeng 2000). To measure color using the SalmoFan, the fish must be placed in natural daylight or use a florescent light. Since color can vary within a fillet, it is always best to ta ke the color reading in
27 the center of the fillet, between the back and the belly. In focus group studies, consumers were shown salmon fillets matching six color hues (different levels of coloration) on the SalmoFan. Color 33 was preferred by a two to one (2:1 ) margin (DSM.Consumer Service 2005). This research indicated that consumers believe and prefer darker salmon colors. The same situation ocurred when they were asked about pricing: they preferred again color 33 because consumers may be willing to spend more for deeply colo red salmon and less money for a color of 22-24. Consumers perceive that redder salmon is equa ted to fresher and higher quality. (Color is identified by numbers from 20 lightest to 34 darkest) Odor Attributes Odor is another parame ter that is used to determine the quality of food products. The odor can be used as a decision making tool to accept or reject salmon or any other seafood product. Generally, when seafood has a fishy, spoiled or putrid odor it is reje cted. These odors arise mainly during the bacterial spoila ge of seafood, and are related to the odors of certain chemicals such as, trimethylamine, dimethylamine, a mmonia or hydrogen sulfide (Stansby, 1962). Fresh seafood exhibits a clean and natural odor (Perki ns, 1992). Fresh salmon has no detectable odors; however some researches define the fresh odor as that of sliced cucumbers (Dore, 1990). In the frozen state, the odor is not completely detectable, but stil l some oxidation reactions occur causing a loss in quality (T hanonkaew and others, 2006). Lipid oxidation Lipid oxidation is a ma jor cause of quality deterioration of seafood. It contributes to the formation of off odors and flavors, deterioration of color and text ure, and the production of toxic compounds that arise from lipid oxidation (Ric hards and Hultin, 2002).F ish lipids are very sensitive to the oxidation process because of their high content of polyunsaturated fatty acids (PUFAs) (Tironi and others, 2007). Fatty fish such as salmon contain high level of omega-3
28 polyunsaturated fatty acid known as eicosapent aenoic acid (EPA) and docosahexaenoic acid (DHA) (Tironi and others, 2007). Even though still l ittle is known about th e actual pathway of lipid oxidation that causes rancidity in fa ts, this may occur by several mechanisms and contributors such as heme pr oteins in fish muscle hem oglobin (Hb) and myoglobin (Mb) (Grunwald and Richards, 2006). A major pathwa y follows the free radical mechanism which includes three stages: in itiation, propagation, and termination. During initiation, ca talysts such as heat, metal ions, and irradiation cause lipid mol ecules to form lipid free radicals (R*). These react with oxygen to form per oxyl radicals (ROOH) and new free ra dicals (R*), resulting in self propagating chain reactions. When free radicals in crease in a system, they interact to form nonradical end products thus terminating th e chain reaction (Fraser and Sumar, 1998). Oxidation of lipids in fish occurs in two ways, enzymatic and non-enzymatic. Enzyme mediated oxidation of fish lipids involves lipase s which are present in the skin (Ke and Ackman, 1976), blood and lymph (Kanner and Kinsella, 1983a ), and microsomal fractions from both lean and fatty fish tissue. The main enzymes involved in the hydrolysis of fish lipids are triacyl lipase, phospholipase A2, and phospholipase B (Audley and others, 1978; Bilanksi and Lau, 1969). The hydrolytic products such as free fatty acids, lysophospholipids, glycero-phosphocholine and phosphoric acid may not noticeably influence the fi sh flavor; however, some are responsible for the formation of peroxides and hydroperoxides. H ydroperoxidation of fish lipids results in the development of rancid flavors and odors in fish meat, and may lead to fading or color deterioration in the skin of the red fishes. There are some endogenous enzymes that can cause deterioration at the same time as microorganisms on fish muscle proteins during chilled storage. These proteins can undergo different modifications due to the presence of so me proteases, such as collagenases, lysosomal
29 cathepsins and calpains which can produce changes in texture, myofibrillar proteins and connective tissue degradation. As a result, proteo lysis can lead to organoleptic changes and acceleration of microbial growth. Proteolysis in fish may occur by proteinases in the muscle (cathepsins) and the intestinal tract (trypsin s). Myosin heavy chain and actin are the main structural proteins in fi sh flesh. Lin and Park (1996) studied the effect of cathepsins in Pacific whiting, stored at a low temperatur e, finding that they were res ponsible for the degradation of myosin heavy chain, more rapidly than actin. They found that more than 70 % of myosin heavy chain was degraded when fish was stored at 0C for 72 hours. Other proteolytic enzymes like trypsins are resp onsible for solubilizati on of proteins in the muscle tissue due to improper hand ling of fish. Autolysis of fish muscle proteins results in the formation of peptides and free amino acids wh ich are used as nutrients by microorganisms, allowing them to grow and produce amines that a ffect the safety of the fish meat. Also, freezing and thawing may result in membrane damage causing the lysosomal enzymes (actylglucosaminidase) activity to increase, which has been found to affect the texture of the fish as well as flavor (Nilsson and Ekstrand, 1995) The rancid off flavor in salmon is caused by formation of volatile oxidation products such as aldehydes and ketones (Milo and Grosch 1996; Refsgaard a nd others, 1998). Some of these volatile compounds have very intense odors and flavors, and even in small concentrations, can affect the sensory quality. The ra ncid off flavor of salmon is mainly caused by an increase in 2, 6-nonadienal with a cucumber odor, 3-hexenal with a green odor and 3, 6-nonadienal with a fatty odor (Milo and Grosch, 1996). These three al dehydes are formed from n-3 unsaturated fatty acids by oxidative process (Grosch, 1987). This is an important cause of quality loss during fish processing and storage, especia lly in fatty fish like salmon.
30 Non-enzymatic oxidation of fish lipids is caused by catalysis of hematin compounds (hemoglobin, myoglobin and cytochromes) which also results in the formation of hydroperoxides (Fraser and Sumar, 1998). There are some mech anisms by which hemoglobin and myoglobin can promote lipid oxidation. Most prob lems associated with heme pr oteins are due to their heme group which contains an iron mol ecule that can be bound to different gas ligands in its reduced state (Kristinsson and others, 2005). Each heme prot ein in fish with red meat autoxidizes to the met form, from the reduced Fe2+ state to the oxidized Fe3+ state, which reacts with H2O2 or lipid peroxides that generates ferryl he me protein radicals that can abstract a hydrogen atom from polyunsaturated fatty acids and hence initia te lipid oxidation ( Kanner and Harel, 1985; Grunwald and Richards, 2006). The oxidation of the heme groups to form metamyoglobin contributes greatly to undesirable color changes in aquatic foods. This is because the oxidized heme proteins have a stale brown color rather th an the desirable fresh red color of reduced OxyHb/Mb. This especially applies to the dark musc le of fish due to its higher levels of heme proteins (Kristinsson and others, 2005). In a ddition, the free fatty acids formed from the hydrolysis of fish lipids have been found to interact with both sarcoplasmic and myofibrillar proteins causing denaturation and some detrimental effects on the textural quality of frozen stored fish (Anderson and Ravesi 1969). Other primary oxidation products such as free radicals and hydroperoxides and secondary ones such as aldehydes, dialdehydes, alcohols, carbonyls, and epoxides oxidation products can r eact with other cellular component s such as proteins, peptides, free amino acids, phospholipids, and nucleic aci ds, causing not only browning but changes in texture due to the crosslinking of polypeptides chains of min ced sea salmon (Tironi and others, 2007).
31 The effects of oxidized unsatur ated fatty acids bound to proteins and the formation of insoluble lipid-protein complexes due to the presence of me tal ions such as iron (transition metals) in cuttlefish texture, were examined af ter multiple freeze-thaw cycles. This oxidation process led to discoloration, drip losses, and texture changes (toughened texture) because of the highly reactive hydroxyl radical th at promoted deterioration in functional properties of myofibrillar proteins (solubility, water holding ca pacity and gel strength) (Decker and others 1993; Thanonkaew and others, 2006). Site specific metal catalyzed oxidation of amino acids in proteins occurred via hydroxyl free radi cals, which were produced from H2O2 at specific ironbinding sites on proteins (Liu and others, 2000). Myosin may be altered by the interaction with different types of lip ids or lipid oxidation products during frozen storage causing considerable changes in prot ein functionality and texture. This denaturation of proteins invo lves alteration of their secondary and tertiary structure due to breakage of both covalent (disulphide) and non covalent (electro static, hydrogen and hydrophobic) bonds that stabilize the native prot ein (Saeed and Howell, 2002) .Some of these changes include loss of enzyme activity, polym erization, and insolublization. The fatty acid character of lipid molecules exer ts a surfactant effect on protei n surfaces, leading to hydrophobic interactions and protein unfolding, thus exposing interior groups for reaction. Myofibrillar proteins are very sensitive to denaturation because of the presence of polyunsaturated fatty acids (PUFAs) in fish musc le that undergoes lipid oxidation (Tironi and others, 2004). The breakdown products of lipid ox idation such as malonaldehyde (MAD) were used to study its effect on myof ibrillar protein in sea salmon, in this case myosin. Malonaldehyde reacted with the -amino groups of myosin. This reaction c ontributed to denaturation and protein modifications in frozen products producing conformational changes and decreasing protein
32 solubility and functionality (Braddock and Dugan, 1973). A decrease in the -amino groups and in the protein solubility was associated with an increase in the amount of MDA measured. The formation of aggregates of high molecular weight in which myosin was involved could also be observed (Tironi and others, 2004). Textural Deterioration Freezing is among the most importan t techni ques for long term preservation of fish muscle. It prevents microbial spoilage and mini mizes the rate of biochemical reactions in the muscle. However, several structural and physicoche mical changes still take place that result in changes in muscle texture. These are enhanced by the disruption of muscle integrity affecting myofibrillar proteins and reducing protein functionality. This allows a close contact among cellular compounds and the access of oxygen ( Hu ltin and others;1992,Rodriguez-Herrera and others, 2006.) Functional and textural properties of fish muscle depend on myofibrillar proteins that constitute approximately 66-67 % of the total proteins in fresh fish, particularly myosin and actomyosin (Kristinsson and Rasco, 2000a, b). Th ese proteins are highly susceptible to denaturation due to processing, freezing, and frozen storage, causing intramolecular conformational rearrangements and intermol ecular aggregation. H ydrophobic interactions, hydrogen bonds and disulphide bridges are primarily involved in these pro cesses because of an increase in intermolecular cross-links (Herrera and Mackie, 2004). Acto myosin plays a major role in determining the quality of fish muscle beca use it is easily affected during frozen storage, where it becomes progressively less soluble and the flesh beco mes tougher (Thakar and others, 1991). Frozen storage of myofibrilla r proteins induces a decrease in functional properties such as solubility, gelling capacity, and wa ter holding capacity. Fish muscle proteins are less stable in frozen storage (changes in the stability of fish myosin) than those from land animals. Since fish
33 are supported by a mass of water, th e muscle fibers require less stru ctural support so their muscle tends to have less connective tissue than muscles from land animals, resulting in a more tender texture and more heat sensitivity, especially fi sh muscle proteins from cold water species (Kristinsson and Rasco, 2000 a). Changes in solubility and viscosity of he rring proteins affected by freezing and frozen storage were studied as a function of pH 2.7 a nd 11 after up to 6 months frozen storage. The evaluation of quality was based on protein degradation, changes in solubility, viscosity and lipid oxidation. Protein solubility at pH 2.7 decreased during frozen storage for 6 months and was 10% lower after 6 months compared to the beginning (P<0.05). Viscosity measured at pH 2.7 increased after 3 months of frozen storage. At pH 11, the solubility became approximately 15% lower after 6 months of frozen storage and visc osity increased significan tly after 1 week of frozen storage. Changes found in solubility and viscosity indicated protein degradation due to freezing and frozen storage of herring fillets. Th e increased TBARS (P<0.05) showed that the lipid oxidation might be the cause for negative changes during frozen storage (Geirsdottir and others, 2007). Several theories have described alterations in the protein structur e that cause protein denaturation during freezing and frozen storage. Temperatures of -20C or lower bring significant deterioration of texture of frozen fish such as toughness, chewiness, rubberiness, or stringiness (Bigelow and Lee, 2007; Sikorski and others, 1976). During extended frozen storage, ice crystals continue to grow. Large ice crystals formed ex tracellularly by slow fr eezing can cause greater damage to cells than small intrace llular ice crystals resu lting from rapid freezing. This leads to an increase in salt concentration in the liquid phase resulting in prot ein denaturation, loss of protein
34 functionality and texture hardening due to a de hydration of the cell and autoxidative changes (Badii and Howell, 2002). As freezing progresses, proteins are exposed to increased io nic strength in the nonfrozen aqueous phase that leads to ex tensive modification of protein native structure (Lin and Park 1998; Jittinandana and others, 2003). In a dehydrated state, protei n-water interactions in tissue are disrupted, and proteins molecules are exposed to an organic environment that is less polar than water. These changes resu lt in increased exposure of hydr ophobic side chains and, changes in protein conformation (Franks, 1995). Denatura tion or insolubilization of actomyosin during frozen storage is a result of protein aggreg ation caused by the progr essive increase in intermolecular cross-links due to the formation of hydroge n bonds, ionic, hydrophobic and disulfide bonds. Also, this may result from incr eased concentration of tissue salts such as calcium which form ionic cross-links between pe ptide chains (Sikorski and others, 1976). This denaturation can be prevented to some extent by cryoprotectants or cryosta bilizers that are very useful for food proteins as well as preserving and stori ng of living cells. Cryoprotectants Intact fish muscle often suffers substantial losses in quality during frozen storage, resulting in water loss from the thawed muscle Cryoprotectants improve quality and extend shelf life of frozen foods by preventing deleterious changes in myofibrillar proteins caused by freezing, frozen storage, and thawing (MacDonald and Lanier, 1997; Jittinandana and others, 2003).There are many cryoprotectants used in fro zen foods. Generally, sugars and polyols such as a sucrose/ sorbitol mixture are commercially used in minced fish products due to availability and low cost to minimize denaturation of prot ein during freezing. They prevent ice crystal growth and the migration of water molecules from the protein, thus stabiliz ing the protein in its native form (Matsumoto and Noguchi, 1992). However, there is an interest in identifying other
35 cryoprotectants with reduced sweetness and less Maillard browning reaction (Park and Lanier 1987). Other cryoprotectants used include polyalcohols, amino acids, oils, hydrocolloids, nonfish proteins, and starches. Other carbohydrates and polyols such as fructose, lactose and glycerol afford better cryoprotection. Hydrocolloid s such as carrageenan a nd alginates have been proposed as cryoprotective additives. All these ha ve its own specific effects on fish muscle. For example, sorbitol and sodium tripolyphosphate ar e effective on muscle proteins, while sodium alginate and carrageenan have a hi gh affinity for calcium ions. Sodium alginate is widely used in the food industry, preventing interactions in muscle fibers and formation of ice crystals (Bigelow and Lee, 2007). It has been reported that sodi um lactate stabilized tilapia actomyosin in model systems against freeze-thaw. Sodium lactate was more effe ctive than sucrose at the same concentration resulting in a good stabilizer a nd cryoprotectant (MacDonald a nd Lanier, 1994). Phosphates have been reported to enhance protein functionality in fish during frozen storage (Park and others, 1988). The effects of various infused cryoprotective ingredients were evaluated during 6 months frozen storage. Red hake fillets were injected with sorbitol-sodium tripolyphosphate (STPP) at (40, to 60%:3%), 1.5% alginate, or 0.75% alginate with soy protein isolated (SPI) sorbitol-STPP (5% to 10%: 40%:3%). Injection of 10% SPI, 1.5 % or 0.75% alginate, or 5% SPI effectively improved water binding and retarded the freeze indu ced texture changes. The SDS-PAGE profile indicated that inject ion of cryoprotectants improved wa ter binding and retarded protein denaturation and intermolecular inte ractions (Bigelow and Lee, 2007). Combination of water, 8% sucros e/sorbitol, or 1% sodium lactate was investigated with or without 0.5% phosphate and with or without 0.05% MgCl2 in the stabilization of trout
36 myofibrillar protein during-20 C for 90 days storage. Compared with the control, cryoprotectants increased total protein and myofibrillar protein solubility; decr eased surface hydrophobicity, total, free, and disu lfide sulfhydryl conten t; and myosin susceptibility to thermal denaturation. Phosphates minimized frozen storage effects on actin solubility (Jittinandana and others, 2003). The use of maltodextrins has been shown to inhibit formaldehyde production in minced blue whiting during frozen storage compared to sucrose. Maltodextrins show better results, preventing protein alterations at higher temper atures (Rodriguez-Herrer a and others, 2002). Research has also investigated different soy protein products. It has been shown that soy protein isolates may be used in emulsified fish products to improve water-and fat-binding properties (Thorarinsdottir and others, 2001). Soy proteins were less effective than salt and phosphate in water holding capacity in frozen cod fillets. When combined with salt and phosphates, the performance was better than salt and phosphate alone (Thorarinsdottir and others 2004). A study using soy protein helped to improve te xtural properties of fro zen fish fillets. This study demonstrated that injecting a 7.5% SPI so lution improved the texture of giant granadier fillet (Crapo and others, 1999). Others compounds have been tested for cryopr otective effectiveness. However; some of them cannot be used because of high cost, are not permitted by food regulations, or because they impart a sweet taste, making the muscle food undesira ble to the consumer. There is an interest in identifying and evaluating other cryoprotectant s for the control of freeze-induced textural changes. Fish Proteins (hydrolysates) Use of fish proteins in fish products can be an alternative to the compounds m entioned above. These proteins have a range of functiona l properties. They can potentially be used as
37 binders, as agents for water-holding, gelation, fat binding, emulsificati on and foaming. Since large amounts of protein-rich byproducts such as head and frames from seafood industry are discarded or processed into fish meal every year the extraction of functional proteins form these byproducts has received increased attention in the past decade (Kri stinsson and Rasco, 2000). Proteins extracted from fish-processing byproduc ts like heads, frames, and viscera can be obtained and modified to improve their quality and functiona l properties by hydrolysis using proteolytic enzymes (Sathivel and others 2003, 2005.). Adding selected proteolytic enzymes preparations to hydrolyze food proteins is a proc ess of considerable importance. Added enzymes have some advantages over chemicals or endogenous enzymes allowing good control of the hydrolysis and the resulting products. Utilizing these enzymes, fish protein hydrolysates (FPH) can be prepared by cleaving spec ific peptide bonds. This woul d increase the number of polar groups resulting with the peptides having new or improved functional ( better absorption) and sensory properties of the native protein without jeopardizing its nut ritional value so these can be injected to fish as cryoprotect ants (Kristinsson and Rasco, 2000). Hydrolysates are defined as proteins that are chemically or enzymatically broken down into peptides of different size. The functional properties of FPH may be improved using specific enzymes and by choosing a defined set of hydr olysis conditions, such as time, pH, and temperature, to partially hydrolyze the proteins to the desired exte nt. This is known as degree of hydrolysis (DH) which indicates th e percent ratio of the numbers of peptides bonds broken to the total numbers of bonds per unit weight. This is on e of the basic parameters that describes the properties of the hydrolysates and needs to be co ntrolled during protein h ydrolysis (Slizyte and others, 2005). Some of the proper ties of hydrolysates are closely related to DH. Breaking peptide bonds results in an increase of amino and carbox yl groups, which increases solubility, decreases
38 molecular weight of proteins and destroys tertia ry structure, affecting the functional properties (Nielsen 1997; Slizyte and others, 2005). Some of these properties depend in part on the water-protein inte raction. Water holding capacity (WHC) refers to the ability of proteins to absorb and retain water against gravitational force within a protein matrix, such as protein gels or beef and fish muscle. ( Fenema 1996). This is very important to the food industry because retaining water in a food system often improves texture. Fish protein hydrolysate s are highly hygroscopic. The presences of polar groups such as COOH and NH2 that increase during enzymatic hydrolys is have a substantial effect on the amount of adsorbed water (Kristinsson and Ra sco 2000 b). A study showed that salmon protein hydrolysates added to salmon mince patties had good water holding capacity, reducing drip loss after freezing. The addition of 1.5% of fish pr otein hydrolysates made from salmon reduced water loss after freezing to 1% compared with 3% for the control. There was no relationship between degree of hydrolysis and water holding capacity ( Kristinsson and Rasco b 2000; Slizyte and others, 2005). It was found th at FHP made with Alcalase, a bacterial enzyme, had better water holding properties in salmon mince patties than egg albumin and soy protein concentrate. A similar study reported that the use of cape lin protein hydrolysates (CPH) improved water holding capacity in minced pork, indicating th at CPH had a strong wa ter-binding capacity (Kristinsson and Rasco 2000 a). Proteins hydrolysates from seal meat were used as a phosphate alternat ive in meat products and its water binding capacity was studied. The cook loss of seal meat was lowest at 3% seal protein hydrolysate, similar to th at of polyphosphates at the same level. When compared with different phosphates, drip loss was lower usi ng seal protein hydrol ysates (Shahidi and Synowiecki, 1997).
39 A number of different proteolytic enzymes can be used for the production of hydrolysates. Alcalase is a commercially available enzyme prep aration that has been widely used in the production of protein hydrolysates due to its th ermostability (50C) and high optimal pH (pH 8.5) that minimizes the growth of microorganisms .It is classi fied as a bacterial enzyme (endopeptidase) and is a food grade preparation. Alcalase or papain produced a hydrolysate with a markedly reduced bitterness and less fi shy odor (Kristinsson and Rasco 2000 a).A study showed that the use of Alcalas e to optimize processing conditi ons to produce capelin protein hydrolysates exhibited superior protein recovery (70.6%), a lower lipid content, and excellent functional properties compared with the alkalin e protease Neutrase and Papain (Shahidi and others, 1995). Alcalase was found to be the most cost effective enzyme out of five enzyme preparations tested to hydrolyze salmon muscle proteins (Kri stinsson and Rasco 2000 a). Studies performed on the hydrolysates produced from sard ine concluded that Alcalase and Papain, at optimum pH and temperature, both gave hydrolys ates with high solubility. Hydrolysates made with Alcalase at higher DH% showed a decrea se in high-molecular weight fractions and increased solubility (Quaglia a nd Orban 1987). The enhanced solubil ity of hydrolysates is due to the smaller molecules and the new exposed i onizable amino and carboxyl groups. Peptides bind water molecules by their ability to form hydrogen bonds between their hydrophilic polar amino acids side groups and the water molecules. Hydrolysis exposes some hydrophobic groups to the surface and converts them to hydrophilic groups by generating two end carbonyl and amino groups. Smaller peptides from my ofibrillar protein hydrolysis have proportionally more polar residues, with the increased ability to form hydrogen bonds with water, increasing protein solubility (Kristinsson and Rasco 2000 b).
40 Although enzymatic hydrolysis of fish proteins allows us to develop desirable functional properties, it can generate bi tterness when added to the produ ct. Fish protein hydrolysates bitterness can be avoided by controlling the degr ee of hydrolysis, thus minimizing the amount of bitter tasting peptides produced during the en zymatic treatment (Ney 1979; Pedersen 1994; Liaset and others, 2003). When a commercial protease was used in Aristichthys nobilis, a connection between the degree of hydrolysis a nd the intensity of bitterness was found. As the degree of hydrolysis increased so did the bitte rness, and after 5 hours hydrolysis samples were described as very bitter (Yu and Fazidah 1994). A later study reported that Papain-hydrolyzed herring had higher bitterness scores than Alcalas e-hydrolyzed herring. Also, Alcalase hydrolyzed samples at higher DH were less bitter than Papain-hydrolyzed samples at the same %DH (Hoyle and Merritt 1994). In order to obtain fish protein hydrolysates of high palatability, the right enzyme must be chosen to produce non-bitter hydrolysates. Am ong the commercial proteolytic enzymes, Alcalase, Flavorzyme 500L, Palatase 2000L Prot ex 6L, Neutrase, and Protamex are known to produce non-bitter hydrolysates (Liaset and othe rs, 2003.). In this study, we decided to use Protamex, a bacterial protease, to obtain hydrolysates from fresh Atlantic salmon frames that would be injected to fresh salmon fillets as cryoprotectants. Effects of Carbon Monoxide on Fish Muscle Refrigeration, freezing and frozen storage are im portant techniques for long term preservation of fish. However, carbon monoxide in fish proce ssing and modified atmospheric packaging is widely used as a supplement for i ce or refrigeration storage to delay spoilage, extend the shelf life, and to preserve and enhanc e the color properties and especially the redness of fish and seafood products (Mancini and Hunt 2005; Sorheim and others 2006). The appearance and color of fresh, and especially those products di rected to the frozen seafood
41 market, is one of the major parameters consumers use to judge the quality of a product because the customer is not able to tast e or smell the actual product since it is either vacuum packed or frozen. Therefore a fresh color and appearance are very important. For salmon a uniform, bright pink, orange or red color is desired for consum er acceptance. The degree of redness (a* values) is one of the most important indicator of quality and freshness in species rich in red muscle such as tilapia, Spanish mackerel, mahi-mahi, tuna and swordfish (Danyali, 2004; Demir and others, 2004; Ross 2000). Since salmon is a fatty fish species, lipid oxidation during frozen and refrigerated storage is a concern. Salmon slices packed under carbon dioxide atmosphere have shown some protection from rancidity, but not as much as expected. Preservati on of the quality and extension of the shelf life of salmon is needed. Color is an attribute that i ndicates freshness and good quality and should be preserved. In other species like Tuna,the color arises primarily from the oxygenated and reduced forms of heme proteins. When these proteins oxidize, an undesirable brown color is produced rather than the desi rable fresh red color of reduced Oxy-Hb/Mb. Oxidized heme proteins are believed to be more pro-oxidative than reduced heme proteins in most circumstances, leading to ra ncidity problems in fish muscle which is rich in unstable polyunsaturated fatty acids (Gorelik a nd Kanner 2001; Kristinsson and others, 2005). The use of carbon monoxide is an alternativ e to extend the shelf life of salmon. Carbon monoxide is a colorless, odorless an d tasteless gas that has about the same density as air. The main objective of the use of carbon monoxide is to maintain the red color of fresh seafood. Studies have demonstrated that CO treatment lead s to less lipid oxidation in fish muscle. Carbon monoxide binds to heme proteins and forms a stab le complex maintaining heme proteins in their reduced state (Kristinsson and others, 2005). A ccording to Kristinsson and others (2005),
42 carboxyhemoglobin was very stable to oxidation even at extreme pH values and temperatures. The same authors also demons trated that the HB-CO comple x had decreased pro-oxidative activity in a model system and may thus extend product shelf life with respect to rancidity. Carbon monoxide attaches to hemoglobin sim ilarly to oxygen but with a binding constant that is 210-270 folds stronger (Demir and othe rs, 2004) displacing any oxygen present in the heme. Because of its great binding affinity to carbon monoxide, carboxy-hemoglobin will accumulate, giving the muscle a bright red color. This binding leads to a conformational change in hemoglobin and myoglobin which makes it very resistant to autoxida tion and discoloration (Kristinsson and others, 2005). Autoxidation of the heme groups to the met fo rm is a critical step in the lipid oxidation process. Met-Hb/Mb reacts with peroxides a nd stimulates production of chemical compounds that initiate and propagate lipid oxidation (Shahidi and Botta 1994). Color depends on the oxidation states of the iron atom in the protein heme group and the type of gas ligands in its reduced state (O2, CO, NO) that are bound to the iron atom On the surface of fresh muscle, oxygen is bound to the ferrous iron yielding oxyh emoglobin/myoglobin which gives the muscle a bright red color. Over time, the hem oglobin will oxidize to form methemoglobin (Fe3+) because oxygen is released from oxyhemoglobin to form ferric (Fe3+) heme iron and the superoxide anion (O2-) (Richards and others, 2002).The formation of methemoglobin gives an undesirable brown color. This can be prevented by keeping fish at very low temperatures (-50 to-80C). However, these practices are expensive. A better practice would be to expos e the muscle to CO to achieve color stability and reduce oxidati on. A study showed that tilapia fillets treated with CO had a significant stabilizing effect on the hemoglobin protein structure (Kris tinsson and others, 2002; Mony and Kristinsson 2004) This treatment also significantly stabilized the red color, reduced
43 microbial growth and developed less lipid oxid ation products compared to untreated tilapia (Kristinsson and others;2003, Garner and Kris tinsson 2004). Carotenoids should be protected from excessive heat, extreme pH conditions, and exposure to light, and oxygen and lipiddegrading enzymes. These factors have negative effects on the stab ility of carotenoids and result in fading of salmonid flesh duri ng frozen storage and processing. Several studies have been conducted to eval uate the effect of gases containing carbon monoxide on the oxidative quality and color of fish muscle. Ludlow and others (2004), Kristinsson and others (2008), tr eated yellowfin tuna steaks w ith various CO treatments at different concentrations and FS (Filtered Smoke) treatment for 48 hr, followed by 30 days of freezing, and subsequent cold storage (4C) afte r thawing. The gas treatments of 18% and 100% CO led to reductions in the formation of secondary lipid oxidation products (TBARS) during freezing and subsequent cold storage of the steak s, possibly due to increased stability of the heme proteins. Stabilization of heme proteins to oxidation is thus expected to reduce the oxidation of lipids. When hemoglobin and myoglob in bind to carbon monoxide, they are very effectively stabilized and remain in the redu ced state and do not easily oxidize, even during abusive conditions (Mantilla 2005). Other studies demonstrated that higher CO levels produce greater color increase and better color stability at different exposure time. A survey conducted on the CO concentration in tilapia exposed to CO for 60 minutes at room temperature and untreated tilapia showed th at the blood colored parts of treated tilapia were bright red contrasting with the dark brown color of the untreated tilapia (Ishiwata and others 1996).Tuna steaks treated with 99.5% CO for 4 hr showed a signif icant increase in a* value (redness) compared to untreated tuna. There were no significant differences between L*(lightness) and b*(yellowness) values between the treated and cont rol sample (Chow 1998).
44 Demir and others (2004) reported that exposure to 4% CO increas ed a* values and preserved color for up to 12 days in refrigerated storage. The effect of filtered wood smoke containing CO in Mahi Mahi has been examined. Results showed that treating fish with filtered smoke increased a* values of the muscle and stab ilized it during frozen storage at -25 C for 30 days. However, a* values decreased rapidly on cold storage at 4C for 8 days. The FS process significantly improved microbial stability as well as stability toward lipid oxidation compared to untreated samples, particularly after thawing (Kristinsson and others, 2007). The effects of CO and FS in Spanish mackerel fillets were also investigat ed. Fillets were treated and kept in the gas environments for 8 days at 4 C. Results showed increase in redness and color stability during storage due to the binding of CO to heme proteins. Lipid oxidation was retarded and a delay in microbial growth was observed (Garner and Kristinsson 2004). Electronic Nose The odor of seafood products has been widely used as one of the ma in indicators of quality since ancient times. Chemical anal yses are not used very often by the salmon industry due to the complexity and length of the methods. Recent de velopments in sensor technology and electronic noses (e-nose) have allowed elect ronic noses to objectively evalua te the odor of food samples. The e-nose unit can correlate the odor of salmon f illets with storage time and with grades from sensory evaluation. The advantages of the methods to assess the quality of salmon are rapid response, no sample preparation, no requirement for chemicals, and are simple to use compared to other analysis tools such as GC, GC-O, GC-MS or sensory ev aluation panels. (Du and others, 2002). It can be used for quality control assessment, freshness evaluation, and process monitoring (Korel and Balaban, 2002b). The electronic nose mimics the human nose. It consists of an array of electronic chemical sensors with partial specific ity and an appropriate pattern -recognition system, capable of
45 recognizing simple or complex odors (Gardner and Bartlett; 1993, Scha ller and others, 1998). The e-nose system tries to simulate the olfactor y process with fewer sensors and with software designed to analyze the responses from the sensor s. Each chemical sensor represents a group of olfactory receptors and produces a time-dependent electrical signal in response to an odor. (Luzuriaga 1999). There are different types of materials used to manufacture sensors for the odor detection. The types of sensors that are being used in commercial electronic noses are semiconductor metal oxides, conducting polymers, and surface acous tic wave sensors (Bar tlett and others, 1997). Other types of sensors are biosen sors, enzymes sensors, electroly tic sensors, and platinum hot wire devices. Also, lipid layers phthalocyanins, and piezoelectr ic materials have been used (Korel and Balaban, 2002b). The me tal oxide sensors commonly used are made with a platinum heater coil coated with alumina. As current passes through the coil, the metal oxide heats up. The reaction between the vapor and the metal oxide causes a change in resistance at a fixed temperature. This change in resistance can be measured and related to the odor being monitored. The electronic nose system consists of sensors, electronics pumps, air conditioner, flow controller, and software for hardware monitori ng, data pre processing, a nd statistical analysis. These systems must be designed for long-term usage with high repeatability (t he ability to obtain the same pattern for a sample on the same array over short intervals of time) and reproducibility (the ability of different sensor batches or different instrument s to produce the same pattern for the same sample) (Schaller and others, 1998). The enose pump is used to pull a sample from the headspace of the material being analyzed and the sensors provide a set of measurements or resistances, which give a specifi c fingerprint of the volatiles pres ent in the headspace at the time of the sniff. The e-nose is used in conjunction with a pattern-recognition algorithm, which allows
46 recognition of different patterns of the traini ng data set, which allo ws on site detection capabilities without much hardware dependency. Some of these applications in clude identifying spilled chemicals, classification of stored grain, water analysis, qua lity of coffee and rancidity in oil (Stetter, 2006). Also the data set obtained from the e-nose can be analyzed using discriminant functions and neural networks (Korel and Balaban, 2002a). The e-nose allows detection of a wide ra nge of volatiles compounds making this technology very versatile (Hodgins 1997). In seafood, spoilage is an important criterion when we need to determine the overall quality of seafood products. Resear ch conducted using an AromaScan electronic nose demonstrated that th e e-nose analyzer could clearly discriminate among 4 different classes of freshness (fresh, early decomposed, very decomposed, and rancid) in Mahi-Mahi and scallops (Du and others, 2001 ). A quality assessment study was performed on salmon fillets under various storage conditions us ing the electronic nose. The responses were compared with microbial counts and histamine production. The results showed that the e-nose could differentiate among fillets stored at differe nt temperature during the spoilage process (Du and others, 2002). The e-nose was used to evaluate the spoilage of shrimp (Balaban and Luzuriaga 1996), fresh tuna at different te mperatures (Newman, 1998), and haddock and cod freshness (Olafsson and others 1992) a nd tilapia (Korel and others, 2001). The e-nose has been used with coffee to differentiate origins and aromas (Delaure and others, 1996.), detecting adulteration of whiskey and wine, and controlling beer fermentation. In dairy products, e-nose has been us ed to differentiate the quality of aged parmesan cheese and the aroma profile of Swiss cheese (Harper and others 1996). In fruits and vegetables, volatiles of fresh squeezed orange juice have been studied as well as in garlic and carrots (Bazemore and
47 others, 1996). The meat industry has also benefited from the e-nose by es timating the quality of beef and detecting adulteration of ground beef with pork (Turhan and others, 1998). Machine Vision The application of treating fish with carbon monoxide or with other gases containing CO is now widespread globally. Carbon monoxide is used to preserve, to stabilize, and enhance the red color of seaf ood products. The CO binds to the he me proteins with great affinity replacing O2 from the heme. CO is able to maintain heme proteins in their reduced state having a positive impact on fish quality (Kristinsson and ot hers, 2005). The CO binding changes the UV-vis spectra of the heme proteins, resu lting in a cherry red color whic h is very stable on both frozen and refrigerated storage. Small quantities of carbon monoxide (>0.5%) are used today in modified atmospheric packaging to preserve the redness of fres h meats such as ground beef (Sorheim and others 1999; Hunt and others 2004). However, a s hort term treatment with higher percentages of carbon monoxide up to 100% is used in the seafood industry to maintain the color of certain seafood products pr ior to frozen storage. Color is an important quality attribute of foods and affects consumer acceptance. Color changes that occur during storage can be used as an indicator of quality deterioration. Color in foods has been measured by visual inspection an d tristimulus colorimetry (Fortner and Meyer 1997). Color in some fish species such as tila pia varies throughout th e fillet, and a single colorimeter value is not a good representation of the actual colors. Several color readings at different locations are needed and averaged to obtain a result which will be an approximation only and the valuable color distribution data in lost (Balaban and others 2005). A solution can be the use of computer vision for f ood color evaluation and quantification. A typical computer vision system consists of the illumination set up for the acquisition of images, a camera for capturing the images and a co mputer. After images are captured, they are
48 sent to the computer for furt her processing. The computer is used for designing the algorithm that enables feature extraction, segmentation, quantification and cl assification of images, and the objects or regions of interest as opposed to whole images can be evaluated (Misimi and others 2007). The image is digitized by using image proce ssing techniques, and divided in small regions called pixels. Each pixel has the information of grey levels for a black-and white image, or the levels of the three primary colors (red, blue a nd green RGB) for a color image (Luzuriaga 1999). This information can be analyzed by computer so ftware describing all colors present in a food sample and the amounts of each color (Martinez and Balaban 2006). There are two important steps in the comput er vision algorithm design: image processing and image analysis. Image processing involves a series of image operations that enhance the quality of image in order to remove defects su ch as geometric distortion, noise, and nonuniform lighting. Image analysis distinguishes the objects from the background giving quantitative information used for decision making ( Brosnan and Sun 2004). Computer vision is being used in the agri cultural and food industr y ,grading and sorting products by analyzing colors in maturity stage (Okamura and others, 1993), defect detection in fruits, separating fresh market vegetables based on visual quality, analyzing color, shape, and blemishes of citrus (Miller and Drouillard 1997). In the seafood industry, machine vision has b een used for quality assessment and sorting of Atlantic salmon fillets according to their color level (the flesh redness of salmon) (Misimi and others, 2007). Korel and others (2001) used machine vision for quality assessment of raw tilapia fillets. Also it has been used in calculations to obtain average L, a* and b* values and ratios before and after CO treatments in tilapia ( Mant illa 2005), quality assessmen t of color of shrimp and salmon ( Luzuriaga 1999), texture and color prim itives analysis at different thresholds and
49 contour analysis at different thresholds (Bal aban, 2008) in food products by this nondestructive method. Research Objective The objectives of this research were to quan tify the effects of CO in Atlantic salmon spp. and the use of protein inject ion (salmon hydrolysates) as cr yoprotectants. An enzymatic hydrolysis process was used to extract functi onal proteins from salm on using Protamex (a bacterial protease.) The first hypothesis tested wa s that the treatment of salmon with CO would minimize lipid oxidation. The sec ond hypothesis tested was that the use of fish proteins (salmon protein based) injection as cr yoprotectants would minimize freeze-t haw damage to muscle after processing, freezing, storing, and then thawing. The effect on color, CO uptake, microbial load, lipid oxidation, texture, water holding capacity, and odor were evaluated for control and treated samples.
50 Table 2-1. Intrinsic characteristic s of wild and farmed salmon Size (kg.) Average [range] Total fat Omega-3 Primary product Species [low, high] (100 g) (100 g) Form Harvest Season King 8.6 [1.8, 18] 11.5 1.8 Fresh/frozen May-Sept. Sockeye 2.7 [1.8, 4.6] 7.5 1.1 Fresh/frozen/can ned May-Sept. Coho 4.6 [1.8, 8.2] 5.7 1.2 Fresh/frozen July-Sept. Chum 3.6 [.9, 5.5] 5.3 0.8 Canned June-Oct. Pink 1.37 [.9, 2.7] 5.3 1.7 Canned July-Aug. Farmed Atlantic 1.8-2.7 10.9 1.8 Fresh/frozen Jan.-Dec. Farmed Coho 1.8-2.7 7.7 1.2 Fresh/frozen Jan.-Dec. Source: USDA nutrient database; Al aska Manufacturers Association. Table 2-2. Fresh SalmonPremium quality indicators Eyes Clear, bright, convex eyes, black pupil, and clear cornea Gills (if present) Bright, pink-red, free of slime Skin Shiny, bright belly and sides, dark dorsal area, no superficial scars or scrapes Body Stiff, straight, firm and elastic. Resilient when pressed lightly Flesh color Varies with sp ecies but should be uniform, bright pink, orange or red Smell Ocean-fresh, slight seaweed scent Scales Adhere tightly to skin, bright silvery cast, few missing Belly Cavity Belly lining inta ct; flesh tight to the bone, no cuts or tears, no discol oration of membranes. Source DSM nutritional products 2005.
51 Figure 2-1. Relationship between redness (a*) and carotenoid con centration (mg/kg) in Atlantic salmon fillets.
52 Figure 2-2. Relationship between redness (a*), yellowness (b*) lightness (L*) and carotenoid concentration (mg/kg) of Atlantic salmon fillets (Based on data from Bjerkeng et al., 1997a).
53 Figure 2-3. The Roche Salm oFan.
54 CHAPTER 3 MATERIALS AND METHODS Fresh salmon Fresh Atlant ic salmon ( Salmo salar, whole fish, eviscerated, and chilled) that were farm raised in Chile were purchased from a certified distributor in Mi ami, FL. A time record of the salmon (harvest, packaging, and receiving of the fish at its distributor in Miami) was obtained to make sure fresh samples were used. The fish we re no more than 48hr old when they arrived at our lab. During transportation, whole fish were packaged individually in plastic wrap and were shipped in Styrofoam boxes with cold packs and i ce. Fourteen salmon were used in this research with an approximate weight of 4.1 kg for each fi sh. Immediately after receiving, the salmon were filleted (Figure 3-1) and equal size fillets were ob tained (Figure 3-2). Afte r filleting, the skin was removed (Figure 3-3) and samples were separate d into 3 lots: one for control, one for CO treatment and one for protein injection combined with CO treatment. All samples were kept on ice and refrigerated until further treatment Determination of Protein by Biuret Reaction Fresh salmon fra mes, obtained from the previous ly filleted fish and kept in refrigeration, were ground at 4C in a Scoville grinder (Pro fessional Meat Grinder MG 800, Waring Pro. East Windsor, NJ) with 6 mm holes. To 1 gr. of min ce was added 10 ml of 0.1N NaOH in a beaker and homogenized with a Bio Homogenizer (M 133/1231 O 2 speed. Biospec Products, Inc, Bartiesville, OK) at setting 2 for 1 minute. From the homogenized solution, 100 L were taken and placed in a test tube to which 900L 0.1N NaOH was added. After this, 4 ml of Biuret reagent (Lab. Chem. Pittsburgh, PA) was added and the solution was incubated for 30 minutes at room temperature. The Biuret reag ent is used for a reaction between cupric chloride and proteins at alkaline pH, which leads to the formation of color complex with absorption maxima at 540
55 nm. At the same time a blank solution was prepar ed by mixing 1 ml of 0.1N NaOH plus 4 ml of Biuret reagent. Using a spectrophotometer (A gilent spectrophotometer 8453, University of Florida) the absorbance was read at 540nm. A standard cu rve using BSA (Bovine Serum Albumin) (Sigma Chemical Co., St. Louis, MO) (mg/ml) was used to quantify protein levels. The amount of protein found was 1.85 BSA (mg/ml). The process flow is de picted in Figure 3-7. By calculating the protein concentration, we were able to determine the amount of mince, volume of distilled water and the amount of enzyme needed to prepare the fish protein hydrolyzates. Preparation of Fish Protein Hydrolyzates Atlantic salmon fram es were th awed at 4C for 6hrs and minced in a Scoville grinder with 6 mm holes. The hydrolysis conditions were si milar to those documented by Kristinsson and Rasco (2000 a). The substrate was prepared by mixing 216.2 g. of frame with 2000 ml of deionized water and homogenized with an Ultra-turrax 19 homogenizer (IKA T 19 basic,Wilmington, NC ) for 2 minutes. The mixture was placed in a plastic container and was put into a water bath at 40C. The mixture was con tinuously stirred and the pH of the mixture was adjusted to 7.5 using 1 N NaOH. The pH was mo nitored to maintain a constant pH of 7.5 for about 4 minutes. The enzyme solution, prepar ed by diluting 22.16 g. of Protamex (Bacillus protease complex developed for th e hydrolysis of food proteins. Novozymes, North America) in 10 ml of water, was added to the mince with co ntinuous stirring. The pH was adjusted again to 7.5 by adding 1 N NaOH. This was monitored until it remained constant for about 4 minutes. The amount of base added was recorded and used to calculate the DH (Degree of hydrolysis). The DH is calculated from the volume and molarity of base used to maintain constant pH and is expressed as the percent ratio of th e numbers of peptide bonds broken (h) to the total numbers of bonds per unit weight (Kristinsson and Rasco, 2000). After the pH remained constant, the protein
56 solution was transferred to a Ziploc bag and imme rsed into a water bath at 95C for 10 minutes to inactivate the enzyme. Then the reaction mixture was cooled down at room temperature for 10 minutes and put on ice in a refrigerator. Th e process flow is depicted in Figure 3-8. Protein Injection Whole f illets for injection were selected and la beled then were weighed to obtain the initial weights. The initial weight of a whole fillet was approximate ly 940 g. The fish fillets were injected with the protein soluti on prepared above to obtain a 12 % increase in weight. After the injection, fillets were wei ghed again and the weight ranged between 1.040 kg to 1.050 kg. The injection was done manually using a stainless steel air pressure injector (Alloy Products Corporation, 5 liters capacit y, Bedford, Massachusetts) which has 20 needles (gauge 18) on a 5.08 x15.24 cm platform (Figure 3-4). The injections were made in a consistent pattern on every selected fillet. In order to achieve this, variab les controlled during inje ction of protein solution were: number and location of injections, the angl e (90) and depth of needle to the muscle, quantity and homogeneity of solution. Injec tion allows relatively uniform dispersion of cryoprotectant throughout the ti ssue (Bigelow and Lee, 2007). CO Treatment of Salmon Fillets Fillets were labeled and divided into 3 groups : one served as the contro l, one for CO treatment and CO treatment of samples already injected with hydrolysat es. Each group had 3 fillets. For CO treatment, samples were placed in a box built with Polyca rbonate Lexan sheets of dimensions 57.15x 45.72 x 30.48 cm with wall of 1.27 cm thicknesses. All f illets for treatment were put on plastic netted shelves to allow exposure to CO of th e entire fillet from both sides (Figure 3-5). A perforated tube was installed inside the gas cham ber to assure good circulation of CO (Figure 3-6). The box was flushed with 100% CO for 15 minutes by passing 748 L of CO. The gas was flushed seven times the volume of the box to assure the desired concentration.
57 Experimental procedures employed safety protocol s to prevent potential es cape of CO. Standard Operation Procedures were developed to assure safety and prevent any exposure to CO. The experimental designed is outlined in Figure 3-9. The box had connections for gas and compressed air. Two valves, one input and one output, were located at opposite sides at the t op surface. When a gas containing CO was flushed out from the box it passed through a Hopcalite cata lyst tube (A manganese-dioxide copper-oxide mixture) to convert it to CO2. For this purpose, a catalytic converter was built with enough volume to convert CO into CO2 and maintain a low pressure dr op in the system. Hopcalite was packed into the tube after it was welded to shape. Glass wool was placed at both ends to keep the granules in the tube and prevent the escape of Hopcalite dust into the ch amber. Since Hopcalite degrades with moisture, a moisture trap was insta lled before the gas entered the catalytic tube (a modified commercial car catalytic converter). We operated the cat alyst tube at 95C to assure complete conversion of CO to CO2 (Mantilla 2005). Two CO mon itors were used; one at the entrance of the tubing and the ot her at the exit of the gas. Th e volume of gas passing through the box was measured with an Alicat Sc ientific flowmeter (M-1SLPM-D (CO2), Tucson, Arizona). After the box was flushed with the desire d concentration of CO, it was kept under refrigeration for 48 hours. Control samples were kept under refrigera tion for 48 hrs. After treatment, whole fillets were cut into ten pieces from head to tail, divided, and labeled according to their different analysis in e qual size pieces (12x5 cm). The fish were handled as aseptically as possible during all processing steps by working on sterilized surfaces and with sterile gloves and tools, then vacuum packed in se parate sterile food saver bags (E VIIR, Walmart stores) and stored for 30 days at a freezing temperature of -30C. The samples were then thawed and refrigerated
58 for 1 week at 4C. Three fillet portions (replicates) were used for each analysis and each fillet portion came from a different fish. All analyses except color were performed in triplicate. Color Analysis by Machine Vision System A digital Color Mach ine Vision System (CMV S) described by Luzuriaga and others (1997b) was used to grab images and measure the average L* (light ness), a*(redness and greeness) and b* (yellowness and blueness) values of salmon fillets. This system consisted of a light box with illumination from D 65 fluorescent lamps (Lumichrome F15 W 6500K, Germany), a Nikon D200 color digital camera (N ikon D200 Digital Camera,Nikon Corp., Japan) located inside the light box, connected to a co mputer, with a USB connection, to acquire the images and processing images with Color Expert data analysis software (Lens Eye) written in Visual Basic by Dr. Murat Balaban (2006). The system was set up following the procedures detailed by Luzuriaga and othe rs (1997b); Luzuriaga, (1999); and Martinez and Balaban (2006). RGB (Red, Green and Blue) values were calcu lated. The L* value measures lightness and darkness ranging from 0 (pure black ) to 100 (pure white). The a* value represents redness, where a negative (-) a* value corresponds to greenness and a positive (+) a* value redness. A positive (+) b* value represents yellowness and a negative (-) b* value blueness (Wallat and others 2002). A red, Labsphere (North Sutton, NH) color refere nce tile was used for color calibration (L*= 49.60, a*=52.87, and b*=25.73). The color analyses we re performed on controls as well as treated fish fillets. Individual pieces of salmon fillets were placed in the light box, which was illuminated with front lighting fluorescent lamps. Pictures of the salmon fillets for each treatment were taken and saved in a computer file. The whole fillet surface was used to analyze color. Color analysis was done on previously cleaned pictures to avoid any inte rference with the color analysis. All the analyses were performed at Da ys 0, 2, 32, and 39 and the same fish fillets were used to analyze their color change of the 3 treatments at each storage time.
59 Texture Analysis Texture profile analysis (TPA) (Bourne, 1978) was performe d on control and treated samples using an Instron Universal Material Tester Model 4411 (Instron Corp., Canton, MA) at room temperature and previously stored at 4C for not more than 1 hr. prior to TPA analysis. Three samples of rectangular shape (2 cm x 2 cm x 1.5 cm) were cut from the midsection of each salmon fillet and analyzed using a #15 cylindrical-shape probe (38 mm in diameter). The test consisted of two successive compressions to a value of 70% of the unloaded specimen height at 100 mm/min speed and 100 Newton compression loa d. Texture analysis parameters (hardness, springiness cohesiveness, adhesiveness, and chewiness) were calculated for each sample using Blue Hill Software (Norwood, MA). All the analyses were performed in triplicate. Lipid Oxidation Lipid oxidation was performe d by analyzing for thiobarbituri c acid-reactive substances (TBARS) using the method of Lemon (1975). For th is test, salmon muscle was excised from the fillet portion and ground in a Scovi lle grinder (Sunbeam products, Boca Raton, FL., USA.). Five g. of ground muscle were mixed with 15 ml of the extracting solution (trichloroacetic acid (TCA)) for 30 seconds in a small stainless stee l blender. Then the homogenate was filtered through Whatman #1 filter paper. The filtrate was used for the TBA (thiobarbituric acid) reaction by mixing 2 ml of filtrate and 2 ml of TBA in a test tube with a screw cap. The tubes were vortexed for 10 seconds at room temperature. A blank was made by mixing 2 ml of TCA solution and 2 ml of TBA soluti on. Tubes were tightly capped and heated in boiling water for 40 minutes, followed by cooling in ice, opening the tubes, and allowing to stand for 5 minutes at room temperature. After the reaction was completed extracts were taken and their absorbance at 530 nm against the blank was determined us ing a spectrophotometer (Agilent 8453). The TBARS values were calculated from a standa rd curve prepared using malondialdehyde (MDA)
60 and the values expressed as mol MDA/kg of tissue lipids. All the analyses were done in triplicate from day 0 to day 39 every 4 days. Water Holding Capacity A Sorvall RC-5B Refrigerated Superspeed Centrifuge (Dupont Instrume nts, Wilmington, DE) with SM-24 rotor type was used, following the procedures described by Hussain (2007), to study water-holding capacity of cont rol and treated salmon fillets. As outlined in this procedure, a single sheet of Whatman # 3 filter paper was pl aced inside the centrifuge tubes to absorb excessive moisture released during centrifugation. Filter paper was folded to the shape of the centrifuge tube. Salmon fillets were minced at 4C in a Scoville grinder ( Hamilton Beach, Washington, NC) with 6 mm holes. Approximately 10 g of minced muscle was weighed accurately, placed inside the filt er paper and immediately centrifuged at 269 x g (1500 rpm) for 15 minutes at 4C. After centr ifugation, the samples were taken out and weighed again, excluding the moisture absorbed by the filter paper. The weight loss after centrifugation was divided by the initial weight and expressed as WHC (water holding capacity). Analyses were done in triplicate. Quantification of CO in Muscle The method describ ed by Miyazaki (1997), and Mantilla (2005) was used to determine the concentration of CO in salmon muscle. Salmon f illets were minced, 6 g. accurately weighed and were transferred into a 60 ml headspace bottle. Th ree (3) drops of 1-octanol (antifoaming agent) and 12 ml of 10% sulfuric acid were added to de nature the heme proteins and release CO. The mixture was shaken for 10 sec, and then incuba ted for 5 minutes at 40 C. After incubation, the tubes were shaken at room temperature for 15 mi nutes. Samples were kept frozen and analyzed at day 0 and day32 for control and treated samples in triplicates. The anal yses were carried out by taking 100 l of the head space gas and injecting into an Agilent (6890 N, Natural GC
61 system) GC 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 The injector port temperature was 100C, column 35C, methanizer temperature 375C, and detector temperature 250C. The carrier gas was Helium (grade 5, Air Products,Gainesville, FL) with a flow rate of 27mL/min. The reducing gas used was hydrogen (94% purity, Air Products, Gainesville, FL). The retention time and area of the CH4 peak were compared to those obtained with a calibration CO gas. CO levels were then calculated base d on a standard curve constructed by injecting different known levels of 100% CO. Aerobic Microbial Growth Microbial analysis (Total Pl ate Count TPC) was performe d using aerobic plate count Petrifilm TM (3M Laboratories, St. Paul, MN ., USA) according to the official 990.12 AOAC method (AOAC, 1995a) before and after treatment, at days 0, 2, 32 and 39 for control and treated samples. The dilutions were made using pre-f illed sterile disposable diluent bottles (Fisher Scientific). Triplicate inoculations were c onducted for each dilution and Petri-films were incubated at 35C for 48 hrs. To obtain aerobic microbial coun ts, only films having between 25 to 250 colonies were read. Microbial counts were expressed as log10 colony forming units (cfu) per g of sample. Freshness (Odor) by Electronic Nose For this study a Cyranose 320 (Smiths Detect ion, NJ) containing 32 thin-film carbon-black polymer sensors was used to sniff the headspace of the salmon samples. The Cyranose was used following the procedures outlined by Martinez (2007) and Luzuriaga (1999).In order to quantify the sensor responses to differences in odor of sa lmon samples, the sensor resistances data were recorded in real time by the Cyranose data acqui sition software. Five repl icates were performed
62 for each sample. The data was analyzed usi ng Statistica 7.0 Software with multivariate discriminant analysis (Korel and Ba laban, 2002 b;Oliveira and others, 2005). A 45 g. piece of salmon fillet was taken out of re frigeration prior to analysis to let the sample equilibrate to room temperature for 45 minutes. The samples were put in an odorless petriplate and placed in a sample holder de vice designed by Dr. Murat Balaban and Luis Martinez (University of Florida, Gainesville, Fl., 2005) composed of two glass sample holders, one for baseline air and another for the samples being analyzed, two moisture traps ( Alltech hydro-purge II), one activated carbon capsule (Whatman Carbon-Cap) both purchased from Fisher Scientific, and a compressed air tank (Air Products, Gainesville, FL). Every day prior to the experiments the elec tronic nose was turned on and compressed air was passed through the sensors for about 10 minutes. Between each sample, the sample holder was flushed with compressed air for 5 minutes to eliminate odors from the previous sample, allowing consistent and accurate readings between samples. The sensor head was then purged for 5 minutes with compressed air while the sample volatiles were equilibratin g in the headspace of the holder. The settings for th e e-nose were: 20 sec. baseline pur ge, 60 sec sample draw reading, 10 sec. for snout removal and 45 sec. air intake purge at high speed. The sensor response data was acquired for 5 minutes for each replicate. Five sniffs were performed per sample with an analysis time for each sample of 20 minutes. All the data generated by the 32 sensor resistances for each step of the sniff were analyzed to obtain the maximum difference from the baseline to the highest resistance point of the sample exposure step within the sniff ( R/R).For each reading at 5 minutes exposure, the sensors resistances of the samples were recorded for each run for the duration of the sniffing and used for data analys is. To be able to analyze this data with discriminant function, eight out 32sensors were chosen for each sample. The sensors that showed
63 a higher R/R value than others, which means that these sensors were more sensitive to the salmon odors than others, were selected and their data was analyzed. Statistical Analysis Results were analyzed statistically using analysis of variance (ANOVA) to look for significant d ifferences between the 3 treatments at each storage day employing the statistical analysis system (SAS) computer program (version 9, Cary, NC). Each analysis was performed in triplicate. Means were separated using Duncans multiple range test (P<0.05).
64 Figure 3-1. Salmon filleting process Figure 3-2. Equal size fillets
65 Figure 3-3. Skin removing process Figure 3-4. Air pressure prot ein injector used in the application of hydrolysates
66 Figure 3-5. CO box gas used for the salmon treatments Figure 3-6. Front view of CO box treatment. Tubing system is depicted in this view.
67 Table 3-1. Nikon D200 camera settings Camera: Nikon D200 Hue Adjustment: 0 Lens : VR 18-200 mm F/3.5-5.6 G White Balance: Direct Sunlight ISO : 100 Zoom: Manual Exposure: Manual Shutter speed: 1/3 s F11 Exposure compensation: -1.0EV Focus: Manual Sharpening: Normal Tone Compensation: Normal Color Mode: Mode I Saturation: Normal
68 Figure 3-7. Outline of the determination of prot ein content in salmon frame by Biuret reaction. GRIND MIX 1g. MINCE 10ml NaOH 0.1N HOMOGENIZE SOLUTION MIX 100L H.sol. 900L NaOH 0.1N ADD 4 ml BIURET REAGENT INCUBATE 30 MIN. AT ROOM T. READ ABSORB. AT 540 nm SALMON FRAME
69 Figure 3-8. Outline of enzymatic hydrolysis process of salmon frame mince Homogenization Adjust pH 7.5 NaOH Enzyme reaction Adjust pH 7.5 NaOH Dilution 2000 ml distiled water Salmon frames Mince 216.2 g Heat inactivation Cool down Refrigeration
70 Figure 3-9. Experimental design fo r the control, CO treated, and CO treated injected samples. Fresh salmon filleted Control samples CO treated Injected hydrolysatesCO treated Vacuum pack and freeze 30 days at -30C. Fillets thawed and maintained in refrigeration 1 week -TBARS -E-nose -Texture analysis -Microbial count -Water holding cap. -Color -CO Day 0 Day 2 Day39 -Color -E-nose -Microbial count -TBARS Day 32 -TBARS -E-nose -Texture analysis -Microbial count -Water holding cap. -Color -CO
71 CHAPTER 4 RESULTS AND DISCUSSION Effect of Carbon Monoxide on Color: A Color Machine Vision System was used to analyze the color and color chan ges of the sam ples during this study. The average a*-value of each sample was determined for control and trea ted samples, before and after treatment over a period of 39 days, 32 in frozen st orage and one week in refriger ation after thawing. The samples were statistically compared using analysis of variance ( ANOVA) to look for significant differences between the 3 treatments means at ea ch storage day (Figure 4-2). Duncans multiple range test (P<0.05) was conducted to determine which sample means where different from each other. The L* values (lightness) and b* values (y ellowness) were also analyzed and are shown in Figures 4-1 and 4-3. The graph shows the average L* values of salmon at day 2 before treatment, and from day 5 through day 32 after treatment and storaged at -30C then one week in refrigeration at 4C.The samples were treated with 100% CO for 48 hours. Treatments with the same letter are not significantly different from each other. All fillets showed a distinctiv e and stable orange-pink color. Kristinsson and others (2005) reported that the application of CO had a positiv e impact by stabilizing heme proteins therefore the red color of the muscle of several fish species. The effect of the two treatments was hardly noticeable compared to the control fillets after treatment (Figure A-1, appendix A). However, th ere were significant differences among the samples before any treatment were applied, when comparing CO treated sample with control and with CO treated-injected sample, which can be e xplained by the way sample fillets were visually sorted and assigned to the treatments; differences in a* values can be seen in table 4-2. Figure 4-2 showed no significant difference (p>0.05) in a* values among salmon fillets after treatment. After treatment, on day 5, CO treated sample showed an increase in a*-values
72 when compared with the CO + protein and the control before treatment. The 100% CO gassing method of fillets sign ificantly increased (p<0.05) a*-val ues from 33.17 to 37.57 from day 2 to day 5 due to an increase in the concentra tion of bound CO in the muscle during 48 hours treatment where carbon monoxide ha d a lot of time to penetrate into the muscle and form the carboxy-myoglobin complex that is responsible fo r the redness of the sample. Upon treatment, significant amounts of CO could remain unbound in the muscle, and during freezing and also thawing the unbound CO could become bound to fr ee reduced heme proteins increasing or maintaining the redness. The a*-values for th e CO + protein samples fillets decreased no significantly (p<0.05) from 38.02 to 35.16 after treatme nt from day 32 to day 39 (Table 4-2). In short we can conclude that there were no significant differences fr om day 5 through day 39 at all. The graph shows the average a* values of salmon at day 2 before treatment, and from day 5 through day 32 after treatment and storaged at -30 C then one week in refrigeration at 4C.The samples were treated with 100% CO for 48 hours. Treatments with the same letter are not significantly different from each other. The redness in the CO treated sample decrease d slightly from day 5 to day 32 but always maintaining a higher a* value compared to the day before treatment and slightly higher than control at each frozen storage time. Danyali (2004) also noted an increase in a* values when CO treated yellowfin tuna steaks were subjected to 30 days of stor age at -25C. The CO+protein samples follow nearly the same pattern shown by CO treated samples by decreasing slightly except for day 32 that showed and increase in a* value. The frozen storage affected to a small extent the a*values of the control and the gassed fillets. On the other hand CO+ proteins sample s had much better stability during freezing period. Control samples were affected the most by the fr eezing and thawing as there a* values decreased
73 from 36.80 to 35.06 which could be a result of incr eased spoilage and oxidation. Gassed fillets were also affected by the freezing, but their a* va lue after thawing was s till higher that the one before treatment. In the final storage after thawing all the treated samples a* values decreased. These results are consistent with results on CO trated tuna, ma hi mahi and Spanish mackerel, which all show an increase in a* value on treatment, but a gradual decline after defrosting (Demir and others, 2004; Ross, 2000; Garner and Kristinsson, 2004) In salmon the b* value (yellowness) is importa nt too for the orange combination color and it would be influenced by the oxidation of lipid s and proteins with the formation of yellow pigments (Thanonkaew and others, 2006). Both of the treatments did have an effect on b*values (yellowness). The results (Figure 4-3 ) were quite similar to the findi ngs of the a*values, where the b* values follow the pattern of the a* values. Before gassing, CO treatments sample s had a b*-value significantly (p<0.05) lower than b*-values from the contro l, and CO + protein samples. After 48 hours treatment, the b* values of the CO treated samples increased significantly from 32.07 to 35.32 while the b* value of the CO + protein samples decreased but not significantly from35.85 to 32.68, and the control remain about the same value. These findings co nfirm that there are changes in the b*values affected by CO treatments, contrary to the reports of others studies (Danyali 2004; Garner 2004; Balaban and others, 2005), were li ttle effects on b* values were reported. Kristinsson and others (2006) reported that no significan t changes have been found in other studies among b* values for CO treated fish. There were no significance diffe rences after the frozen storage when the b* values of the control and CO treated samples d ecreased slightly but the b* value in the CO + protein samples increased during frozen storage. The b* values of control, treated, and treated
74 and injected samples decreased in the fina l week under refrigeration but they showed no significant difference (Table 4-3). The results from the analysis of L* values ( lightness) of the 48 hours control, treated, and treatedinjected samples showed significant differences (before treatment at day2 and after treatment day 5) Figure 4-1. For the control and CO + protein samples, there were significant increase (p<0.05) while the CO treated samples decreased but no significantly. During frozen storage all samples showed no significant diffe rence. By day 32 afte r thawing all samples showed a close L* value between 63.5-64. However, after storage under refr igeration for a week the fillets treated with CO+protein had an increa se in the L* value from 63.5 to 65.2 (Table 4-1). In other study, fish protein hydrolysate when inco rporated in lizard fish surimi showed yellow color and decreased lightness duri ng storage (Khan and others, 2003). Texture Texture analyses were performe d on contro l, CO treated, and CO +injected with cryoprotectants (hydrolysates), obtained from the hydrolysis of salmon frame using a proteolytic enzyme (Protamex), to determine firmness among the samples at each storage time. The injected samples with 10% increase in weight were comp ared to control and CO treated samples. The texture values were evaluated at day 5after treatment, and da y 32 after one month of frozen storage, been the initial hardness of the sa mples different. Figure 4-4 showed no significant difference (p>0.05) in firmness between control and CO treated. However, salmon fillets CO treated and injected showed si gnificantly lower (p<0.05) firmne ss (N) compared to control and CO treated samples at day 5. This decrease in ha rdness may have been due to the 10% injected protein solution. The proteins ha dnt bound completely to the muscle ; also there was an increase in moisture retention compared to the other samp les. These results are difficult to compare with other studies since most of the work done has us ed soy protein isolated, sorbitol, phosphates, and
75 hydrocolloids incorporated to food muscle. After 30 days under frozen storage at -30C, control and both treatments, showed no significant difference (p>0.05), but the c ontrol and the sample treated with CO decreased their hardness while the sample treated with CO and injected with proteins increased its hardness during the freez ing storage probably due to the binding and gelation of the protein inside the fillet muscle. Table B-1 Appendix B. Carbon Monoxide Quantification by GC For our study, in order to determine the am ount of CO retain by the salmon m uscle, CO concentration in the muscle was quantified using a GC equipped with a flame ionization detector (GC-FID). This quantification was based on an injection volume of 100 l and their respective peak area, seen in its chromatogram (Figure C-1, C-2 Appendix C). This technique has been used lately to determine whether a product has been treated with CO to cover the quality of temperature abused product. The CO that was ab sorbed into the tissue during the treatment was released into the headspace of the vial where it was sampled with a syringe and then analyzed with the GC detecting CO at parts per million (ppm) levels. Samples were taken from each treatment at day 2 after treatment and da y 32 after frozen for 30 days at -30C. In Figure 4-5 it can be clearly seen that there was a difference in CO concentration between the CO treatments and th e control after treatment but not after the freezing and thawing process. Control samples had low levels of CO (0.12 ppm). It was expected to find some CO in the muscle since endogenous CO is produced duri ng the metabolism of protoheme (Ishitawa and others, 1996). An increase in CO concentra tion on extended storage has been reported previously, and is one of the indicators used by th e Japanese health authorities that fish has not been treated (Ishitawa and others, 1996). If fish has been treated w ith CO, the level is expected to decline which was the case for th e CO treated salmon in this study.
76 After treatment a significant (p <0.05) increase in CO concentration was noted for both CO + protein and gassed samples (Figure 4-5). CO concentration increased to 3.12 ppm and 1.62 ppm for the gassed treated and the CO + protein fillets respectively Table C-2, C-3 Appendix C. After thawing, the concentration of CO in the ga ssed fillets remained considerably higher (~0.5 ppm) than the level for the CO + protein fillets (0.15 ppm) and that of the control (~0.1ppm). The initial pick is maybe due to the additional gas trapped in the extracellular matrix of the samples (Mantilla, 2005). The greater amount of bound and released CO fr om the treated samples lead to a higher concentration of CO and greater a* value (redne ss). On day 32 after frozen storage, the amount of CO between treatments decreased (it was not significantly different) but still the CO treated samples showed a higher amount than the control and CO+protein, indicati ng that CO was still bound to the heme group of that sample. Also si nce oxidation occurred to some extent for COHb samples subjected to frozen storage, we can explain that a considerable amount of CO was lost from Hb at -30C. These results agreed with the study of Kristinss on and others (2005) in tilapia fillets. Water Holding Capacity Water holding capacity of proteins added to mu scle food is of great importance to the food industry because retaining water in a food system improves text ure. This depends on how well the proteins bind and hold water in the food sy stem. According to Kristinsson and Rasco (2000 a) fish proteins hydrolysates ar e highly hygroscopic and have excel lent water holding capacity. If the fish is put under stress before being slaughter ed, this will greatly influence its final pH and thus its water holding capacity (Terlouw, 2005). As the muscle pH decreases post-mortem, the number of negative charges decreases on the muscle proteins and they are moved closer to their isoelectric point.
77 Muscle has its lowest water-holding capacity at the isoelectric point of the myofibrillar proteins (Foegeding and others 1996). Water holding capacity analyses were performed on minced salmon muscle from control, treated with CO, and treated+injected with hydrolysates at 10% injection level. Statistical analysis s howed significant difference (p<0.05) among the two treatments and the control. Figure 4-6 shows that the CO + protein samples had the highest initial water holding capacity at day 2 and after 1 month of frozen storage. At day 2, fillets injected with hydrolysates improved water-holding capacity w ith weight loss significantly less as seen in Figure 4-6 .It showed a weight of 8.76 grams. CO treated samples al so showed significant difference with the control at day2. The use of en zymes on proteins helped the formation of more peptides during the hydrolysis. These peptides plus amino acids formed the hydrolysates. Since we use a bacterial protease that worked as an endo-exo protease, it was able to brake protein in more polar groups COOH and NH2 (charged groups), that increased during enzymatic hydrolysis, having a substantial effect on the am ount of absorbed water because these charged groups bind to water. This can explain the sign ificant difference between injected samples and the others where a higher water holding capacity was obtained and the others. Water holding also is exponentially related to the pr otein content of the muscle, as the protein content increases, water holding capacity increases. At day 32, after freeze/thaw cycle, there were no significant differences between control and CO but CO+injected samples still showed a si gnificant difference compared to the other two treatments maintaining its weight from day 2. They only lost about 1.2 grams from their original weight (10 g.).Table B-2 Appendix B. The increase in water binding can be attributed to the cryoprotective actions of the cryoprotectant inject ed (hydrolysates) into the salmon fillets. Also these injected cryoprotectants are responsible fo r the improvement of te xtural properties by
78 reducing freeze-induced shrinkage of myofibrils The good water binding ability of hydrolysates helped to reduce the amount of free water availa ble for ice crystallization, reducing protein intermolecular interaction, resulting in less freeze -induced shrinkage of muscle myofibrils during frozen storage. These results agreed with the study of Kristinsson and Rasco (2000a) who utilized fish protein hydrolysat es obtained from salmon muscle, using several enzymes, and added to minced salmon patties reducing wate r loss after freezing for 48 days. The use of Alcalase (Proteolytic enzyme) showed the best water holding properties than egg albumin and soy protein concentrate ( Kristinsson and Rasco ,2000a) Lipid Oxidation Lipid oxidation was studied using the TBAR S method to monitor le vels of secondary oxidation products formed from the degradation of lipid hydroperoxides, during the oxidation process of polyunsaturated fatty acids. The TBA values were measured as MDA content. As can be seen from the results of the present study, 100% CO treatment very effectively leads to less lipid oxidation in the fish muscle ( Figure 4-7). The difference between the treatments was particularly evident for the samples subjected to CO treatment compared to the control (untreated), then freezing for 30 days. There is a trend towards an increase in TBA values up to a certain point during the frozen storage period, foll owed by a decrease in th ese values, and then a lower increase rate after thawed and refriger ated.As seen in Figure 4-7 untreated samples developed significantly (P<0.05) more seconda ry oxidation products after treatment (6.42 molMDA/kg) as shown by high TBARS compared to treated samples (4.67 molMDAl/kg) at day 9,except at day 20 where TBA values of th e two groups, control and CO treated had about the same values. From day 24 to day 42 as s een in Figure 4-7 the control group developed significantly (p<0.05) more second ary oxidation products in a decrea sing pattern as compared to the others two groups.(Table C-1,C-4.Appendix C). This is likely due to exposure to higher
79 levels of oxygen for those samples combined with lower stability of heme proteins (Hultin, 1994). All treatments showed an increased lipid oxidation on freezing, which is in agreement with other studies on frozen s eafood where control treated tuna had more TBARS than other treatments throughout frozen storage. A gradual increase in oxidation was seen for samples treated with 18% CO, filtered smoke (FS) and 10 0% CO during cold storage. All treatments except 100% CO showed decreases in TBARS at day 8, which is commonly seen for lipid oxidation. This is in agreement with results ob tained by Babji and othe rs. (1998) that showed similar increases in TBARS values during stor age followed by declines in TBARS. Igene and Pearson (1979) suggested that decreases in TBARS values in stored foods might be due to interaction between malondialdehyde and proteins. These results suggest that CO treatment can be effective in retarding lipid oxidation and are in agreement with previous studies by Kr istinsson and others. (2003 a& b), Garner and Kristinsson (2004) and Danyali (2004) that reported that CO and F iltered Smoke treatment led to a significant reduction in TBARS compared to un treated fillets. From day 48 to day 55 at refrigeration temperature, the differences be tween samples were not significant but always showing less oxidation on treated samples. Carb on monoxide stabilizes heme proteins providing protection from oxidation, possibly reducing pro-oxidative activ ity. Garner and Kristinsson (2004) found a good correlation between CO binding to heme protein in Spanish mackerel and oxidative stability, which strength ens this theory. Increased stab ilization of lipid oxidation has also been reported for beef lo ins (Luno and others., 2000). The protein injected samples showed a minor increase in the lipid oxidation compared to control samples. This can be explained by the interaction of injected protein preventing aggrega tion of muscle proteins thus exposing interior groups for reaction. Results of this study with salmon seem to suggest a link between lipid
80 oxidation, heme protein ligand binding/oxidation stat e, and color stability. Data suggest that treatment with 100% CO may be an effective method to stabili ze fish against lipid oxidation. Deterioration of many fishes sp ecies is strongly related to li pid oxidation and reactions of degradation products (Hultin,1994). The key prooxidants in fish muscle are Hb and Mb (Undeland and others 2004). The oxidized forms of heme proteins catalyze lipid oxidation and break down lipid hydroperoxides to give rise to secondary lipid oxidati on products responsible for rancidity. For this reason, stab ilization of heme proteins to oxidation was expected to reduce oxidation of lipids. In our study, we were able to demostrate that when Hb and Mb were bound to CO, they were stabilized to some extent and remained in the reduced state and were not easily oxidized. These results agreed w ith Kristinsson and others (2005) that showed that tilapia Hb bound to CO had significant less pro-oxidative activity. Microbial Analysis Microbiological analyses of fresh salmon (bef ore gassing) confirmed presence of bacterial counts of ~2.9 in log210 CFU/g in all fish samples analyz ed. Figure 4-8 shows the microbial levels of untreated salmon which was subjected to the same frozen storage period as the CO and CO + protein treated samples. All samples showed a increase of at least 1 log in microbial count after treatment at 4 0C but the CO and CO + protein treatmen ts had higher increases in microbial counts of almost 2 logs perhaps due to contamina tion in the treatment chamber or in the protein injected to these fillet Table D-1 Appendix D. Microbial counts remained about the same af ter freezing and storage. Freezing for 30 days increased the microbial count around 1 log in th e CO treated samples and the control (Figure 48). But the CO plus protein showed a minimal re duction of the microbial count. During the last week of storage on ice control samples remain sl ightly lower increasing in less than one log but the CO and CO + protein samples increased in aerobic microbial counts slightly more that one
81 log. This agreed with data published for other frozen and thawed fish species (Crynen, 2007). The gas treatments didnt lead to a reduction in aerobic bact erial counts. Freezing suppresses microbial growth and reduces numbers of microorganisms in seafood products (Huss, 1995). Control samples showed lower c ounts of bacteria than those s een for the treated samples. However, although control samples had lower ba cterial levels throughout the frozen storage compared to treated and injected samples, the bact erial levels did still increase, not significantly, on storage for control and treated samples. E-Nose Results A Cyranose 320 (Smiths Detection, New Jers ey, NJ.) with 32 thin-film carbon-black polymer sensors was used to analyze the headspa ce of the fish samples before treatment, after treatment and after storage. The treated fish fillets and the controls used for the tests were sniffed separately five times. An equilibration time in the same chamber of approximately 5-7 minutes was used for each sample. Between samples, the sample holders were flushed with pure air (airgas) for approximately 5 minutes until no odor was detected. The sensor resistances were recorded for each run for the duration of the sniffing, starting from the baseline purge, through th e sample sniff and the sensor purges. All the data generated by the 32 sensor resistances for each step of th e sniff were analyzed to obtain the maximum difference from the baseline to the highest resi stance point of the sample exposure step within the sniff ( R/R). This was done by Cyranose Analysis software written by Dr. Murat Balaban (University of Florida, Gainesville, FL.). All the R/R values for each sensor for each of the samples were put in a spreadsheet in Excel Also within each sample, an average R/R value, standard deviation and % error were calculated for each sensor (Data in Appendix B). The R/R values for each sensor were plotted by the Cyra nose Analysis software. Some sensors showed a higher R/R value than others, which means that thes e sensors are more sensitive to the fish
82 aroma than others. This was also reported in the coffee study by Martinez (2007). To be able to analyze the R/R values with discriminant func tion, eight sensors with the highest R/R values for each sample were chosen (Figure 4-9). Each sensor chosen was also matched with th e % error calculated in Excel to make sure that the most sensitive sensors also have low error %.The sensors chosen for discriminant function analysis were sensors 6, 18, 20, 23, 26, 28, 29, and 31 Figure B-1,2,3 Appendix B. No significant differences can be seen between the treatment and the control (no cluster separation). The R/R for each of the 8 sensors chosen were very similar, which was confirmed by the Root 1 vs. Root 2 graph based on the un standardized canonical scores of each sample, as we can see in Figure 4-10. The squared Mahalanobis distances calculated by Statistica 7.0 also show how distant each sample group is from the other (Tab le B-3). The discriminant function analysis summary, the F values and the p-levels for each sample are shown in Tables B-4, B-5 Appendix B. This analysis plot of unstandardized canonical scores showed no separation between control and treated samples (Figure 4-10). Salmon fillets stored at the same temperature showed clusters very close each other. These re sults, no separation of different treatments, can be explained by the lower production of secondary oxidati on products measured by TBARS (mainly malondialdehyde) responsible for the o ff-odors developed from lipid oxidation. Strong rancidity can be detected at 20mo l/kg tissue TBARS (Richards and Hultin 2000). Based on our results the maximum values for TBARS in our study were 9.43mol/kg tissue at day 20. Since all samples were maintained at fr eezing temperature and two groups were treated with CO there was a reduction in lipid oxidation and production of off odors undetectable for the e-nose sensors. The microbial grow th levels increase but not signi ficantly for the spoilage thus production of off odors that can be to some extent sensitive for the electron ic nose. In the other
83 hand, this can be confirmed by the results of Du and others 2002, Olafsdottir and others 2005, where salmon samples were subjected to different storage refrigeration te mperatures. The results were significantly different as seen by the separation of treatments. The clusters of salmon fillets stored at different temperatures with different microbial loads separated from each other. The clusters with the closer bacterial num bers were closer to each other.
84 Figure 4-1. Lightness (L*values). Statistical an alysis of the color of salmon samples over 39 days. Table 4-1. Means separation. L* valu es for control and treated samples Day Treatment 2 5 3239 Control 62.93b 64.43a 63.8a63.42a CO Treat. 64.86a 63.52a 64.1a63.42a CO+Prot. 62.82b 63.86a 63.82a65.18a Table 4-2. Means separation. a* valu es for control and treated samples Day Treatment 2 5 3239 Control 36.74ab 36.8a 35.06a35.42a CO Treat. 33.17b 37.57a 35.37a34.37a CO+Prot. 38.02a 35.16a 37.79a33.65a Table 4-3. Means separation. b* values for control and treated samples Day Treatment 2 5 3239 Control 34.86ab 34.98a 34.18a33.19a CO Treat. 32.07b 35.32a 33.5a32.66a CO+Prot. 35.85a 32.68a 34.82a31.44a
85 Figure 4-2. Redness (a*values). Stat istical analysis of the color of salmon samples over 39 days. Figure 4-3. Yellowness (b*values). Statistical analysis of the color of salmon samples over 39 days. The graph shows the average b* values of salmon at day 2 before treatment, and from day 5 through day 32 after treatment.
86 Figure 4-4. Statistical analysis of salmon firmness during 30 days stored at -30C. The graph shows the differences in firmness between control, CO treated, and CO+protein injected to give 10% weight increase with a protein solution used as cryoprotectant. Treatment means with the same letter are not significantly different from each other.
87 Figure 4-5. Concentation of CO (ppm) in salm on fillets after 48 hours treatment with 100% CO. in refrigeration, then frozen for 30 days. Treatment means with the same letter are not significantly different from each other. Figure 4-6. Water Holding capacity based on inject ed weight of salmon fillets injected with hydrolysates to obtain a 10% in jection level. Control and tr eated samples were frozen for 30 days, then thawed and analyzed again. Treatment means with the same letter are not significantly different from each other.
88 Figure 4-7. Statistical analysis in lipid oxidati on of salmon fillets untreated and treated with 100%CO and injected proteins. Fillets portio ns were treated, except control, for 48hrs in refrigeration followed by freezing for 30 days at -30C, then thawed and storage at 4C for one week.
89 Figure 4-8. Aerobic microbial grow th (log CFU/g) in untreated and treated salmon fillets. Fillet were subjected to 100% CO fo r 48 hours under refrigeration, frozen for 30 days at 30C, then kept under refriger ation for one week at 4C.
90 Figure 4-9. R/R values for each sensor for the contro l, treated, and treated-injected salmon fillets.
91 Figure 4-10. Scatterplot of root 1 vs. root 2 of unstandard ized canonical scores for control, treated samples with 100% CO, a nd CO treated+injected samples.
92 CHAPTER 5 SUMMARY AND CONCLUSION The use of carbon monoxide to treat fish m usc le is now widespread and a very useful technology to avoid the oxidation of the heme proteins and mainta in an appealing and attractive fresh color of seafood muscle. Heme proteins play an important role in the quality of aquatic foods. The autoxidation of these pr oteins contributes to undesirable color changes, odor changes and texture deterioration. Since fish muscle is high in polyunsaturated fatty acids these are highly susceptible to oxidation. Today it is known that CO helps to retain the color of muscle foods as well as leads to development of less lipid oxid ation by replacing the oxygen in the blood and muscle tissue. This effect makes CO very in teresting and attractive for the meat and seafood industry. This oxidative process that can be prevented by treatment with CO can help to some extent during frozen storage because ot her alterations can occur simulta neously on the muscle texture due to freeze-induced perturbati ons. Generally, freezing is a wide ly used method of preserving fish muscle for a long period. A number of factor s such as a decrease in the amount of liquid water available, mechanical damage resulting from ice crystal formation and lipid oxidation products are thought to be involve d in the denaturation and aggreg ation of myofibrillar protein during freezing and frozen storage, causing loss of physicochemical and functional properties. This study demonstrated that treating salmon fille ts with CO and previously injected with fish protein hydrolysates had a protective effect agai nst lipid oxidation and a positive effect on the color of the fish fillets and stabilized it during frozen storag e been significantly different at day 5 after treatment. A larger increase in a* values was seen from day 2 to day 5. The combined effect of CO and injected proteins and subsequent fr ozen storage at -30C leads to less lipid oxidation on treated samples compared to c ontrol (p>0.05). The effectiveness
93 of using salmon frame hydrolys ates was demostrated on water holding capacity, reducing the water loss on treated and injected samples becau se of more water was bound to proteins. These cryoprotectants ( hydrolysates) can improve the quality of salmon fillets by preventing freezeinduced denaturation of muscle proteins. The incorporation of CO ca n be confirmed by the concentration of this gas in salmon tissue by the GC analysis that showed a high concentration of CO at day 2 after treatment. It is shown during this study that CO proce ssing did not affect the growth or inhibition of microorganism but the increase of them was not significant. An electronic nose was used to determine di fferences in odor between the treated and control samples during frozen storage and refr igeration. However, no spoilage was detected confirmed by the discriminant function analysis that showed no separation between samples. This allows us to conclude that the use of CO combined with the incorporation of cryoprotectants can improve the qual ity and extend the shelf life of salmon fillets. The use of CO among different countries is regulated as a food ingredient or additive. However, its benefits when using in muscle foods must be considered as an alternative to maintain the quality and freshness of fresh seafood. The same consideration must be taken wh en using natural fish protein as cryoprotectants.
94 APPENDIX A VISUAL ANALYSIS A (A) Control samp les frozen for 30 days. B (B) Samples treated with CO and frozen for 30 days then thawed a week after. A-1. Salmon fillet pictures analyzed by a machine vision system.
95 SOP for the Salmon gas treatment using Carbon Monoxide 1. Procedure must be done in a ventilated area, by a fume hood 2. Previous to use of CO make sure that th ere is not leak from the CO box (previously tested) 3. Previously test Hopcalite catalyst tube 4. Verified flow meter adjusted for CO gas 5. Prepare the Hopcalite by warming up the catalytic converter up to 95C 6. Clean and sanitize CO box and transfer the fresh fillets into the box then seal it 7. Hook up all the in lets and outlets 8. Turn on CO monitors. 9. Start flushing CO in order to saturate the box 10. Monitor pressure gauge. If pressure goes ove r the established limit reduce the gas flow. 11. Monitor temperature and the amount of gas been flushed into the box by volume. 12. If CO monitors go off put on the gas ma sk, close CO valve immediately and evacuate the area 13. After the determined volume of gas has b een reached, stop flushing CO and close the inlets and outlets valves. 14. Turn off Hopcalite unit. 15. Disconnect inlets and outlet 16. Let the CO box and the treated samp les for 48 hrs.in refrigeration 17. After 48 hours connect again inlet and outlets. 18. Turn on Hopcalite unit and wait fo r temperature to reach 95C. 19. Start flushing and make sure all CO left in the box is converted to CO2 20. Open the box and aseptically remove the samples 21. Clean and sanitize the CO box
96 APPENDIX B PHYSICAL ANALYSES Table B-1. Mean Separation. Results for firmne s s in control, CO treated, and CO+protein salmon samples Hardness (N) Springiness(mm) Cohessiveness(F) Gumminess(N) Treatment Day2 Day 32 Day2 Day 32 Day2 Day 32 Day2 Day 32 Control 19.67a 17.09a 2.61a3.7a0.051b0.089a0.82b 1.31b CO Treat. 17.28a 11.55a 2.54a4.66a0.11a0.15a1.84a 1.05b CO+Prot. 11.4b 16.46a 2.3a4.68a0.057a0.21a0.58b 3.03a Table B-2. Mean Separation. Resu lts for WHC. Differences in wa ter lost between control and treated samples. Water loss from 10 g. of sample Water Holding capacity (water g.loss) Treatment Day 2 Day 32 Control 7.94c 8.34b CO Treat. 8.16b 8.31b CO+Prot. 8.72a 8.69a
97 Table B-3. Squared Mahalanobis distances for all treatments Table B-4. Discriminant func tion analysis F-values for control and treated samples Table B-5. Discriminant func tion analysis p-levels for control and treated samples
98 DR/RSensors 0.00 0.05 0.10 0.15 0.20 1234567891011121314151617181920212223242526272829303132 Figure B-1. R/R values for each sensor in one of the control samples at day 5 after treatment. DR/RSensors 0.00 0.05 0.10 0.15 1234567891011121314151617181920212223242526272829303132 Figure B-2. R/R values for each sensor in CO treated samples after treatment.
99 Figure B-3. R/R values for each sensor in CO treated+protein samples.
100 APPENDIX C CHEMICAL ANALYSIS Table C-1. ANOVA analysis in lipid oxidation of salm on fillets untreated, treated with 100%CO, and treated-inj ected with proteins TBARS mol MDA/kg Treatment Day 0 9 12 20 22 24 26 28 29 Control 4.77a 6.42a 5.15a9.23a4.28a 8.39a5.78a 7.81a 4.97a CO Treat. 2.86b 4.67b 3.79b9.43a3.65a 5.60b4.99ab 5.40b 4.07a CO+Prot. 5.46a 5.81ab 3.87b6.68b3.29a 5.79b4.34b 5.05b 4.30a TBARS Treatment 32 35 39 42 45 48 51 55 Control 5.51a 5.56a 5.90a3.96a6.53a5.15a3.80a5.06a CO Treat. 4.29b 3.13b 3.43b2.51b4.92b4.23a3.85a4.68a CO+Prot. 3.53b 2.49b 4.33b2.86b4.49b5.30a3.01a4.42a
101 Table C-2. ANOVA Analysis GC(CO levels)ppm CO/g fish Treatment Day2 Day 32 Control 0.12c 0.053a CO Treat. 3.12a 0.45a CO+Prot. 1.62b 0.15a Concentration of CO (ppm) in the muscle of c ontrol, treated, and treated+protein salmon fillets
102 Figure C-1. Chromatogram of the injection of 100l of the headsp ace analysis over each sample, from top to the bottom control, CO treated, and treated and injected at day 2 after treatment. The peak represents ca rbon monoxide. The area under the peak is proportional to the quantity of this compound.
103 Figure C-2. Chromatogram of the injection of 10 0l of the headspace anal ysis over each sample, from top to the bottom control, CO treated, and treated and injected at day 32 after 30 days at frozen temperature. The peak represents carbon monoxi de. The area under the peak is proportional to the quantity of this compound
104 Table C-3. GC data results. Concentration of CO (ppm) in the muscle of control, treated, and treated+protein salmon fillets Treatment day Peak 1 Peak2 Peak3 Avg peak %CO ppmCO gCO/gfish ppm CO/gfish control 0 7.04 6.66 6.68 6.793 -0.00013 -1.265 -1.0380E-08 -0.010380926 control 0 9.29 9.73 9.44 9.486 0.000401 4.013 3.29371E-08 0.032937121 control 0 9.49 9.36 9.23 9.36 0.000377 3.765 3.08999E-08 0.030899886 control 2 11.07 10.64 10.56 10.756 0.00065 6.503 5.33631E-08 0.053363081 control 2 10.94 12.07 12.07 11.693 0.000834 8.338 6.84279E-08 0.068427897 control 2 9.56 9.94 9.65 9.716 0.000446 4.464 3.66363E-08 0.036636311 CO Treat. 2 249.23 246.05 248.2 247.826 0.047116 471.160 3.86626E-06 3.866262622 CO Treat. 2 162.58 164 164.46 163.68 0.030623 306.232 2.5129E-06 2.512895318 CO Treat. 2 190.21 192.91 195.1 192.74 0.036319 363.190 2.98028E-06 2.980279891 CO+Prot 2 125.6 126.57 126.94 126.37 0.023311 233.105 1.91282E-06 1.91282242 CO+Prot 2 141.36 142.99 143.27 142.54 0.02648 264.798 2.17289E-06 2.172891537 CO+Prot 2 55.92 55.42 55.65 55.663 0.009452 94.520 7.75616E-07 0.775616461 control 32 14.34 14.21 14.8 14.45 0.001374 13.742 1.12765E-07 0.112764562 control 32 10.35 9.77 9.64 9.92 0.000486 4.863 3.99066E-08 0.039906609 control 32 20.17 20.37 20.24 20.26 0.002513 25.129 2.06209E-07 0.206209309 CO Treat. 32 23.79 23.84 23.37 23.666 0.003181 31.806 2.61E-07 0.261000206 CO Treat. 32 22.39 22.67 22.71 22.59 0.00297 29.696 2.43684E-07 0.243683709 CO Treat. 32 59.81 59.53 59.18 59.506 0.010205 102.053 8.3743E-07 0.837430456 CO+Prot 32 14.45 14.33 14.48 14.42 0.001368 13.683 1.12282E-07 0.112282059 CO+Prot 32 11.17 11.18 11.03 11.126 0.000723 7.228 5.9314E-08 0.059313952 CO+Prot 32 25.08 25.08 24.88 25.013 0.003445 34.446 2.82659E-07 0.282659229
105 Table C-4. Lipid Oxidation values obtained ov er 45 days of study for untreated and treated salmon fillets. Control (Untreated) 100% CO treated 100% CO+ protein treated Time (day) Average (TBA umol/MDA kg Stdev Average (TBA umol/MDA kg) Stdev Average (TBA umol/MDA kg) Stdev 0 4.766498867 0.6310872.8616620.775.4606330.99 9 6.56 1.3134474.6732981.425.8152911.76 12 5.15 0.2927113.7896090.483.8724220.75 20 9.225305882 1.1510139.4297573.476.6746881.42 22 4.281037574 1.1869973.6516222.623.2872981.07 24 8.392606826 1.1242085.59832315.7930961.11 26 5.779240813 1.0236364.9929631.34.3364040.78 28 7.811699187 1.9237715.4043911.445.0449771.1 29 4.974026826 1.3118744.0749740.864.299930.44 32 5.513638054 1.3395994.2882050.953.5302820.58 35 6.031946013 1.6877213.1349220.42.4867311.14 39 5.945020973 0.9135353.433411.334.1139421.1 42 3.955989339 1.4360442.5060790.462.8551830.55 45 6.688879346 1.7841674.9211651.444.4934810.62 48 5.309297328 2.1765554.2281591.615.3015032.37 51 3.449378911 0.7164013.8526021.723.0103380.74 55 5.20903435 2.5853454.6819062.544.4175943.32
106 APPENDIX D MICROBIAL ANALYSES Table D-1. Microbial resu lts CFU per (g) average Day Treatment 0 23239 Control 2.90E+02 3.33E+03 2.20E+04 5.00E+04 CO Treatment 2.90E+02 3.07E+04 7.63E+04 1.20E+05 CO + Protein 2.90E+02 7.21E+042.80E+042.80E+05
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118 BIOGRAPHICAL SKETCH Max Ochsenius was born in Santiago, Chile. He attended to the Univ ersity of Santiago Chile where he graduated in food science. In fall 2003 he attended the University of Florida where he graduated in August 2005 with a B.S. in food science. In summer 2005 he did an internship as an undergraduate with Tyson Foods. He started his m asters degree in food science in January 2006 under Dr. Murat O. Balabans s upervision at the University of Florida and graduated in May 2009. He was a member of the F ood Science dairy products judging team for 2 years. Max is listed in Who is Who Among Students in Americ an Universities and Colleges He received the Yeoman assistantship award in 2007. Max has participated in several poster presentations at Annual IFT meetings in the la st 3 years. He has al so participated poster presentations at the Pacific Fish eries Technologists meeting in 2008.