Vol. 71, Nr. 2, 2006—JOURNAL OF FOOD SCIENCE S83Published on Web 2/10/2006

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Quality Characterization of FarmedAtlantic Halibut During Ice StorageCCCCCHRISTELLEHRISTELLEHRISTELLEHRISTELLEHRISTELLE G G G G GUILLERMUILLERMUILLERMUILLERMUILLERM-R-R-R-R-REGOSTEGOSTEGOSTEGOSTEGOST, , , , , TTTTTRINERINERINERINERINE H H H H HAAAAAUGENUGENUGENUGENUGEN, R, R, R, R, RAAAAAGNARGNARGNARGNARGNAR N N N N NORORORORORTTTTTVEDTVEDTVEDTVEDTVEDT, M, M, M, M, MAAAAATTTTTSSSSS C C C C CARLEHÖGARLEHÖGARLEHÖGARLEHÖGARLEHÖG,,,,,BBBBBJØRNJØRNJØRNJØRNJØRN TTTTTOREOREOREOREORE L L L L LUNESTUNESTUNESTUNESTUNESTADADADADAD, A, A, A, A, ANDERSNDERSNDERSNDERSNDERS K K K K KIESSLINGIESSLINGIESSLINGIESSLINGIESSLING, , , , , ANDANDANDANDAND A A A A ANNANNANNANNANNA M M M M MARIAARIAARIAARIAARIA B B B B B. R. R. R. R. RØRÅØRÅØRÅØRÅØRÅ

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: A quality index method (QIM) was dev: A quality index method (QIM) was dev: A quality index method (QIM) was dev: A quality index method (QIM) was dev: A quality index method (QIM) was developed for fareloped for fareloped for fareloped for fareloped for farmed Amed Amed Amed Amed Atlantic halibut, and together with instrtlantic halibut, and together with instrtlantic halibut, and together with instrtlantic halibut, and together with instrtlantic halibut, and together with instrumen-umen-umen-umen-umen-tal, chemical, sensortal, chemical, sensortal, chemical, sensortal, chemical, sensortal, chemical, sensoryyyyy, and bacter, and bacter, and bacter, and bacter, and bacteriological analysisiological analysisiological analysisiological analysisiological analysis, quality changes of halibut stor, quality changes of halibut stor, quality changes of halibut stor, quality changes of halibut stor, quality changes of halibut stored on ice for 26 d was eved on ice for 26 d was eved on ice for 26 d was eved on ice for 26 d was eved on ice for 26 d was evaluated.aluated.aluated.aluated.aluated.TTTTTwo grwo grwo grwo grwo groups of fish woups of fish woups of fish woups of fish woups of fish wererererere fed diets that differe fed diets that differe fed diets that differe fed diets that differe fed diets that differed only in the soured only in the soured only in the soured only in the soured only in the source of lipid, wherce of lipid, wherce of lipid, wherce of lipid, wherce of lipid, where 1 diet contained only mare 1 diet contained only mare 1 diet contained only mare 1 diet contained only mare 1 diet contained only marine oiline oiline oiline oiline oilsources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored onsources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored onsources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored onsources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored onsources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored onice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effectice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effectice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effectice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effectice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effecton fron fron fron fron freshnesseshnesseshnesseshnesseshness, (A, (A, (A, (A, (ATP) degrTP) degrTP) degrTP) degrTP) degradation (K-vadation (K-vadation (K-vadation (K-vadation (K-value), texturalue), texturalue), texturalue), texturalue), textureeeee, color, color, color, color, color, or liquid-holding capacity, or liquid-holding capacity, or liquid-holding capacity, or liquid-holding capacity, or liquid-holding capacity. . . . . The QIM scorThe QIM scorThe QIM scorThe QIM scorThe QIM scores incres incres incres incres increased witheased witheased witheased witheased withstorage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut.storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut.storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut.storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut.storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut.The K-value was strongly correlated with storage time (The K-value was strongly correlated with storage time (The K-value was strongly correlated with storage time (The K-value was strongly correlated with storage time (The K-value was strongly correlated with storage time (rrrrr = 0.99), while total bacterial counts increased after 7 to 8 d = 0.99), while total bacterial counts increased after 7 to 8 d = 0.99), while total bacterial counts increased after 7 to 8 d = 0.99), while total bacterial counts increased after 7 to 8 d = 0.99), while total bacterial counts increased after 7 to 8 dof ice storof ice storof ice storof ice storof ice storageageageageage. . . . . The texturThe texturThe texturThe texturThe textureeeee, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, and color w, and color w, and color w, and color w, and color wererererere significantly affected be significantly affected be significantly affected be significantly affected be significantly affected by story story story story storage time durage time durage time durage time durage time during theing theing theing theing theearly perearly perearly perearly perearly period of storiod of storiod of storiod of storiod of storageageageageage, pr, pr, pr, pr, probably due to obably due to obably due to obably due to obably due to rigorrigorrigorrigorrigor stiffness and stiffness and stiffness and stiffness and stiffness and rigorrigorrigorrigorrigor r r r r resolution. esolution. esolution. esolution. esolution. The texturThe texturThe texturThe texturThe textureeeee, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, liquid-holding capacity, and, and, and, and, andcolor did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.

KKKKKeyworeyworeyworeyworeywords: ds: ds: ds: ds: HHHHHippoglossus hippoglossusippoglossus hippoglossusippoglossus hippoglossusippoglossus hippoglossusippoglossus hippoglossus, fr, fr, fr, fr, freshnesseshnesseshnesseshnesseshness, quality, quality, quality, quality, quality, v, v, v, v, vegetable oil, micregetable oil, micregetable oil, micregetable oil, micregetable oil, microbiologyobiologyobiologyobiologyobiology

Introduction

Fish farming has developed into a highly productive and effi-cient global industry producing animal protein. The FAO (2004)

has predicted that by 2015, 39% of all fish for human consumptionwill come from aquaculture or sea ranching. Atlantic halibut (Hip-poglossus hippoglossus L.) is a well-known species from the fisheriesof the North Atlantic Ocean (Haug 1990). Catches of wild Atlantichalibut have decreased in recent years to a level below 5000tonnes, and further growth for this species in the food sector musttherefore come from aquaculture. Atlantic halibut is a promisingspecies for aquaculture, and researchers have concentrated onsolving biological and technological obstacles in juvenile produc-tion (Harboe and others 1994, 1998; Gara and others 1998). An in-creasing supply of juvenile Atlantic halibut could soon result inmany fish reaching market size. However, little is known about thequality characteristics, shelf life, and product differentiation of thisspecies (Norvedt and Tuene 1998; Ruff and others 2002a; Olssonand others 2003b).

The quality of wild halibut differs considerably between fish(Olsson and others 2003b), whereas the quality of farmed halibutis more homogenous. This is because aquaculture can better man-age product quality through breeding, feeding, and slaughter han-dling. Common freshness indicators used in fish include the K-value (which measures the rate of [ATP] degradation), and theconcentrations of biogenic amine, trimethylamine, and volatilecompounds (Olafsdottir and others 1997). Luten (1995) stated, with

reference to plaice, sole, and herring, that determination of the K-value is a more useful freshness indicator than determination oftotal volatile bases, ammonia, trimethylamine, and histamine.These indicators are believed to be more suitable for classifyingborderline cases. In recent years, the quality index method (QIM),a grading system for estimating the freshness and quality of sea-food, has been established for several species like seabream(Huidobro and others 2000) and salmon (Sveinsdottir and others2003), and is widely used in the industry. Oehlenschlaeger (1995)showed that plaice (Pleuronectes platessa) stored on ice for up to 25d, could be marketed up to day 18 and was edible up to day 20 of icestorage. The chemical, microbiological, and sensory parameters offarmed halibut hardly deteriorate at all during the 1st 21 d of icestorage (Akse and Midling 2001).

The demand for high-quality fish oil for aquaculture productionwill exceed supply within a few years, and other sources of fat willbe required in fish diets. The use of vegetable oils has increasedextensively, but will inevitably alter the quality of the flesh, relat-ed to both fat content and fat composition. It is well known that di-etary fat content and the dietary fatty acid profile influence the fatcontent and the fatty acid profile of the muscle in a variety of spe-cies (Thomassen and Rosjo 1989; Nortvedt and Tuene 1998). Thelipid content and composition of the fillet may therefore be manip-ulated in aquaculture species. Lipid oxidation is a major concern inhigh-value seafood products because it leads to the loss of texture,color, flavor, and highly polyunsaturated fatty acid (PUFAs) (Waag-bo and others 1993). The rate of lipid oxidation depends upon thetype and amount of fat in the fillet.

The aim of this study was to evaluate quality changes in Atlantichalibut stored on ice for 28 d. A quality index method was devel-oped for Atlantic halibut, and chemical and bacteriological degra-dation of the fish were measured as well as sensory changes. Inaddition, the effect of different lipids in the feed pre-slaughter wasinvestigated.

MS 20050257 Submitted 5/4/05, Revised 7/24/05, Accepted 11/7/05. AuthorsGuillerm-Regost and Rørå are with AKVAFORSK, Inst. of Aquaculture Re-search AS, P.O. Box 5010, NO-1432 Ås-NLH, Norway. Author Haugen is withInst. of Marine Research, Bergen, Norway. Authors Nortvedt and Lunestadis with Natl. Inst. of Nutrition and Seafood Research, Bergen, Norway. Au-thor Carlehög is with Norwegian Inst. of Fisheries and Aquaculture Research(Fiskeriforskning), Tromsø, Norway. Author Kiessling is with Inst. of Animaland Aquaculture Sciences, Agricultural Univ. of Norway, Norway. Directinquiries to author Trine Haugen (E-mail: Trine.Haugen@imr.no).

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Quality of halibut during ice storage . . .

Materials and Methods

Fish material and treatmentsFish material and treatmentsFish material and treatmentsFish material and treatmentsFish material and treatmentsFarmed Atlantic halibut were reared at the Inst. of Marine Re-

search, Austevoll (Norway) for 1 y on experimental diets. The dietswere based on wheat, decapitated saithe (Pollachius virens), wholeherring (Clupea harengus), squid (Ommastrephes sagittatus), and vi-tamin and mineral mix. In addition, the marine diet contained12.5% herring oil and the soya diet contained 12.5% soybean oil.The gross chemical composition of the diet was 25% lipids (mea-sured as percentage of the dry matter), 60% protein, 7% carbohy-drates, and 8% ash. It was prepared as a moist pellet with a size of18 mm, using microwave technology. Fifty-two fish from eachgroup whose weights were as close as possible to the mean weightof the group were selected and slaughtered. The mean weight ofthese fish was 2438 ± 83 g. Each fish was individually labeled, andits round weight, length, and gutted weight were noted. Forty-twofish from each group were individually wrapped in plastic bags,packed on ice, and immediately sent to AKVAFORSK (Ås, Norway),where they were stored on ice at 4 °C before analysis. Another 10halibut from each group were packed on ice and sent to Fiskerifor-skning (Tromsø, Norway) for sensory analysis.

SamplingSamplingSamplingSamplingSamplingFish were sampled at 14 points after slaughter: 1, 2, 4, 6, 8, 10, 12,

14, 16, 18, 20, 22, 24, and 26 d after slaughtering, 28 d correspond-ing to the estimated maximum storage time. Three fish from eachgroup were sampled for analysis at each time point. The 3 fish wereevaluated using the QIM method, weighed, and then 2 cutlets of athickness of 2.5 cm were removed from 10 cm posterior the base ofthe pectoral fin. The 1st cutlet was used for texture analyses bycompression, and for liquid-holding capacity analyses, whereasanalysis of color by Minolta colorimeter was carried out on the 2ndcutlet. Two pieces of the 1st cutlets of fish sampled on days 2, 4, 8,14, 20, and 26 were frozen in liquid nitrogen and stored at –80 °C foranalysis of ATP degradation. At the same times, muscle samplesfrom 3 fish in each group were pooled, homogenized, and stored at–40 °C for analysis of thiobarbituric acid-reactive substances(TBARS) and vitamin E. Muscle samples with skin (10 g) of a sur-face area of 2.5 cm2 were taken aseptically on days 2, 4, 8, 14, 20, 22,24, and 26, transferred to a sterile plastic bag, and stored at –20 °Cfor microbiological analysis. The samples of the second cutlet takenon days 6, 8, 22, and 24 were pooled, homogenized, and stored at –40 °C for analysis of dry matter, ash, protein, free amino acid, fat,and fatty acids. Samples from the 10 fish taken for sensory analy-sis were taken from the dorsal side on days 6 and 22.

Quality index methodQuality index methodQuality index methodQuality index methodQuality index methodA quality index method was established for farmed Atlantic hal-

ibut (Table 1). The QIM scheme was based upon the objective eval-uation of certain attributes of raw fish (skin, eyes, gills, and flesh)using a demerit point scoring system (from 0 to 3 points) adjustedto farmed Atlantic halibut with new attributes description wherenecessary. The attributes that were evaluated were dark side, whiteside, mucus and texture for appearance, pupils and form of eyes,color, mucus, and odor of gills, and color of flesh fillets (Table 1).

Three trained personnel agreed on 1 score per attribute per fish,and the scores for all attributes were then added to give an overallsensory score, called the quality index.

Flesh quality analysesFlesh quality analysesFlesh quality analysesFlesh quality analysesFlesh quality analysesThe color of the cutlets was measured with a Minolta ChromaMeter

CR200 (Minolta, Osaka, Japan) equipped with light source C and 2°

angle of observation. Two parts of the raw cutlets were measured, andfor each measurement, the L* value (lightness: L* = 0 for black, L* = 100for white), a* value (red/green: +a* = redness, –a* = greenness), and b*value (yellow/blue: +b* = yellowness, –b* = blueness) were recorded,as recommended by the CIE (1976) L*a*b* method. Fillet texture wasevaluated instrumentally using a Texture Analyser TA-XT2 (StableMicro Systems, Surrey, England) equipped with a load cell of 5 kg anda cylindrical plunger (12.5 mm diameter) performing a texture profileanalysis (TPA). The plunger was pressed into the cutlets at a constantspeed of 2 mm/s until it reached a depth that was 90% of the sampleheight. The maximum force obtained during compression was record-ed at 2 locations on each cutlet, and the mean value was used in thedata analysis. The liquid-holding capacity (LHC) of raw muscle wasdetermined using a modified version of the method developed byGomez-Guillen and others (2000). Muscle samples (15 g) were sliced,weighed, and placed in a tube with a weighed filter paper (V1) (Schle-icher & Schuell GmbH, Dassel, Germany). The tubes were centrifugedat 500 × g for 10 min at 10 °C, and the wet paper was weighed (V2) be-

Table 1—Quality index method scheme for farmed Atlan-tic halibuta

QIMDescription score

Appearance Dark Fresh, bright, no discoloration 0 side Bright, but without shine 1

Dull, pale, some green discoloration 2Dull, purple, green, green spoilage tints 3

White Fresh, bright, no discoloration 0 side Rather mat, wound near the tail is yellow 1

Yellow/green discoloration at fins and in the middle 2Yellow and purple discoloration 3

Mucus Clear, not clotted 0Slightly clotted and milky 1Clotted and slightly yellow 2Clotted and yellow 3

Texture Firm, elastic 0Less firm 1Soft 2Very soft 3

Eyes Pupils Clear and black, golden rim around the pupil 0

Rather mat, faint golden rim around the pupil 1Mat, opaque pupil, reddish 2Milky, gray purple 3

Form Convex 0Convex and slightly sunken 1Sunken, swollen, eye socket shrunken 2Flat, sunken in the middle 3

Gills Color Bright, red 0

Slightly discolored, at the end of filaments 1Discolored, brown/yellow, gray 2Yellowish, brown, gray 3

Mucus No mucus 0Milky, clear 1Milky, yellow, slightly clotted 2Clotted, yellow, brown 3

Odor Fresh, seaweed 0Neutral, oily, grassy, metallic 1Musty, yeast, sour milk 2Rotten, rancid, sour, sulphurous 3

Flesh fillets Color Fresh, translucent, bluish, crème 0

Waxy, milky 1Yellow, brownish, discolored 2

QIM score 0-29aQIM = quality index method.

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fore drying at 50 °C to constant weight (V3). The liquid loss as % wascalculated as 100 × (V2 – V1)/S, where S is the weight of muscle sample;the percentage water loss was calculated as 100 × (V2 – V3)/S; and thepercentage fat loss as 100 × (V3 – V1)/S.

Sensory profiling was performed on steam-cooked samples byan external sensory panel (Fiskeriforskning, Tromsø, Norway) con-sisting of 7 assessors selected for their interest, availability, andsensorial capacities of memorizing stimuli and discriminating in-tensities. All assessors received regular training to develop theirsensory performances and their knowledge of marine products.Sessions were conducted in a room designed for sensory analysis,with standardized light (ISO 8589) and equipped with a computer-ized system, Tecator’s Senstec system (Tecator AB, Höganäs, Swe-den). The samples were wrapped in aluminum foil, coded, steameduntil a core temperature of 72 °C was reached, and served immedi-ately in a random order. The samples were evaluated using a con-tinuous scale presented on a computer screen from 0 (low intensity)to 10 (high intensity) for the following 18 attributes: total intensity,fresh, deviant, old, rancid, and green for smell; total intensity, fresh,sour, green, old, deviant, and rancid for taste; whiteness, hardness,juiciness, chewing resistance, and after-taste.

Chemical analysisChemical analysisChemical analysisChemical analysisChemical analysisThe samples were analyzed for dry matter content (105 °C for 24

h), ash (550 °C for 16 h), protein (Kjeldahl N × 6.25) (Kjeltech AutoAnalyser, Tecator AB, Höganäs, Sweden), and fat using the methoddescribed by Folch and others (1957). Fatty acids (FA) were deter-mined after methanolysis whereby all FA were converted to methylesters in a gas chromatograph (Perkin Elmer Autosystem GC (Per-kin Elmer, Boston, U.S.A.) equipped with a programmable split/splitless injector, CP Wax 52 column [L = 25 m, inner dia = 0.25 mm,df = 0.2 �m]), flame ionization detector and 1022 data system). Thecarrier gas was He and the injector and detector temperatures were280 °C. The oven temperature was raised from 50 °C to 180 °C at arate of 10 °C/min, and then raised to 240 °C at a rate of 0.7 °C/min.Individual fatty methyl esters FA composition was expressed aspercentage of FA methyl esters.

TBARS were analyzed following a procedure modified fromSchmedes and Hølmer (1989). TBARS were extracted from the ho-mogenized samples by shaking under nitrogen with achloroform:methanol mixture (2:1) for 30 min at room temperature.Two parts of distilled water was added to make a 2-phase system.An aliquot of the methanol water phase containing the short chainaldehydes was heated (30 min, 100 °C) in the presence of excessthiobarbituric acid in trichloroacetic acid. The resulting complex wasmeasured spectrophotometrically at 532 nm against a malondial-dehyde-standard. The amount of ATP degradation that took placewas determined using the K-value as described by Valle and others(1998). The tocoperols were analyzed according to Lie and others(1994) by normal phase HPLC with fluorescence detection (excita-tion: 289 nm, emission: 331 nm). The homogenized samples (0.5 g)were prepared for analysis by saponification in 4 mL ethanol, 0.5mL saturated ethylenediaminetetraacetic acid (EDTA), and 0.5 mL20% KOH, and extracted in 2 × 2 mL hexane. Ascorbic acid and py-rogallol were added before saponification to prevent oxidation ofthe sample.

Samples destined for the analysis of free amino acid (FAAs) werehomogenized (2 g) and phosphate buffer (pH = 7) was added. Thisprocedure extracted amino acids, peptides, and soluble protein.The protein was then removed by centrifugation and precipitationafter adding sulpho-salicylic acid (50 g/L 1:1), followed by the inter-nal standard (Norleucin DL, Sigma Chemicals Ltd.) based on Cohenand others (1989). The samples were post-derivatized by adding

ninhydrine after the ion exchange column and the free amino ac-ids were determined by spectrophotometer (Biochrom Ltd., Cam-bridge, U.K.) at 440 nm (proline and hydroxyproline) and at 570 nm(main channel) detection. The integration of the single peaks wasdone by the Millenium (Ver. 32) software package.

MicrobiologyMicrobiologyMicrobiologyMicrobiologyMicrobiologyThe aerobic total plate count and the number of H2S-producing

bacteria were determined in triplicate using a pour-plate techniqueapplying Iron Agar Lyngby (Oxoid, Basingstoke, U.K.) according tothe method of The Nordic Committee on Food Analysis (1994).Samples were homogenized for 30 s in a “Stomacher” and then di-luted with distilled water and pour-plated. The bacterial coloniesthat grew on the plates were counted after 3 d of aerobic incubationat 20 ± 1 °C. The aerobic total plate count was obtained by register-ing all bacterial colonies on incubated plates from a suitable dilu-tion of the sample. The number of colonies showing a clear darkcolor represented the bacterial population able to produce H2S.

StatisticsStatisticsStatisticsStatisticsStatisticsAnalysis of variance (ANOVA) was performed by GLM in Unistat

5.0 (UNISTAT 2000) studying the main effects of diet and storagetime and interaction effects. Means were ranked by the StudentNewman-Keuls method. Pearson’s product-moment correlationsbetween different variables were also calculated. The significancelevel was set at 5% unless otherwise stated.

Results and Discussion

FreshnessFreshnessFreshnessFreshnessFreshnessFreshness is one of the most important quality criteria for fish, and

storage time and temperature are the main factors affecting the rateof loss of quality and shelf life of fish (Whittle 1997). The freshness offarmed Atlantic halibut evaluated by QIM decreased with storage time(Figure 1). There was no statistically significant difference in QIMscores for the 2 dietary groups, and consequently the results present-ed in Figure 1 are from all fish. The QIM score increased from day 4until day 26, and the score was highly correlated with storage time (r =0.99). This has also been found for Atlantic salmon (Sveinsdottir andothers 2003) and for seabream (Huidobro and others 2000).

The total QIM score was 0 on day 1 and day 2. The score for fac-tors related to appearance remained low (2) until day 12 and thenincreased rapidly from day 16 (Figure 2a), showing that the skin iswell preserved the 1st 14 d of ice storage. The score from factorsrelated to the eyes increased between days 2 to 4, decreased on

Figure 1—Quality index method scores of farmed Atlantichalibut stored on ice for 26 d

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Quality of halibut during ice storage . . .

day 6, and then increased gradually and continuously from day 8to the end of the experiment (Figure 2b). The score from factorsrelated to the gills did not increase initially, but increased rapidlyafter day 12 (Figure 2c), never reaching the maximum score (9)even after 26 d. The score from the quality of the flesh rose mostslowly, remaining at 0 until day 14 (Figure 2d).

In the present study, QIM scores did not reach the maximumscore at day 26, confirming the results of Akse and Midling (2001)showing the very long storage ability of farmed halibut.

The diet of the fish affected the composition of the fillet (Table

2). Dry matter and fat contents were affected both by the inclusionof soybean oil and also by the storage time. The percentages of fatin the muscle were similar to the previous results from farmed hal-ibut (Norvedt and Tuene 1998; Olsson and others 2003b), but thevariation between the different measuring points was high, vary-ing from 1.6% to 5.7% (Table 2). Only 3 fish from each dietary groupwere sampled at any measuring point, and according to Nortvedtand Tuene 1998, this number may be too low because Atlantic hal-ibut has been shown to have a great individual variation in fat con-tent of the fillet related to both weight and previous weight gain.

Table 2—Proximate composition (expressed as percentage of wet weight) and fatty acid (FA) profiles of farmed Atlan-tic halibut musculature, stored on ice for up to 26 d (n = 3)a

Day 6 Day 8 Day 22 Day 24 ANOVA

Marine Soya Marine Soya Marine Soya Marine Soya Diet Storage

Muscle compositionDry matter 24.9 24.7 26.8 26.5 24.2 24.4 24.8 23.3 * *Ash 1.4 1.6 1.5 1.4 1.3 1.4 1.3 1.4 ns nsProtein 20.1 20.9 20.3 20.6 21.6 20.9 20.7 20.6 ns nsFat 3.5 3.7 5.7 4.9 1.6 2.5 3.3 1.9 * ***FA profileSum saturated 24.1 18.8 23.3 18.9 23.8 20.0 23.9 21.4 *** nsSum monoenes 48.8 36.7 47.6 37.0 45.2 33.3 48.1 35.0 *** *18:2n-6 2.5 25.9 2.6 24.8 2.6 23.4 3.1 25.7 *** ns20:2n-6 0.3 1.0 0.3 0.9 nd 0.9 nd 0.8 *** ns20:4n-6 0.4 0.4 0.3 0.2 0.5 0.5 0.5 0.4 ns *Sum n-6b 3.5 27.4 4.1 26.2 3.4 25.0 3.9 27.2 *** ns18:3n-3 1.1 2.8 1.3 3.4 1.0 2.6 1.2 nd ** ns18:4n-3 2.3 0.7 2.5 0.9 2.1 0.8 2.1 0.8 *** ns20:3n-3 0.8 0.7 0.8 0.7 0.8 nd 0.3 0.7 ns ns20:4n-3 0.5 nd 0.6 nd 0.6 nd 0.6 0.2 ** ns20:5n-3 5.6 3.0 5.9 3.4 6.3 3.8 5.6 3.8 ** ns22:5n-3 0.8 0.7 0.9 0.5 1.1 0.8 1.0 0.6 ** ns22:6n-3 11.0 9.1 10.8 7.9 15.3 13.0 11.8 10.5 ** *Sum n-3 22.2 16.9 22.8 16.8 27.2 20.9 22.6 16.6 *** **n-3/n-6 6.3 0.6 5.6 0.6 8.0 0.8 5.8 0.6 ** ns� of sums 98.6 99.8 97.8 98.9 99.6 99.2 98.5 100.2a*is P < 0.05; ** is P < 0.01; *** is P < 0.001; ANOVA = analysis of variance; ns = not significant; nd = not detected.bSum n-6 include 18:3n-6 and 20:3n-6 (not shown).

Figure 2—Quality indexmethod: appearance (a),eyes (b), gills (c), andflesh (d) scores offarmed Atlantic halibutstored on ice for 26 d

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Table 3—Free amino acids (expressed as mg/g of muscle) of farmed Atlantic halibut stored on ice for 26 da

Day 6 Day 8 Day 22 Day 24 ANOVA

Marine Soya Marine Soya Marine Soya Marine Soya Diet Storage Interac.

AA profileAlanine 0.185 0.204 0.196 0.134 0.197 0.241 0.265 0.228 ns *** ***Arginine 0.014 0.027 0.016 0.021 0.023 0.023 0.021 0.029 *** *** ***Asparagine nd nd nd nd nd nd nd nd — — —Aspartic acid 0.013 0.018 0.014 0.013 0.017 0.019 0.029 0.017 * *** ***Cystein nd nd nd nd nd nd nd nd — — —Glutamic acid 0.053 0.058 0.056 0.041 0.062 0.083 0.089 0.070 ns *** ***Glutamine 0.029 0.021 0.032 0.016 0.017 0.018 0.017 0.023Glycine 0.332 0.280 0.216 0.069 0.160 0.177 0.250 0.231 *** *** ***Histidine 0.043 0.033 0.074 0.039 0.047 0.061 0.052 0.077Isoleucine 0.006 0.006 0.007 0.008 0.016 0.017 0.017 0.018 ns *** nsLeucine 0.011 0.009 0.013 0.010 0.027 0.031 0.034 0.042Lysine 0.047 0.122 0.083 0.117 0.096 0.109 0.096 0.115 *** ** ***Methionine 0.008 0.008 0.008 0.009 0.015 0.017 0.019 0.025 *** *** ***Phenylalanine 0.008 0.009 0.010 0.011 0.020 0.022 0.024 0.030 *** *** **Proline 0.012 0.046 0.056 0.012 0.029 0.060 0.049 0.029 ns *** ***Serine 0.043 0.055 0.037 0.038 0.036 0.034 0.049 0.047 * *** ***Threonine 0.068 0.077 0.082 0.036 0.062 0.111 0.091 0.099 *** *** ***Tryptophan nd nd nd nd nd nd nd nd — — —Tyrosine 0.008 0.009 0.009 0.011 0.018 0.020 0.022 0.026 *** *** nsValine 0.015 0.014 0.015 0.017 0.032 0.037 0.036 0.038 *** *** *a* P < 0.05; ** P < 0.01; ***P < 0.001; ANOVA = analysis of variance; ns = not significant; nd = not detected at a detection limit of 0.002 mg/g.

Neither diet nor storage time affected the percentages of pro-tein and ash in the muscles. The type of oil in the diet affected thefatty acid (FA) profile of the muscles, as did, to a lesser extent, thestorage time on ice. The reflection of the dietary FA profile on themuscle has also earlier been demonstrated in Atlantic halibut byHemre and others (1992) and for other fish species (Greene andSelivonchick 1990; Regost and others 2003b). The sum of n-6 FAswas higher in fish fed the soya diet than it was in fish fed the marinediet. Consequently, the sum of n-3 FAs was higher in the marine oil–fed group than it was in the soybean oil–fed group.

The profiles of free amino acids (FAAs) in the muscles were differ-ent in the 2 dietary groups (Table 3). The amounts of 4 amino acids:alanine, glutamic acid, isoleucine, and proline, were not affected bydiet. The amount of FAAs present increased throughout the storageperiod from day 6 to day 24, with an average increase for all FAAs of93% ± 30% in the soya group and 105% ± 23% in the marine group.This general increase of FAAs during storage has also been reportedin salmon (Hultmann and Rustad 2004) and was mainly attributedto muscle proteolysis. In the present study, this increase was relative-ly high, but the final absolute levels were 1 to 2 orders of magnitudelower than observed in stored by-products from Atlantic salmon andrainbow trout (Nortvedt and others 2002). FAAs serve as a substratefor microbial growth as muscle spoilage progresses. Other researchershave reported that the amounts of FAAs decrease after long storage,as FAAs are converted to biogenic amines. This did not take placeduring the 26 d of storage in our study.

Flesh qualityFlesh qualityFlesh qualityFlesh qualityFlesh qualitySensory analyses of flesh from the 2 groups were performed 6 d

and 22 d after slaughter (Table 4). The sensory panel did not rejectany fish on day 22. The inclusion of vegetable oil in the diet did notsignificantly affect the sensory parameters of the flesh, except forgreen smell. This agrees with the results of Waagbo and others(1993) showing that the sensory characteristics of salmon were littleaffected by FA composition of the diet, for a constant lipid level. Thishas also been showed on brook charr (Guillou and others 1995) andAtlantic salmon (Hardy and others 1987), whereas Regost and oth-

ers (2003a) found an influence of soybean oil on sensory propertiesof turbot. This discrepancy between the aforementioned studiescould be explained by differences in levels of vegetable oil in thediets and in storage conditions between the studies.

The term green smell is a grassy or cucumber like smell, and isusually a contributor to the distinct fishy smell of fish oil and iscaused by the presence of aldehydes (Lin 1994) or more specificnonadienal, decadienal, and/or nonenal (Prost and others 1998).The marine group had a significantly higher degree of green smellat day 6 than the soy fed group, but this difference had disap-peared by day 22. The intensity of smell increased somewhat withstorage time. Rancid smell and rancid taste increased with storagetime, while green smell decreased, indicating that lipid oxidationhad begun at a low rate in the flesh, and the components giving thegreen smell in the marine group had oxidized, or the rancid compo-nents had become predominant. The increases in rancid smell andrancid taste were higher in the group fed exclusively on fish oilthan they were in the group with soybean oil inclusion, and webelieve that this is a result of higher levels of PUFAs in the musclesof the fish oil group. Sour and green tastes decreased significantlywith storage time while the hardness increased. Deviant smell anddeviant taste are smells or tastes that are not familiar or natural tothe product. There was no detected difference between the marindiet and the soya diet neither on day 6 nor on day 22 of storage.This shows that the use of soybean oil as a replacement for part ofthe dietary oil gives no taste or flavor to the product that is unwant-ed and could deteriorate the impression of the product.

The K-value increased with storage time (Figure 3a) showing thatthe nucleotides were degraded. Once again, there was no differ-ence between the 2 dietary groups, and the results presented inFigure 3 are from all fish. The K-value was highly correlated withstorage time (r = 0.99, P < 0.05), as it is for sea bass stored on ice for21 d (Kyrana and Lougovois 2002) and for seabream stored on icefor 23 d (Alasalvar and others 2001).

Dietary treatment did not affect the texture of the flesh. Thetexture, expressed by the maximal force, decreased with the storagetime in the 1st period due to rigor mortis and rigor resolution (Fig-

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Quality of halibut during ice storage . . .

ure 3b). The texture did not change significantly after day 8. Thisresult agrees with the results obtained by Sigurgisladottir and oth-ers (1999) for salmon and those obtained by Alasalvar and others(2001) for seabream. The sensory analyses on the other hand re-vealed an increased hardness between day 6 and day 22. The dis-crepancy between these 2 methods is probably an effect of the liq-uid loss. The sensory method is more sensitive to liquid loss thanthe instrumental measurements, especially when the liquid lossoccurs on the surface of the fillet.

The liquid-holding capacity (LHC) of farmed Atlantic halibutmuscle, expressed as percentage of loss, was not influenced by thecomposition of the oil in the diets. The water losses were low for the1st 4 d, increased on days 6 and 8, and then decreased slightly be-tween days 10 and 16. From day 18, the water losses increasedagain (Figure 3c). This agrees with the results of Olsson and others(2003a) who found an increase in liquid loss from day 0 to day 4and a decrease after 8 d of cold storage. The percentage of waterlost in our study was approximately the same as that found by Ols-son and others (2003c) on halibut but somewhat higher than theloss from salmon muscle (Rora and others 2003). In the presentstudy liquid loss was negatively correlated with the texture of theflesh from day 4 to day 8 (Figure 3b and 3c). The fat losses were verylow, not affected by storage time and amounted to only 10% of thetotal liquid losses. This indicates a relation between the strengthand longevity of the rigor process and water loss from the fillet.

The lightness L* of the cutlets increased significantly during the1st 6 d of storage, and the same was the case for the b* value (yel-lowness/blueness), showing that the cutlets went from blueness forthe 1st 4 to 6 d to yellowness from day 6 and for the rest of the stor-age period. The negative a* value (redness/greenness) showedthat the cutlets had a greenish color, and this value decreased sig-nificantly between day 4 and day 6 (Figure 4).

These early color changes are probably related to muscle structurealterations during rigor and the rigor resolution. The color parametersdid not change significantly after day 6, which agrees with the resultsfrom the sensory evaluation of whiteness (Table 4). Fillets of halibutbecome more yellow as the melondialdehyde concentration increase(Ruff and others 2002a). On the other hand, in our study, the TBARSvalues were very low and mostly under the detection limit in allgroups (0.24 to 1.61 nmol). It seems that, in halibut, lipid oxidation is

Table 4—Sensory profiles for farmed Atlantic halibut after 6 d and 22 d of storage on icea

Day 6 Day 22 ANOVA

Descriptors Marine Soya Marine Soya Diet Storage

Total intensity of smell 3.8 4.4 4.9 4.6 ns *Fresh smell 4.3 3.8 4.0 4.1 ns nsDeviant smell 0.5 0.9 1.0 0.7 ns nsOld smell 1.3 1.7 1.9 1.5 ns nsRancid smell 1.6 1.5 3.8 2.8 ns ***Green smell 3.0 1.8 1.4 1.2 ** ***Total intensity of taste 4.8 4.4 4.9 4.5 ns nsFresh taste 3.3 3.9 3.8 3.9 ns nsSour taste 2.2 2.4 1.0 0.9 ns ***Green taste 2.1 1.2 1.1 1.0 ns *Old taste 2.2 1.2 1.6 1.5 ns nsDeviant taste 0.6 0.5 0.9 0.9 ns nsRancid taste 1.8 1.8 3.1 2.4 ns *Juiciness 5.0 3.6 4.4 4.4 ns *Chewing resistance 3.2 4.6 4.5 4.1 ns nsAfter-taste 4.0 3.6 3.6 3.4 ns nsWhiteness 7.1 7.0 6.9 7.0 ns nsHardness 3.2 3.8 5.4 5.0 ns ***a* P < 0.05; ** P < 0.01; ***P < 0.001; ANOVA = analysis of variance; ns = not significant.

Figure 3—K-values (a), instrumental texture analyses (b),and changes in liquid-holding capacity (expressed as per-centage of water and fat lost) (c) of farmed Atlantic hali-but muscle stored on ice for 26 d

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inhibited before the formation of quality-deteriorating secondaryoxidation products (Ruff and others 2002a), which is in contrast toresults from other flatfish (Ruff and others 2002b, 2003).

Analysis of the E vitamins did not detect any �-tocopherol. Thelength of storage on ice did not affect the concentrations of �-, �-, �-tocopherols. The mean concentrations were 14.2 ± 0.6 �g/g for �-tocopherol, 5.2 ± 0.5 �g/g for �-tocopherol, and 1.8 ± 0.2 �g/g for d-tocopherol, respectively. Diet affected only the concentration of�-tocopherol, the marine group having a mean concentration of15.6 ± 0.8 �g/g, whereas the soya group had a mean concentrationof 12.8 ± 0.7 �g/g. Because the diet was the same, only differing inthe dietary lipids, the reason for this difference in �-tocopherol con-centration in the muscle is unknown.

MicrobiologyMicrobiologyMicrobiologyMicrobiologyMicrobiologyThe activity of microorganisms is a major factor limiting the shelf

life of fresh fish. Dietary treatment did not affect the bacterialcounts between the 2 groups, so the results are presented collec-tively (Figure 5). The aerobic total plate count decreased slightlyfrom approximately 103 to approximately 102 colony-forming unitsper gram (CFU/g) during the 1st 4 d of storage. The number of bac-teria increased during the subsequent 20 d, reaching a maximumvalue of 107 CFU/g, and then leveled off. The number of H2S-pro-ducing bacteria remained constant during the 1st 8 d of storage,increased after that until day 20 to a level of 105 CFU/g, and thenleveled off.

It is generally recognized that halibut deteriorates slower thanmost other fish species. The reason for this is unknown, but one mayspeculate whether halibut have high amounts of antimicrobial pep-tides (AMPs) in the skin mucus or more efficient types of AMPs, forexample, Hipposin, a potent AMP isolated from halibut (Birkemo andothers 2004), and this property might be the explanation for this re-duction in the bacterial count at the end of the experiment. For thisto be the case, it is of crucial importance that the fish is stored withthe skin intact. But the reduction may also be coincidental becausethe counts were executed from different individuals at each samplingpoint. Seawater and marine sediments are shown to harbor highnumbers of microorganisms. Unpolluted seawater is reported toshow bacterial numbers between 104 and 109 cells/mL (Austin 1988),whereas marine sediments often have bacterial numbers reaching1010 cells/mL (Hansen and others 1992). Seawater-dwelling fish willtherefore have microorganisms on all external surfaces and in theirgastrointestinal tract (Cahill 1990). Both autolytic and microbial ac-tivity reduces the quality of seafood during post mortem storage.Microbial activity plays the most important role in such spoilage to-ward the end of the shelf life (Huss 1995). The rate of microbial spoil-age of fish depends on the bacterial flora present and on the storageconditions, such as temperature and the availability of oxygen orother gasses as found in modified atmosphere packaging. Somebacterial groups are particularly associated with spoilage of seafood.The spoilage flora of fish caught in cold marine waters and stored onice under aerobic conditions is usually dominated by Shewanellaputrefaciens, Photobacterium sp., Pseudomonas sp., and representa-tives of the family Vibrionaceae (Huss 1995). Such organisms mayproduce ammonia, biogenic amines such as cadaverine, putrescine,and histamine, or mercaptanes, trimethylamine, skatol, indole, andH2S (Huss 1995). These products are all associated with the “off-odor” of spoiled fish. The ability to produce H2S seems to be an im-portant property among the most active seafood spoiling bacteriafrom cold waters (Gram and others 1987). Organisms that can pro-duce H2S can reduce trimethylamine oxide, which is found in themuscle of marine fish in concentrations ranging from 1% to 5% on adry weight basis (Huss 1995). These bacteria are often called the spe-cific spoilage organisms (SSO) for cold water seafoods (Gram 1992). Thenumber of organisms belonging to the SSO may be quantified usingiron agar (Jensen and Schulz 1980; Gram 1992). For fish stored on ice,the number of SSOs should reach levels of 108 to 109 CFU/g to causespoilage (Huss 1995). In the present study, the number of SSOsreached 105 at the end of the storage period, and we concluded thatthe halibut were not spoiled due to the microbial load.

Conclusions

Farmed Atlantic halibut is well-suited for chilled storage on ice.The texture, liquid-holding capacity, color, and gross chemical

composition for fish stored on ice for up to 26 d changed onlyslightly. The concentration of free amino acids and the amount ofbacteria increased during storage, while TBARS values remained

Figure 4—Changes in color: luminosity (L* value) (a), blue-ness/redness (a* value) (b), and greenness/yellowness (b*value) (c) of farmed Atlantic halibut stored on ice for 26 d(CIE 1976 L*a*b* standard)

Figure 5—Bacterial growth plate count, shown as aerobictotal plate count (TVC) and H2S producing bacteria, onfarmed Atlantic halibut stored on ice for 26 d. CFU = colonyforming units.

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Quality of halibut during ice storage . . .

low. Sensory profiling showed that the quality of the flesh was ac-ceptable for at least 22 d of storage, and that Atlantic halibut can beconsumed after storage on ice for up to 26 d with regard to the mi-crobial load. Further tests prolonging the storage time is necessaryto establish the ultimate period halibut can be stored on ice. TheQIM scheme that we developed for farmed Atlantic halibut gavegood results that correlated closely to storage time, and this meth-od can therefore be used as a freshness indicator.

The dietary lipids fed had no major effect on either of the mea-sured quality traits of halibut during ice storage.

AcknowledgmentsChristelle Guillerm-Regost was funded by a post-doctoral grant(Bourse Lavoisier Ministère des Affaires Etrangères, France). Theauthors would like to express their gratitude to Kjersti Borlaug, MayBritt Iversen, Tone Galluzzi and Elise Midthun for their excellenttechnical assistance.

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