TRACING THE ORIGIN OF THE CHARACTERISTIC BAD ODOUR OF DRIED STOCK FISH
PROJECT BY ADESINA ADEBOWALE T. (2011) 1.0 INTRODUCTION
1.1 Background Information
With the ever growing population and the need to store and transport food from one place to another where it is needed, food preservation becomes necessary in order to increase its shelf life and maintain its nutritional value, texture and flavor. Therefore good food preservation techniques must prevent microbial spoilage of food without affecting its quality and nutritional attributes. Fish are recognized as highly perishable having a relatively short shelf life, therefore fish requires proper handling and preservation to increase its shelf life and retain its quality and nutritional attributes. Fresh fish spoilage can be very rapid after it is caught, immediately a fish is caught it loses its natural resistance to attack by microorganism and also start to undergo both physical and chemical changes that in return bring changes in appearance, taste, smell and texture. During fish spoilage there is a breakdown of various components and formation of new compounds. This new compounds are responsible for changes in odour, flavour and texture of the fish. Fish lipids contain omega − 3 polyunsaturated fatty acid (PUFA), especially Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). These fatty acids play a vital role in human nutrition, disease prevention, and health promotion. Long chain omega − 3 PUFAs cannot be synthesized by humans and must be obtained through the diet. Scientific data indicate that the consumption of fish oil containing omega − 3 PUFAs reduces the risk of coronary heart disease, decreases mild hypertension, prevents certain cardiac arrhythmias and sudden death, lowers the incidence of diabetes, and appears to alleviate symptoms of rheumatoid arthritis. Besides, it appears that omega − 3 PUFAs play a vital role in the development and function of the nervous system (brain), photoreception (vision), and the reproductive system (Alasalvar, et al., Conner, 1997, Dyeberg, 1986, Leaf and Weber, 1988, Sidhu, 2003, Skonberg and Perkins, 2002 and Tapiero, et al., 2002). Dietary trials aimed at reducing the risk of cardiovascular diseases have emphasized the importance of ingesting marine oil and fish products that are rich in omega -3 PUFA and poor in omega -6 PUFA (Singer et al., 1983; Herold and Kinsella, 1986). The beneficial effects have been attributed to an increased ratio of omega -3 to omega -6 PUFA in blood lipids and cell membrane lipids. Stock fish is unsalted fish, especially cod, dried by cold air and wind on wooden racks on the foreshore called ”hjell”s .The drying of food is the world’s oldest known preservation method and dried fish has a storage life of several years. The method is cheap and effective in suitable climate.cod is the most common fish used in stock fish production, while other white fish such as Pollock, haddock, hake, ling and tusk are used to a lesser degree. The fish is prepared immediately after capture. After gutting the fish. It is either dried whole, or split along the spine leaving the tail connected. The fish is hung on hjell from February to May. Stable cool weather protects the fish from insects and prevents an uncontrolled bacterial growth. A temperature just above zero degree Celsius, with little rain is ideal. Too much frost will destroy the fish, as ice destroys the fibers in the fish. The climate of Northern Norway is excellent for stock fish production. After its three months hanging on the hjell, the fish is then matured for another two to three months indoors in a dry and airy environment. During the drying about 80% of the water in the fish disappears which is why it is said that one kilogram of stock fish is equivalent to 5kg of fresh fish. Stock fish is extremely popular and widely consumed in west Africa especially Nigeria, Cameron, Togo and Sierra Leone where it is used in many soups that complement the grain staples fufu and garri even though stockfish is originally native to the Vikings of the scandianavia. The name okporoko for stock fish among the igbo of Nigeria refers to sound the hard fish makes in the pot, and called “kpanla” or “panla” by the Yorubas of southern Nigeria it is a highly favoured delicacy in Ogbono, palm nut soup (banga) and “Equsi” soups as well as in efo riro. It is also used in afang soup, and indeed most other soup like “Edikaikong”. Stock fish is known primarily for two things, its strong fishy taste and even stronger bad odour. Those who cook stockfish usually find their homes filled with the strong fish smell and often wonder how to get rid of the smell when it is cooked; the powerful fishy odour permeates everything including furnishings, clothing and even the breath and sweat of people who consume it which then emits the fishy odour. In cultures that value stock fish, it is not a problem, but it is a cause of conflict in America where people are not used to it. If someone starts cooking stockfish in an apartment complex, the smell soon fills the corridor and enters the room of other tenants. Many works had been done on lipid oxidation being responsible for the rancid odour of fish due to the presence of polyunsaturated fatty acid in fish which are highly susceptible to oxidation but little or no work had been done to determine the origin of the strong stinking odour of dried stock fish which makes this project work very important.
1.2 Justification Judging from the fact that when fresh stockfish of the same species is cooked, a similar strong fish stinking odour that one gets from the dried stockfish does not develop, this suggests that some chemical reactions could have taken place during the drying process that gave rise to the strong odour since fish are highly perishable and its spoilage can be very rapid after it is caught. One possible reaction that may contribute to the bad odour is lipid oxidation which is a major cause of deterioration of fish species such as mackerel, hake, cod etc which are used in the making of stockfish. The drying process of stockf ish could have adversely affected the nutritional attributes of stockfish which resulted in its stinking fishy odour. Once the cause and origin of this rancid odour of the stockfish is known which could be as a result of lipid oxidation then a possible solution can be determined.
1.3 Aim and Objectives
The aim of this project work is to trace the origin of the characteristics bad odour of dried stock fish with the flowing objectives. * To determine the fatty acid composition of fresh and dried stockfish (hake “merluccius species”) with the hope of finding out if there are major difference is the polyunsaturated content. * To sun-dry fresh stock fish (hake “merluccius species”)with and without antibiotics and follow lipid oxidation during the drying process * To perform sensory evaluation on the sun-dried stockfish and the imported dried stockfish as it relates to the characteristic bad odour of the imported dried stockfish * To figure out from the data collected if lipid oxidation has any role to play in the imported dried stockfish odour.

CHAPTER TWO

2.0 LITERATURE REVIEW
2.1 Historical Background Of Fish Fish are aquatic vertebrates that are typically ectothermic (previously cold-blooded), covered with scales, and equipped with two sets of paired fins and several unpaired fins. Fish are abundant in the sea and in fresh water, with species being known from mountain streams (e.g., char and gudgeon) as well as in the deepest depths of the ocean (e.g., gulpers and anglerfish). They are of tremendous importance as food for people around the world, either collected from the wild or farmed in much the same way as cattle or chickens. Fish are also exploited for recreation, through angling and fish keeping, and are commonly exhibited in public aquaria. Fish have an important role in many cultures through the ages, ranging as widely as deities and religious symbols to subjects of books and popular movies.The term "fish" is most precisely used to describe any non-tetrapod chordate, (i.e., an animal with a backbone), that has gills throughout life and has limbs, if any, in the shape of fins. Unlike groupings such as birds or mammals, fish are not a single clade but a paraphyletic collection of taxa, including hagfishes, lampreys, sharks and rays, ray-finned fishes, coelacanths, and lung fishes.A typical fish is ectothermic; has a streamlined body that allows it to swim rapidly; extracts oxygen from the water using gills or an accessory breathing organ to enable it to breath atmospheric oxygen; has two sets of paired fins, usually one or two (rarely three) dorsal fins, an anal fin, and a tail fin; has jaws; has skin that is usually covered with scales; and lays eggs that are fertilized internally or externally. Fish come in many shapes and sizes. This is a sea dragon, a close relative of the seahorse. Their leaf-like appendages enable them to blend in with floating seaweed To each of these there are exceptions. Tuna, Swordfish, and some species of sharks show some warm-blooded adaptations, and are able to raise their body temperature significantly above that of the ambient water surrounding them. Streamlining and swimming performance varies from highly streamlined and rapid swimmers which are able to reach 10-20 body-lengths per second (such as tuna, salmon, and jacks) through to slow but more maneuverable species such as eels and rays that reach no more than 0.5 body-lengths per second. Many groups of freshwater fish extract oxygen from the air as well as from the water using a variety of different structures. Lungfish have paired lungs similar to those of tetrapods, gouramis have a structure called the labyrinth organ that performs a similar function, while many catfish, such as Corydoras extract oxygen via the intestine or stomach. Body shape and the arrangement of the fins is highly variable, covering such seemingly un-fishlike forms as seahorses, pufferfish, anglerfish, and gulpers. Similarly, the surface of the skin may be naked (as in moray eels), or covered with scales of a variety of different types usually defined as placoid (typical of sharks and rays), cosmoid (fossil lungfishes and coelacanths), ganoid (various fossil fishes but also living gars and bichirs, cycloid, and ctenoid (these last two are found on most bony fish. There are even fishes that spend most of their time out of water. Mudskippers feed and interact with one another on mudflats and are only underwater when hiding in their burrows. The catfish Phreatobius cisternarum lives in underground, phreatic habitats, and a relative lives in waterlogged leaf litter.Fish range in size from the 16 m (51 ft) whale shark to the 8 mm (just over ¼ of an inch) long stout infant fish. Many types of aquatic animals commonly referred to as "fish" are not fish in the sense given above; examples include shellfish, cuttlefish, starfish, crayfish and jellyfish. In earlier times, even biologists did not make a distinction - sixteenth century natural historians classified also seals, whales, amphibians, crocodiles, even hippopotamuses, as well as a host of aquatic invertebrates, as fish. In some contexts, especially in aquaculture, the true fish are referred to finfish to distinguish them from these other animals.(www.fishpedia.com)
2.2 Spoilage of Fish
Fish are recognized as being highly perishable, having a relatively short shelf life, which is defined as the length of time from the day of catch that fresh fish can be in the marketplace unspoiled (Regenstein and Regenstein 1991). Therefore fish requires proper handling and preservation to increase its shelf life and retain its quality and nutritional attributes. Quality is defined as the aesthetic appearance and freshness or degree of spoilage which the fish has undergone (FAO). Immediately as fish is caught, it looses its natural resistance to attack by microorganisms and also starts to undergo both physical and chemical changes that in return bring changes in appearance, taste, smell and texture.

2.1 Forms of Fish Spoilage “Spoilage” can be defined as a change in fish or fish products that renders them less acceptable, unacceptable or unsafe for human consumption (Hayes 1985). Fish undergoing spoilage has one or more of the following signs: slime formation; discolouration; changes in texture; off-odours; off-flavours and gas production. The development of these spoilage indicators in fish and fish products is due to a combination of microbiological, chemical and enzymatic and physical phenomena (Huis in’t Veld 1996).

2.2.1 Microbiological Spoilage
Life fish is normally considered to be sterile, but microorganisms are found on all the outer surfaces (skin and gills) and in the alimentary tract of live and newly caught fish in varying numbers. A normal range of 102-107cfu (colony forming units)/cm2 on the skin and between 103 and 109cfu/g in the gills and intestines has been observed (Liston 1980). When fish dies, its entire body resistance mechanisms breakdown, giving way to microorganisms or the enzymes they secrete to invade or diffuse into the flesh where they react with the complex mixture of natural substances present. During storage a characteristic flora develops, but only a part of this flora, known as the specific spoilage organisms (SSO), contribute to spoilage. The SSO counts reach a minimal spoilage level where the fish is sensorially rejected (Figure1). Temperate fish have psychrotrophic (cold-tolerant) bacteria of the genera Pseudomonas, Moraxella, Acinobacter, Shewanella Flavobacterium, Vibrio, Photobacterium and Aeromonas as part of their natural flora whereas tropical fish normally have non-psychrotrophic (mesophilic) spoilage bacteria that make tropical fish spoil much faster than temperate water fish in the absence of ice.
Pseudomonas and Altermonas putrefaciens are probably the major bacterial species that cause fundamental spoilage of usually iced fish. These can use the non-protein nitrogen compounds present in the fish such as trimethyl amine oxide (TMAO) that result in several volatile odoriferous compounds such as trimethyl amine oxide, TMA (Regenstein and Regenstein 1991). These volatile compounds are responsible for the off-odours and off-flavours characteristic of spoiled fish. In several studies, Photobacterium phosphoreum and Shewanella putrefaciens have been found in packed cod with the former most likely the SSO of packed cod stored on ice (Dalgaard 1995).
Bacteria are able to decompose proteins, other nitrogen containing compounds to ammonia, hydrogen sulphide, which produce an unpleasant and disgusting flavour (Herbert and Shewan 1975). Trimethyl amine oxide (TMAO), mostly found in marine fish, is broken down to trimethyl amine (TMA), dimethyl amine (DMA) and ammonia (NH3), which are responsible for off-odours in fish undergoing spoilage.
The main spoilage test of metabolite(s) produced during fish storage or distribution to obtain a quantitative fish quality index is total volatile bases (TVB). It measures the total content of TMA+DMA+ ammonia plus other basic nitrogenous compounds associated with fish spoilage. TVB and TMA values of 30 mgN and 15 mgN/100 g are the rejection spoilage levels respectively (Regenstein and Regenstein 1991). The fishy odour of TMA when it reacts with lipid is generally detectable when the TMA level reaches 4-6 mgN/100 g.
Microbiological quality evaluation of fish aims to quantify the hygienic quality of fish, including temperature abuse and the possible presence of pathogenic microorganisms in the fish. Total aerobic bacteria, also called total plate count (TPC); specific spoilage organisms (SSO) and various pathogenic bacteria are examined using appropriate agar media. Quality levels are based on the plate counts for acceptance or rejection of fishery products for human consumption. With representative sample units not less than five, plate counts below 5 x 105 are considered of good quality; between 5 x 105 and 107 marginally accepted quality (sample units with plate counts between 5 x 10 5 and 107 not exceeding three) and plate counts at or above 107 are considered unacceptable in quality (ICMSF 1986).

2.2.2 Oxidative Spoilage
Lipid oxidation is a major cause of deterioration and spoilage for the pelagic fish species such as mackerel and herring with high oil/fat content stored fat in their flesh (Fraser and Sumar, 1998). Lipid oxidation involves a three stage free radical mechanism: initiation, propagation and termination (Frankel, 1985; Khayat and Schwall, 1983). Initiation involves the formation of lipid free radicals through catalysts such as heat, metal ions and irradiation. These free radicals which react with oxygen to form peroxyl radicals. During propagation, the peroxyl radicals reacting with other lipid molecules to form hydroperoxides and a new free radical (Fraser and Sumar, 1998; Hultin, 1994). Termination occurs when a buildup of these free radicals interact to form non-radical products. Oxidation typically involves the reaction of oxygen with the double bonds of fatty acids. Therefore, fish lipids which consist of polyunsaturated fatty acids are highly susceptible to oxidation. Molecular oxygen needs to be activated in order to allow oxidation to occur. Transition metals are primary activators of molecular oxygen (Hultin, 1994). In fish, lipid oxidation can occur enzymatically or non-enzymatically. The enzymatic hydrolysis of fats by lipases is termed lipolysis (fat deterioration). Duringthis process, lipases split the glycerides forming free fatty acids which are responsible for: (a) common off-flavour, frequently referred to as rancidity and (b) reducing the oil quality (Huis in’t Veld, 1996; FAO, 1986). The lipolytic enzymes could either be endogenous of the food product (such as milk) or derived from psychrotrophic microorganisms (Huis in’t Veld, 1996). The enzymes involved are the lipases present in the skin, blood and tissue. The main enzymes in fish lipid hydrolysis are triacyl lipase, phospholipase A2 and phospholipase B (Audley et al., 1978; Yorkowski and Brockerhoft, 1965). Non-enzymatic oxidation is caused by hematin compounds (hemoglobin, myoglobin and cytochrome) catalysis producing hydroperoxides (Fraser and Sumar, 1998). The fatty acids formed during hydrolysis of fish lipids interact with sarcoplasmic and myofibrillar proteins causing denaturation (Anderson and Ravesi, 1969; King et al., 1962). Undeland et al. (2005)reported that lipid oxidation can occur in fish muscle due to the highly pro-oxidative Hemoglobin (Hb), specifically if it is deoxygenated and/or oxidized. They found that the addition of acids, which lower the pH, can accelerate lipid oxidation through deoxygenated Hb.
2.2.3 Autolytic Enzymatic Spoilage Shortly after capture, chemical and biological changes take place in dead fish due to enzymatic breakdown of major fish molecules (FAO,2005). Hansen et al. (1996) stated that autolytic enzymes reduced textural quality during early stages of deterioration but did not produce the characteristic spoilage off-odors and off-flavors. This indicates that autolytic degradation can limit shelf-life and product quality even with relatively low levels of spoilage organisms. Most of the impact is on textural quality along with the production of hypoxanthine and formaldehyde. The digestive enzymes cause extensive autolysis which results in meat softening, rupture of the belly wall and drain out of the blood water which contains both protein and oil (FAO, 1986). A number of proteolytic enzymes are found in muscle and viscera of the fish after catch. These enzymes contribute to post mortem degradation in fish muscle and fish products during storage and processing. There is a sensorial or product associated alteration that can be contributed by proteolytic enzymes (Engvang and Nielsen, 2001). During improper storage of whole fish, proteolysis is responsible for degradation of proteins and is followed by a process of solubilization (Lin and Park, 1996). On the other hand, peptides and free amino acids can be produced as a result of autolysis of fish muscle proteins, which lead towards the spoilage of fish meat as an outcome of microbial growth and production of biogenic amines (Fraser and Sumar, 1998). Belly bursting is caused by leakage of proteolytic enzymes from pyloric caeca and intestine to the ventral muscle. The proteases have optimal pH in the alkaline to neutral range. Martinez and Gildberg (1988) reported that the rate of degradation by proteolytic enzymes was reduced when the fish was kept at 0°C and a pH of 5.
2.3 Factors That Influence the Rate of Fish Spoilage
2.3.1 Effect of Time/Temperature Conditions on Microbial Growth
The most crucial factors determining the quality of fishery products are time and temperature tolerance. Proliferation of microorganisms requires appropriate high temperatures, while at lower temperatures close to 0ºC, their activity is reduced, thereby extending the shelf life of fish products. Temperature is the single most important factor affecting post-harvest quality of the products. It is often critical to reach the desired short-term storage temperature rapidly to maintain the highest visual quality, flavour, texture, and nutritional content of fresh fish.
The rate of spoilage is dependent upon the holding temperature and is greatly accelerated at higher temperatures, due to increased bacterial action. The shelf life at different storage temperatures (t°C) has been expressed by the relative rate of spoilage (RRS), defined by the equation (Spencer and Baines 1964):

2.3.2 Effect of Hygiene on Fish Quality During Handling
Apart from the microorganisms that fishes have at the time of capture, more is added via unhygienic practices and contaminated equipment such as storage facilities. This was demonstrated by studies that compared the quality and storage life of completely aseptically treated fish (aseptic handling), washed fish, iced in clean plastic boxes, with clean ice (clean handling) and with un-washed fish, iced in old, dirty wooden boxes (normal handling). A considerable difference was found in the bacterial contamination of the three batches, the latter heavily contaminated with a reduction in storage life compared with the other samples (Huss et al. 1974).
The design of a fish hold is of great importance as far as hygiene in the hold is concerned. Hold design should enable the purge (drip loss) to be collected easily. The amount of purge was suggested to be higher at 5-7ºC; at which temperature there is greater spoilage since the purge is a very good medium for bacterial growth (Hermansen 1983).
2.3.3 Rough Handling
Rough handling will result in a faster spoilage rate. This is due to the physical damage to the fish, resulting in easy access for enzymes and spoilage bacteria. Physical mishandling in the net, such as very large catches, fishermen stepping on fish or throwing boxes, containers and other items on top of the fish, may cause bruises and rupture of blood vessels.
When fish is in rigor mortis (a complicated series of chemical changes that result in stiffening of the fish’s muscle shortly after death), rough handling can cause gaping (Huss 1995).

2.3.4 Initial Bacterial Load
The microflora on tropical fish often carries a slightly higher load of Gram-positives and enteric bacteria but otherwise is similar to the flora on temperate-water fish (Liston 1980). Basically, bacteria populations on temperate fish are predominantly psychrotrophic reflecting water temperatures of about 100C while fish from the tropics have largely mesophilic bacteria (Gram and Huss 1996).

2.3.5 Method of Capture
The fishing gear and method employed determines the time taken between capture and death. Fish caught in gillnets struggle much to escape, and in so doing, they are bruised by the net which increases exposure to microbial entry and subsequent deterioration. Fish caught by hook and line methods, on the other hand, die relatively quickly and therefore bruises and stresses are likely to be minimal. Physical mishandling in the net due to long trawling nets and very large catches accelerates spoilage. The large catches in the net are compacted against each other resulting in the fish getting bruised and crushed (especially small sized fish) by the heavy trawl net.
2.3.6 Mode of Storage
In bulk-storage, the weight of the pile may crush the fish at the bottom, leading to a loss of weight (yield) as well as other physical damage. It has been reported that when haddock is kept in a short, deep pile of about 3 ft, the bottom fish lose 15% of their weight compared to a normal weight loss of 3-8%, which is entirely due to biochemical changes that cause a loss of water holding capacity leading to drip (Regenstein and Regenstein 1991). Crushing of the fish by ice or other fish can seriously affect the quality of fish by releasing enzymes from the gut into the fish muscle thereby accelerating autolytic processes.
2.4 Fats in Fish
Fats and oils are composed of different fatty acids called TRIGLYCERIDES. The major sources of fats and oils are plants and animals (Gulzer & Zuber., 2002). Fatty acids are the building blocks of fats and there are many different types of fatty acids, some of which can be made by the body and some which cannot. The so-called essential fatty acids are vital substances that cannot be made by the body. So a dietary supply is essential.
Essential fatty acids are polyunsaturated fatty acids and are the parent compound of the omega-6 and omega-3 fatty acids (Bethesdan, 2005). The human body can produce some fatty acids but not essential fatty acids. The fatty acid of fish has a high content of polyunsaturated (omega-3) fatty acids which are important in lowering blood cholesterol level and high blood pressure.
Fish oil contains very little alpha-linoleic acids but is rich in omega-3 derivatives EPA and DHA. Fish are at the top of a food chain based on phytoplankton(algae) that manufacture large amount of EPA and DHA. Nonetheless, fish can be high in toxic methyl mercury (Leaf and Cober, 1987). Fish oil also contains other essential PUFA’s which act in the same way as linoleic and arachidonic acids which have benefits in growing children.
2.4.1 Omega-3 Fatty Acid and Nutrition
Omega-3 oils have been called “the miracle food of the century”. Research shows the risk kind can help prevent heart disease, maintain blood pressure and cholesterol levels and give almost immediate relief from joint pain, migraines, depression and many other conditions. For this reason, omega-3 fatty acids must be obtained from food because of its essentiality to human health. It is important to maintain an appropriate balance of omega-3 and omega-6 in the diet as these two substances work together to promote health.
Omega-3 fatty acid plays an important role in the life and death of cardiac cells because they are essential fuels for mechanical activities of the hearth (Honore et al, 1994; Reiffel an Mc Donald, 2006).There are three major types of omega-3 fatty acids that are ingested in foods and used by the body, they are; alpha linoleic acid(ALA), eicosapentaenoic acids(EPA) and docosahexaenoic acid(DHA). These type of omega-3 fatty acids have been discovered to reduce inflammation and help prevent certain chronic disease, such as heart disease and arthritis(Beluzz, et al.2000).
Study shows that omega-3 fatty acids like DHA and perilla-oil suppress cancer (Atkenson,et al.,1997; Akano, et al,.1988). also support for the idea that DHA is critical for brain development came from an experiment which studied the effect of adding DHA(in form of fish oil) to infant formula. At both 16 and 30 weeks of age the breast fed and supplement-formula-fed infants showed significantly better visual acuity than placebo-formula-fed infants(Makride, et al., 1993; Borch ,et al.,1998) .It is very important to maintain a balance between omega-3 and omega-6 fatty acids in the diet. A healthy diet should consist of roughly one to four times more omega-6 fatty acids than the omega-3 fatty acids. A typical American diet tends to contain 11 to 30 times more omega-6 fatty acids and many researcher believe this imbalance is a significant factor in the rising rate of inflammatory disorders in the united states(Danao and Shinaina, 1999).

2.4.2 Importance of Omega-3 Fatty Acid to the Health
There is promising preliminary evidence that omega-3 fatty acids supplementation might be helpful in cases of depression and anxiety. Studies report highly significant improvement from omega-3 fatty acids supplementation alone and in conjunction with medications. The evidence is strongest for hearth disease and problems that contributes to hearth disease (Haper and Jacobson, 2001). Possible uses of omefa-3 fatty acids include; * Omega-3 fatty acids help nerves cells communicate with each other, which is essential in maintaining good mental health and prevention of depression.(Edward et al., 1998). * Omega-3 fatty acids from fish oil help lower triglycerides and raise high density lipoprotein(HDL) which benefits diabetes patients (Friedberg et al., 1998) * Abundant omega-3 polyunsaturated fatty acids in erythrocyte membranes were associated with a reduced risk and treating of breast cancer (de Dickers., 1999). * Omega-3 fatty acids help decrease inflammatory skin disorder and inflammatory bowel disease. * Studies of hearth attack survivor have found that daily omega-3 fatty acids reduce the risk of hearth attacks and strokes and protection against heartbeat abnormalities (Dewailly et al., 2001). * Several studies suggest that diets or supplements rich in omega-3 fatty acids lower blood pressure significantly in people with hypertension (Appel, 1999). * Fish oil supplements containing EPA and DHA have been shown to reduce low density lipoprotein(LDL) cholesterol and triglycerides (Al-Harbi et al., 1995). * Omega-3 fatty acids from fish help overweight people to acieve better control over their blood sugar and cholesterol levels (Hibbeln and Salem, 1995). 2.4.3 Sources of Omega-3 Fatty Acid
Fish oil and plant oils are the primary dietary sources of omega-3 fatty acids (de Dekere et al., 1998). Omega-3 fatty acids are found in fish such as cod, salmon, halibut, sardines, tuna, mackerel, and herrings. ALA is found in flaxseed oil, walnut, canola (rapeseed oil). Other foods that contain omega-3 fatty acids include shrimp, clams, light chunk tuna catfish, cod and spinach.
2.5 STOCKFISH PRODUCTION
2.5.1 Origin of Stockfish
The terms "dried cod" and "stockfish" express only two different ways to treat the same fish, the codfish or "gadus morrhua", of the Gadidians family, osseus fish pertaining to the suborder of the Anacantinis, and these treatments are due to well defined environmental and climatic conditions for its preservation. The Gadidians family counts 140 species, grouped in 15 genders. Norway, Iceland, Greenland, the Baltic Sea and Newfoundland are extremely rich of this fish, because the sea waters in these lands are very cold and limpid; while the vulgar variety, the whiting, can be found in the Mediterranean Sea, but with different characteristics. ("Cod", EncyclopediaBritannicaonline2008) The one we are more interested in is the Gadus Morrhua of the Teleosteis order, brown or dark green coloured, with little yellow spots on the back and a white side band on all its body, and with a brownish abdomen, This fish can be up to 1-1,50 meters. long, and can reach the respectable weight of 50 kilograms. The most practiced places for catching it are the Lofoten islands, on the Northern Coast of Norway, in the shores of which you can see thousands of fishing boats between the months of December and April. The fishing is accomplished with fish-hooks and fishing-nets. As lure, fishermen use calamaries or slices of other fish. ( Extraordinary Animals: An Encyclopedia of Curious and Unusual Animals. Greenwood Press. 2007).
2.5.2 History of Stockfish Trade
Cod has been an important economic commodity in international markets since the Viking period (around 800 AD). Norwegians traveled with dried cod and soon a dried cod market developed in southern Europe. This market has lasted for more than 1000 years, enduring the Black Death, wars and other crises and is still an important Norwegian fish trade ( Barrett et al .,2000). The Portuguese began been fishing cod in the 15th century. Clipfish is widely enjoyed in Portugal. The Basques played an important role in the cod trade and allegedly found the Canadian fishing banks before Columbus' discovery of America. (Kurlansky, Mark 1997)The North American east coast developed in part due to the vast cod stocks. Many cities in the New England area located near cod fishing grounds.
Apart from the long history cod differ from most fish because the fishing grounds are far from population centers. The large cod fisheries along the coast of North Norway (and in particular close to the Lofoten islands) have been developed almost uniquely for export, depending on sea transport of stockfish over large distances (G.Rolfsen 1966). Since the introduction of salt, dried salt cod (clipfish or 'klippfisk' in Norwegian) has also been exported. By the end of the 14th century the Hanseatic League dominated trade operations and sea transport, with Bergen the most important port.William Pitt the Elder, criticizing the Treaty of Paris in Parliament, claimed that cod was "British gold"; and that it was folly to restore Newfoundland fishing rights to the French.Pacific Cod is currently enjoying strong global demand. The 2006 Total Allowable Catch (TAC) for the Gulf of Alaska and Aleutian Islands was 260,000 tons (Kurlansky mark, 1997). 2.5.3 Varieties of Stockfish
Due to its popular use in Nigeria, this are the varieties of stock fish in the nation and also found in the region of the west; White cod, Black cod(Paranotothenia microlepidota;), Apama, Oosan, and Ocheze which is also referred to as the king of the cods (Nigeria market, 2009).
In relation to the different types of cod in terms of literature, the following are types of cod stockfish found in the world;
Black Cod - Paranotothenia microlepidota; This saltwater fish, which is not a true cod, has a soft textured flesh and a mild flavor. Its high fat content makes it a good fish for smoking. Also called "sablefish."
Pollack - This low to moderate fat fish has firm, white, flesh with a delicate, somewhat sweet flavor. Pollack is often used to make imitation crab meat. Also known as "Coalfish" or "Saithe," this saltwater fish is a member of the cod family.
White cod- A small gray and white saltwater fish sometimes called the "silver hake." This low-fat fish, which is related to both the "cod" and the "hake," has a tender white fine-textured flesh and a flaky, delicate flavor.
Lingcod - A North American Pacific coast fish with a mildly sweet flavor and a firm, lean texture.
Haddock - Melanogrammus aeglefinus; A North Atlantic fish, the smaller cousin to the cod. The haddock has firm white flesh that is mild in flavor. Smoked haddock is called "finnan."
Hake - This low-fat saltwater fish, related to the cod, is found in the Atlantic and North Pacific. It's flesh features a white, delicate flavor.
King cod- The skull has a distinct top or crown giving it the name "king cod" or kongetorsk in Norwegian. In Norway this rare fish was earlier considered to be able to forecast the weather and was commonly used for that purpose.
2.5.4 Stockfish in Diet
Even though stockfish is originally native to the Vikings of the Scandinavia, it is extremely popular and widely consumed in the whole of west Africa, especially in Nigeria, Cameroon, Togo and sierra leone. It is called okporoko by the igbos of Nigeria and kpanla or panla by the Yorubas of the southern Nigeria. It is a highly flavored delicacy in ogbono, palmnut fruit(banga and egusi soup as well as in efo riro. It is also used in afang soup, and indeed most other soups like edikaikong. It is a prestigious and highly regarded gift to include in a visit to in-laws. With the advent of snack stockfish as well as stockfish chippings, it can be easily included in jollof rice and salad meal. 2.5.6 Effect of Drying on the Stockfish
Stockfish refers to fish dried in the sun on rocks or racks of wood in order to preserve the fish. The head and gut of the fish are removed before drying. This method of fish preservation is so powerful and effective that fish can last for up to 12 years without going bad, and yet retain its full nutritional content! Any fish can be "stocked", but only a few types of fishes are now used in making stock fish. They include Cod fish (most widely used), Tusk fish, Haddock, Pollock or Saithe and Ling fishes.
Fish preserved this way are extremely nutritious, and truly healthy. Unlike fresh fish cod that is made up of 80% water for example, cod stockfish is made up of only about 14% water, and 80 percent protein, which can then be said that 1 kg of stock fish is equivalent to 5kg of fresh fish.

CHAPTER THREE
3.0 Materials
The dried stock fish and the raw fresh frozen fish both of the same species (merluccius species) will be procured from Araada market in ogbomoso. Processing and drying of the fresh raw fish will be carried out in owodunni food processing laboratory, Department of food science and Engineering, LAUTECH, ogbomoso.
3.1 Method
3.1.1 Sample Preparation
The dried stock fish will be weighed and cut into pieces. The cut pieces will be pounded in a mortal and pestle, and then grounded in a milling machine into a fine texture. It will then be reweighed and recorded . samples for fatty acid analysis and lipid oxidation test will be weighed and taken for the analysis and the test.
The raw fresh frozen fish will be left to thaw, after which it will be weighed and washed. After washing, it will be dissected into two equal halves in order to remove the inedible portion and sliced thining. The neatly sliced fish will be divided into two batches, one of the batches will be treated with an antibiotics () while the other bathes will be without antibiotics. Both batches will be neatly spread on a tray and put in the sun in an airy environment for about four days. At daily interval, samples from the two batches will be taken for peroxide value and iodine value test. After the completion of the drying of the stimulated stock fish, sensory evaluation will be done on it.

Raw fresh fish
Thawing
Weighing
Washing
Slicing

Antibiotics Without Antibiotics Spreading on Tray Spreading on Tray Drying Drying Dried Stock Fish Dried Stock Fish

FLOW CHART FOR THE STIMULATION OF DRIED STOCK FISH

3.2 Fatty Acid Analysis 3.2.1 Extraction
Extraction of the fish lipids will be done according to the method of Bligh and Dyer [5] with some modification by Kinsella et al [6]. Representative samples of fish tissue (50g) will be homogenized in a blender for 2 minutes with a mixture of methanol (100 ml) and chloroform (50 ml). Then 50 ml of chloroform will be added to the mixture. After blending for an additional 30 seconds, 50ml of distilled water will be added. The homogenate will be stirred with a glass rod and filtered through a Whatman no.1 filter paper on a Buchner funnel under vacuum suction. 20 ml chloroform will be used to rinse the remainder. The filtrate will be allowed to settle to separate into the organic and aqueous layers. The chloroform layer containing the lipids was transferred into another beaker. Finally, a known amount of BHT (of about 0.02 g) will be added to the lipid solution as an antioxidant [6]. The solution will then be evaporated to a constant weight in a tared 100 ml round-bottom flask with a rotary evaporator at 40 oC. The determination of lipid content will be done separately for the dried stock fish, stimulated dried stock fish and the fresh raw fish.
3.2.2 Preparation of Fatty Acid Methyl Esters (Methylation) Fatty acid methyl esters will be prepared from the extracted lipid samples. 20mg of lipid sample will be placed into a screw capped tube; 1 ml of 0.5m methanolic potassium hydroxide added, capped and heated in a water bath for 5 minutes at 80 ºC. The tube will be removed from the water bath and cooled. 1ml of Boron trifluoride (14% in methanol) will be added and heated in a water bath at 80 ºC for 10 minutes. The tube will then be cooled slightly before the addition of 1ml of water and 1ml hexane. The solution will be vortex vigorously for 30 seconds and after settling, the content of the tube formed two separate layers. The top hexane layer was removed and put into a tube containing a small amount of sodium sulphate. After swirling to remove any water from the solvent, the hexane layer was transferred to a vial for gas chromatographic analysis.
3.2.3 Gas Chromatographic Analysis
The fatty acid methyl esters of the dried stock fish, stimulated dried stock fish and the fresh raw fish will be separated by the Buck Scientific, Model 910, Gas Chromatograph, equipped with a flame ionization detector and a Restek capillary column (RT-2560, 100 m x 0.25 mm I.D, 0.25 micron dry film). The oven temperature will be programmed to start at 1000C, held 4 minutes, then raised to 2400C at a rate of 30C/min and subsequently maintained at 2400C for 15 minutes. The injector and detector temperatures were set at 2200C and 2500C respectively. The carrier gas was Helium at a flow rate of 40 psi. Fatty acids were identified by comparing the retention times with those of 37 components contained in the Restek Food Industry FAME standard.
3.3 Lipid Oxidation Test
3.3.1 Iodine Value The iodine value during the process of stimulation of dried stock fish will be done at daily interval and also for the dried stock fish and will be determined by wiji’s method. 8g of iodine trichloride will be dissolved in 200ml of glacial acetic acid, 9g of iodine in 300ml of carbon tetra chloride. The two solutions will be mixed and diluted to 1000ml with glacial acetic acid. About 0.2-0.5ml of the extracted oil will be carefully measured with the aid of a dropping pipette into a glass stoppered flat bottom flask of about 250ml. The approximate weight of the oil to be taken will be determined by dividing 20 by the highest expected value. 10ml of carbon tetrachloride will be added to the oil in the flask and its content will be dissolved. 20ml of wiji’s solution will be added, mixed and the stopper which will have been previously moistened with potassium iodide will be inserted and allowed to stand in the dark for 30 minutes.15ml of potassium iodide solution and 100ml of water will be added, mixed and the mixture will be titrated with 0.1M thiosulphate solution using starch as indicator just before the end point (titration value will be equal to a,ml). The blank will be carried out simultaneously omitting the oil (titration value will be equal to b,ml).
Calculation
Iodine value = (b-a)ml 1.269 Weight (in g) of sample
If b-a is greater than b/2, The test will be repeated using smaller amount of sample. The result will be recorded in wijis 3.3.2 Peroxide Value
According to ( metilenbacher, 1960). The test will be carried out in subdued daylight. 1g of oil will be weighed out into a clean dry boiling tube after which 1g of powdered potassium iodide and 20ml of the solvent mixture ( 2 volume of glacial acetic acid + 1 volume chloroform) will be added. The tube will be placed in a boiling water bath so that the liquid boils within 60 seconds, the content will then be poured quickly into a titration flask containing 20ml potassium iodide solution, the tube will be washed twice with 25ml portions of water and added to the titration flask which will be titrated with 0.002M sodium thiosulphate using starch as indicator and recorded as (a, ml). A blank determination will also be carried out and the blank titre will be recorded as (b, ml).
Peroxide value = 2(a-b) milliequivalent of peroxide Weight of sample(kg)
3.4 Statistical Analysis of Data
Statistical analysis of all data will be done with the Statistical Analysis System (SAS) package (version 8.2 of SAS Institute Inc., 1999). Statistical significant differences (p≤0.05) in all data will be determined by Analysis of Variance (ANOVA) procedure while Least Significant Differences (LSD) will be used to separate the means

REFERENCES
Al-Harbi M.M, Islam M.W, Al-Shabanah O.A, Al-Gharably N.M. (1995). Effect of acute administration of fish oil. Omega-3; 33(7):555-558
Alasalvar, K.D.A. Taylor, E. Zubcov, F. Shahidi and M. Alexis, Differentiation of cultured and wild sea bass (Dicentrarchus labrax) total lipid content, fatty acid and trace mineral composition, Food Chemistry 79 (2002), pp. 145–150.
Anderson, M.L. and E.M. Ravesi, 1969. Reactions of free fatty acids with protein in cod muscle frozen and stored at -26°C after aging in ice. J. Fish Res., 26: 2727-2736.
Appel L.J, Non pharmacologic Therapies that reduce blood pressure; a fresh perspective 1999, 22(22(suppl.III):III1-III5.) Audley, M.A., K.J. Shetty and J.E. Kinsella, 1978. Isolation and properties of phosphilase A from Pollock muscle. J. Food Sci., 43: 1771-1775. DOI: 10.1111/j.1365-2621.1978.tb07410.x
Belluzzi A, Boschi S, Brignola C, Munarini A, Cariani C, Miglio F. polyunsaturated fatty Acids and inflammatory bowel disease. Am J Clin Nutr 2000:71(suppl):n339S-342S.
"Cod", Encyclopedia Britannica online 2008
Conner, W. E, The beneficial effects of ω − 3 fatty acids: cardiovascular disease and neurodevelopment, Current Opinion in Lipidology 8 (1997), pp. 1–3.
Dalgaard.P.1995. Qualitative and quantitative characterization of spoilage bacteria from packed fish. International Journal of Food Microbiology 26(1995) 319-333.
Danao-Camara TC, Shintani TT. The dietary treatment of inflammatory arthritis: case Report and review of the literature. Hawaii med J. 1999:58(5): 126-131.
De-Deckere EAM. Possible beneficial effect of fish and fish omega-3 polyunsaturated fatty acids in breast and colorectal cancer. Eur J Cancer prev. 1999;8:213-221.
Dewaily E, Blanchet C, Lemieux S, et al. Omega-3 fatty acids and cardiovascular disease risk factors among the inuit of Nunavik. Am j Clin Nutr. 2001;74(4):464-473.
Dyeberg, J. Linolenate polyunsaturated fatty acids and prevention of atherosclerosis, Nutrition Reviews 44 (1986) (4), pp. 125–134.
Edward R, Peet M, Shay J, Horrobin D. omega-3 polyunsaturated fatty acid levels in the diet and in the red blood cell membranes of depressed patients. J Affect Discord 1998;48(2-3):149-155.
Engvang, K. and H.H. Nielsen, 2001. Proteolysis in fresh and cold-smoked salmon during cold storage: Effects of storage time and smoking process. J. Food Biochem., 25: 379-395. 10.1111/j.1745-4514.2001.tb00747.x
Extraordinary Animals: An Encyclopedia of Curious and Unusual Animals. Greenwood Press. 2007. FAO (Food Agricultural Organisation).Quality and changes in fresh fish. http://www.fao.org/docrep/V7180E/V7180e09.htm FAO., 1986. Fisheries Technical Papers-T142. The production of fish meal and oil. Fisheries Industries Division, Food and Agriculture Organization of the United Nations, Rome, Italy. http://www.fao.org/DOCREP/003/X6899E/X6899E00.HTM FAO., 2005. Post-harvest changes in fish. In: FAO Fisheries and Aquaculture Department, Food and Agriculture Organization, Rome, Italy. http://www.fao.org/fishery/topic/12320/en Frankel, E.N., 1985. Chemistry of free radical and singlet oxidation of lipids. Progress. Lipid Res., 23: 197-221. Fraser, O. and S. Sumar, 1998. Compositional changes and spoilage in fish. Nutr. Food Sci., 5: 275-279.
Friedberg CE, Janssen MJ, Heine RJ, Grobbee DE. Fish oil and glycemic control in diabetes: a meta-analysis. Diabetes care. 1998;21:494-500.
G. Rolfsen (1966). "Norwegian fisheries Research.". FiskDir. Skr. Ser. HavUnders. 14(1): 36. Gram and Huss H.H.1996. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33(1996) 121-137 Hansen, T.L., T. Gill, S.D. Rontved and H.H. Huss, 1996. Importance of autolysis and microbiological activity on quality of cold-smoked salmon. Food Res. Int., 29: 181-186. DOI: 10.1016/0963-9969(96)00003-8
Haper CR, Jacobson TA. The fats of life: the role of omega-3 fatty acids in the prevention of coronsry hearth disease. Arch Intern Med. 2001;161(1802185-2192.
Hayes,P.R 1985. Food Microbiology and hygiene.Elsevier,London,pp 80-139.
Herbert,R.A and Shewan,J.M.1975. Precursors of volatile sulphides in spoiling North Sea Cod(Gadus morhua).Journal of Science Food and Agriculture,26,1195-1202. Hermansen1983.In: Regenstein C.E and Regenstein M.J 1991. Introduction to fish technology. Page 64. Published by Van Nostrand Reinhold, New York.
Herold, P.M and Kinsella, J.E. 1986. Fish oil consumption and decreased risk ofcardiovascular disease: A comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43: 566.
Hibbeln JR, Salem N, Jr. Dietary polyunsaturated fatty acids and depression; when cholesterol does not satisfy. Am J Clin Nut. 1995;62(1);1-9.
Huis in,t Veld.1996. Microbial and biochemical spoilage of foods: an overview. International Journal of Food Microbiology,33(1996)1-18.Elsevier Science B.V. Hultin, H.O., 1994. Oxidation of Lipids in Seafoods. In: Seafoods Chemistry, Processing Technology and Quality, Shahidi, F. and J.R. Botta (Eds.)., 1st Edn., Blackie Academic and Professional, London, UK., ISBN: 10: 0751402184, pp: 49-74.
Huss,H.H.,Dalgaard,D.,Hansen,L.,Ladefoged,H.,Pedersen,A., and Zittan,L.1974. The influence in catch handling on the storage life of iced cod and plaice. Journal of Food Technology.9, 213-221.
Huss, H.H. (editor) 1995. Quality and Quality Changes in Fresh Fish. FAO Fisheries technical paper 348, FAO Rome, Italy.
International Commission on Microbiological Specifications for Foods (ICMSF).1986. Sampling plan and recommended microbiological limits for Seafood. At: www.fao.org/docrep/X5624E/x5624e08.htm Khayat, A. and D. Schwall, 1983. Lipid oxidation in seafood. Food Technol., 37: 130-140. King, F.J., M.L. Anderson and M.A. Steinberg, 1962. Reaction of cod actomyosin with linoleic and linolenic acids. J. Food Sci., 27: 363-366. DOI: 10.1111/j.1365-2621.1962.tb00107.x
Kurlansky, Mark (1997). Cod: A Biography of the Fish That Changed the World. New York: Walker. ISBN 0-8027-1326-2.
Leaf, A and P.C. Weber, Cardiovascular effects of n − 3 fatty acids, New England Journal of Medicine 318 (1988), p. 549. Lin, T.M. and J.W. Park, 1996. Protein solubility in Pacific whiting affected by proteolysis during storage. J. Food Sci., 61: 536-539. DOI: 10.1111/j.1365-2621.1996.tb13151.x Liston, J.1980. Microbiology in fishery science. In: J. J. Connell (editor),Advances in Fishery Science and Technology. Fishing News Books, Farnham,England ,pp.138-157. Martinez, A. and A. Gildberg, 1988. Autolytic degradation of belly tissue in anchovy (Engraulis encrasicholus). Int. J. Food Sci. Technol., 23: 185-194.DOI: 10.1111/j.1365-2621.1988.tb00566.x
Regenstein M.J and C.E Regenstein. 1991. Introduction to Fish Technology. Published by Van Nostrand Reinhold, New York.
Sidhu, K. S. Health benefits and potential risks related to consumption of fish or fish oil, Regulatory Toxicology and Pharmacology 38 (2003), pp. 336–344.
Singer, P., Jaeger, W., Wirth, M., Voigt, S., Naumann, E., Zimontkowski, S. Hajdn, and Goedicke, W. 1983. Lipid and blood pressure-lowering effect of mackerel diet in man. Atherosclerosis 49: 99.
Skonberg, D. I and B.L. Perkins. (2002. Nutrient composition of green crab (Carcinus maenus) leg meat and claw meat, Food chemistry 77, 401–404.
Spencer R.and Baines C, R.1964.The effect of temperatures on the spoilage of wet fish 1.storage at constant temperature between-1C and 25C.Food Technology campaign 18,769-772.
Tapiero, H., G. Nguyen Ba, P. Couvreur and K.D. Tew, Polyunsaturated fatty acids and eicosanoids in human health and pathologies, Biomedicine Pharmacotherapy 56 (2002), pp. 215–222. Undeland, I., G. Hall, K. Wendin, I. Gangby and A. Rutgersson, 2005. Preventing lipid oxidation during recovery of functional proteins from herring (Clupea harengus) fillets by an acid solubilization process. J. Agric. Food Chem., 53: 5624-5634. DOI: 10.1021/jf0404445 Yorkowski, M. and H. Brockerhoft, 1965. Lyso lecthinase of cod muscle. J. Fish Res., 22: 643-652.