Food Chemistry 134 (2012) 1399–1408

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Food Chemistry

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Effects of pre-slaughter stress on proteolytic enzyme activities and muscle qualityof farmed Atlantic cod (Gadus morhua)

Lisbeth Hultmann a, Tran Minh Phu a,b, Torbjørn Tobiassen c, Øyvind Aas-Hansen c, Turid Rustad a,⇑a Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norwayb Department of Aquatic Nutrition and Products Processing, Can Tho University, Can Tho, Viet Namc The Norwegian Institute of Food, Fisheries and Aquaculture Research, Nofima, Tromsø, Norway

a r t i c l e i n f o

Article history:Received 17 October 2011Received in revised form 1 February 2012Accepted 7 March 2012Available online 16 March 2012

Keywords:Atlantic codStressIced storageQualityCathepsinCollagenaseSlaughterFish welfare

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.03.038

⇑ Corresponding author. Tel.: +47 73 59 33 20; fax:E-mail address: turid.rustad@biotech.ntnu.no (T. R

a b s t r a c t

Farmed Atlantic cod were subjected to a combination of stressors in a holding tank before being killed,pre rigor filleted and stored in ice. At slaughter, a higher level of stress was confirmed by blood physiologyanalyses. This was further associated with significantly reduced muscle pH and somewhat elevated mus-cle collagenase-like activity in the stressed fish, whereas no differences in cathepsin-like activities werefound. After 5 days of iced storage, the stressed fish had significantly lower water holding capacity,reduced hardness and yellowish colour compared to the control group, and no differences in the otherparameters investigated. Independent of pre-slaughter stress, the activities of cathepsin B- and B/L-likeenzymes increased and activities of cathepsin D/E- and collagenase-like enzymes decreased with storage.

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1. Introduction

Atlantic cod (Gadus morhua) is becoming increasingly importantfor fish farming in Norway, and the production has increased from1200 metric tons in 2002 to 21,200 metric tons in 2010 (StatisticsNorway, 2011). The global financial crisis in combination withstrong competition from other farmed white fish species (likePangasius) has put cod farming in a temporary more difficult situ-ation. In order to maintain a high fish quality, it is necessary to in-crease the understanding of pre and post mortem biochemicalprocesses in fish. There are many factors that influence the qualityof fish, both during cultivation, handling and slaughter, and duringprocessing and storage (Haard, 1992; Hultmann, Rørå, Steinsland,Skåra, & Rustad, 2004; Sigholt, Erikson, Rustad, Johansen,Nordtvedt, & Seland, 1997). In addition, handling procedures preslaughter are important in terms of fish welfare (Brown, Watson,Bourhill, & Wall, 2010; Damsgård, 2008; Digre, Erikson, Misimi,Lambooij, & van de Vis, 2010).

When fish is subjected to stress, a diversity of physiological andbiochemical responses may occur (Barton, 2002). Crowding stresscaused an increase in plasma cortisol, lactate and H+ concentra-tions in rainbow trout and Atlantic salmon (Thomas, Pankhurst,

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+47 73 59 12 83.ustad).

& Bremner, 1999), increased plasma cortisol in rainbow trout(Lefèvre, Bugeon, Aupérin, & Aubin, 2008), and increased plasmacortisol and glucose in European sea bass (Lupatsch, Santos,Schrama, & Verreth, 2010). The acute stress response in Atlanticcod (Olsen, Sundell et al., 2008) has been compared with salmon(Olsen et al., 2002). The magnitude of the response (includingblood physiology, tissue damaging indicators and oxidative stress)was less than in salmonids. However, the cod returned moreslowly to basal levels of plasma cortisol, lactate and glucose, sug-gesting that cod were under the effect of the negative results ofacute stress for a longer time compared to salmon. Fish in goodnutritional status seems to be more resistant towards stress thanfood deprived fish (Lupatsch et al., 2010; Olsen, Sundell et al.,2008; Olsen et al., 2002).

Handling stress and/or exercise before slaughter of fish has beenfound to result in a more rapid decline in muscle pH, accompaniedby a more rapid onset of rigor mortis (Digre et al., 2010; Erikson, Di-gre, & Misimi, 2011; Kristoffersen, Tobiassen, Steinsund, & Olsen,2006; Morzel, Chambon, Lefevre, Paboeuf, & Laville, 2006; Sigholtet al., 1997; Thomas et al., 1999). This may affect the muscle pro-teins and thereby quality characteristics like water holding capac-ity. A combination of handling stress and exercise may increasethe degree of protein denaturation and thereby increase the accessof proteolytic enzymes to protein substrates, leading to more rapidmuscle softening, which is not beneficial in fish muscle. Handling

Protease activity sample on the day of slaughter, ~ 2 g

Protease activity sample after 5 days of storage, ~ 50 g

Protein solubility sample after 5 days of storage, ~ 4 g

Fig. 1. Sampling positions.

1400 L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408

stress in farmed Atlantic salmon resulted in softer fillets, accompa-nied by a lower texture score in the sensory test (Sigholt et al.,1997). Pre-slaughter crowding stress, especially long-term stress,led to increased cathepsin B activity and cathepsin L gene expressionin Atlantic salmon muscle, and increased the percentage of myofi-bre-myocommata detachments during storage (Bahuaud, Mørkøreet al., 2010). Several studies have shown that there is a strong cor-relation between physiological stress in fish, pH drop and impor-tant quality parameters like gaping, water holding capacity andcolour for many fish species (Kristoffersen et al., 2006; Morzel,Sohier, & Van de Vis, 2003; Stien et al., 2005). Fillets from stressedAtlantic cod were softer during the rigor period, and less transpar-ent post rigor than the unstressed fillets (Stien et al., 2005).

In the present study, the main objectives were to determine ef-fects of a combination of pre-slaughter stressors on muscle qualityin Atlantic cod. More specifically, the immediate effects on musclepH and activities of selected proteolytic enzymes were investi-gated, followed by effects on a number of defined quality criteriaafter 5 days of iced storage.

2. Materials and methods

2.1. Fish and experimental protocol

Farmed Atlantic cod were reared in sea cages at Tromsø Aqua-culture Research Station (Tromsø, Norway) and slaughtered inOctober 2009. At the start of the experiment, cod were carefullygathered in one part of the sea cage by a sweep net and 22 controlfish were subsequently dip-netted, killed by a blow to the head andsampled for blood (within 2 min, as described below) before beingtransferred to a tank with sea water and bled for 30 min. Musclesamples for analyses of enzyme activities were taken by samplingwhite muscle from the dorsal part of the fish, just in front of thedorsal fin and put in three cryotubes (1 ml). Cryotubes were imme-diately frozen in liquid nitrogen and transferred to a container withdry ice. They were transferred to a �80 �C freezer and stored at thistemperature till analysed. The length and the weight of the fishwere recorded, as well as weight of gonads, liver and guttedweight, after which each fish was filleted pre rigor with individu-ally labelled fillets being stored in ice for five days at 4 �C (freshice added daily). Stressed fish (n = 22) were netted from the seacage and placed into a 500 l holding tank with a surplus of flow-through seawater and subjected to a combination of stressors.The stressors applied consisted of 1.5 h of confinement and crowd-ing stress (225 kg fish/m3) combined with repeated (30 s every15 min) disturbance consisting of chasing by a rod causing forcedswimming activity and knocking on the tank wall creating a sounddisturbance. The stressed fish were subsequently netted and killedand further processed as described for control fish (above). The dayof slaughter is considered as day 0. At the end of day 4 of storage,the left-side fillets from 10 cod from each of the two groups (con-trol and stress) were sent to Department of Biotechnology, NTNUin Trondheim by airplane for analysis of proteins and proteolyticactivities. The samples were kept in ice during transportationand until sample collection was carried out on day 5. Samples(4 g) for protein fractionation were cut from the dorsal part ofthe fish (Fig. 1), stored on ice in plastic bags and analysed the sameday. Samples for protease activity (50 g) were put in ziplock bags,frozen and stored at �40 �C until analysis. On the same day, theright-side fillets from the same 10 fish from each of the two groups(control and stress) were used for further biochemical analyses (asdescribed below). These fillets were selected based on gender, sothat there were five female and five male fish in each group. Ofthe remaining 12 right-side fillets in each group, six were usedfor sensory analyses (described below).

2.2. Biological characteristics

Fulton’s K-factor, gonadosomatic index (GSI) and hepatosomat-ic index (HSI) were calculated as given in the equations below:

Fulton0sK-factorðg=cm3Þ ¼ whole body weightðgÞðfork length ðcmÞÞ3

� 100

GSI ð%Þ ¼ ½Gonad weight ðgÞ=whole weight ðgÞ� � 100

HSI ð%Þ ¼ ½Liver weight ðgÞ=whole weight ðgÞ� � 100

2.3. Blood analyses

Blood was withdrawn from the caudal vasculature using a hep-arinized syringe and immediately analysed for whole blood lactateand glucose levels using hand-held meters (LactatePro™, ArkrayInc., Japan; c.f. Brown, Watson, Bourhill, & Wall (2008) and Free-Style Lite�, Abbott Laboratories, Illinois, USA, respectively). Forblood lactate, the detection limit of the meter was 0.8 mmol/l, withvalues below the limit indicating a rested, unstressed cod (Brownet al., 2008). As a conservative measure in the analyses and statis-tics, values below the detection limit (5 of 22 fish in the controlgroup only) were set to 0.8 mmol/l. The remaining blood wastransferred to a heparinized vacutainer and placed on ice andtransported to the laboratory and centrifuged (3000 rpm, 10 min,4 �C) on the same day, after which plasma was stored at �40 �C.The plasma cortisol concentrations were measured in diethyl-etherextracted plasma using a commercially available enzyme-linkedimmunosorbent assay (ELISA) kit for cortisol (Neogen�, Lexington,KY, USA). Plasma cortisol and blood glucose analyses were per-formed in duplicate whereas blood lactate analyses consisted ofsingle measurements. Immediately following slaughter, the bloodpH was measured in the pericardium by a WTW pH meter (pH330, Wissenschaftlich-Technische Werkstatten GmbH, Weilheim,Germany) equipped with a Hamilton double pore glass electrode(Hamilton Bonaduz AG, Bonaduz, Switzerland).

2.4. Muscle pH, water content and water holding capacity

The muscle pH at slaughter was measured through an incisionin the skin anterior to the dorsal fin in the loin part of the fish di-rectly after the measurement of blood pH, using the same equip-ment. For determination of muscle pH after iced storage, theright-side fillets were coarsely chopped (3 � 5 s) using a StephanBlender with cooling. The minced muscle was kept on ice untilanalysis. The pH was determined in duplicate in a 1:1 (w:v) mix-ture of minced muscle and 0.15 M KCl by a PHM 80 pH meter(Radiometer) equipped with a combined glass-electrode. The

L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408 1401

water content was determined by drying minced muscle (in dupli-cate) at 103 �C for 16–18 h. The water holding capacity (WHC) wasdetermined in each fish using the centrifugation method describedby Ofstad, Kidman, Myklebust, and Hermansson (1993). Mincedmuscle (15 g) was put in 42 mm diameter centrifugal tubes andcentrifuged at 5 �C for 15 min at 324g (using a Beckman GS-6R cen-trifuge (Beckman, PaloAlto, CA, USA)). WHC is given as fraction ofwater bound after centrifugation (% of total water). The analyseswere run in quadruplicate.

2.5. Solubility properties of proteins

Protein was extracted from white muscle by a modification ofthe methods of Anderson and Ravesi (1968) and Licciardello,Ravesi, Lundstrom, Wilhelm, Correia, and Allsup (1982) as de-scribed by Hultmann and Rustad (2002). The extraction was donein a cold room. Each fillet was extracted once. Water-soluble pro-teins were extracted from white muscle (Fig. 1) in sodium phos-phate buffer (0.05 M, pH 7.0). After centrifugation, the saltsoluble proteins were extracted from the sediment with phosphatebuffer with KCl (0.05 M, 0.6 M KCl, pH 7.0). The amount of proteinin the extracts was determined after centrifugation (10 min, 7900g,4 �C) with BioRad protein assay using bovine serum albumin as astandard (Bradford, 1976), and the analyses were run in triplicate.

2.6. Extracts for determination of proteolytic activities

The day 0 samples were extracted by Precellys 24 homogenizer(Bertin Technologies, France). White muscle (300 mg, frozen sam-ples, from protease sample, Fig. 1) was homogenized in 1.5 ml colddistilled water at 5000 rpm for 20 s in two cycles, separated by a10 s break (using 1.4 mm zirconium oxide beads, Bertin Technolo-gies, France). The homogenization was repeated once to ensurehomogeneity. After centrifugation (30 min, 16,000g, 4 �C), the ex-tract was decanted into microcentrifuge tubes (1.5 ml) and ali-quots were frozen and stored at �80 �C until analysis.

The extractions of proteolytic enzymes of day 5 samples (fromprotease samples, Fig. 1) were performed on ice using cold distilledwater (4 �C) and were done once per sample. White muscle (5 g,frozen samples) was homogenized at 24,000 rpm for 30 s in20 ml cold distilled water using an Ultra-Turrax t25 homogenizer.Homogenization was repeated once more with 30 s break time.After centrifugation (20 min, 14,600g, 4 �C), the supernatant wasmade up to 25 ml with cold distilled water. Aliquots were frozenand stored at �80 �C until analysis.

Before analysing the proteolytic activities, the extracts werethawed in cool tap water for 10 min, and centrifuged (10 min,7900g, 4 �C). The protein content in the extracts was determinedwith the BioRad protein assay using bovine serum albumin as astandard (Bradford, 1976). The analyses were run in triplicate.

2.7. General proteolytic activity

The general proteolytic activity was determined by a modifica-tion of the method by Barrett and Heath (1977). Buffer at pH 5.5and 1 h incubation at 30 �C were found to be appropriate condi-tions for determination of general proteolytic activity of farmedcod muscle. The incubation mixture consisted of 1.2 ml phosphatecitric acid buffer pH 5.5 (McIlvaine, 1921), 0.4 ml enzyme extract,and 0.4 ml of substrate (1% haemoglobin). The reaction was ar-rested by addition of 2.0 ml 5% (w/v) trichloroacetic acid (TCA),and the mixtures were shaken and left for 30 min to cool to roomtemperature. The zero samples (unincubated samples) were pre-pared as described above, except that TCA was added before theenzyme solution, and the zero samples were filtered after beingleft for 30 min at room temperature. After filtration (Blackband

filter, Schleicher and Schuell 589, Germany), the content of pep-tides and amino acids was measured according to Lowry, Roseb-rough, Farr, and Randall (1951). The proteolytic activity wasexpressed as mg TCA soluble peptides liberated per g wet weightper hour, given as the arithmetic mean of three individual mea-surements. The content of TCA soluble peptides in the zero sampleswas used as a measure of the endogenous amount of amino acidsand short peptides in the samples. The contribution from haemo-globin to the amount of TCA soluble protein was measured byreplacing protein extract with buffer in the assay, and was foundto be small. It was, however, subtracted from the results. In calcu-lation of results in units per gram wet weight for the samples at theday of slaughter, the water content of the samples were taken intoaccount.

2.8. Specific proteolytic activities

The activities of cathepsin B-, B/L- and collagenase-likeenzymes were measured against synthetic fluorogenic substr-ates. Samples were diluted with distilled water to a proteinconcentration of 2.25 mg/ml. The activities of cathepsin B- and B/L-like enzymes were measured against the synthetic fluorogenicsubstrates N-carbobenzoxy-arginine-arginine-7-amido-4-methyl-coumarin (Barrett & Kirschke, 1981) and N-carbobenzoxy-phenyl-alanyl-arginine-7-amido-4-methylcoumarin (Inubushi, Kakegawa,Kishino, & Katunuma, 1994), respectively (Sigma Chemical Co., St.Louis, MO, USA). The activity of collagenase-like enzymes wasmeasured against a synthetic fluorogenic substrate, N-succinyl-glycine-proline-leucine-glycine-proline-7-amido-4-methylcouma-rin (Bachem, Bubendorf, Switzerland) (Kinoshita, Toyohara,Shimizu, & Sakaguchi, 1992). Stock solutions of substrates(3 mM) were prepared in DMSO, and stored at �20 �C. On theday of use they were diluted to 0.09375 mM with distilled water.The specific proteolytic activities were determined at 4 �C (in acold room) as described by Hultmann et al. (2004) with some mod-ifications. Enzyme extract (100 ll) and buffer (150 mM Bis–Tris,30 mM EDTA, 6 mM dithiothreitol, pH 6.0 or pH 7.0 for cathepsinB- and B/L-like enzymes and 150 mM Bis-Tris, 7.5 mM CaCl2, pH6.0 or pH 7.0 for collagenase-like enzymes) (100 ll) were mixedtogether and left for 10 min before adding the substrate (100 ll).The reaction mixture was incubated for 10 min before 3 ml of 1%SDS in 50 mM Bis-Tris (pH 7.0) was added to stop the reaction. Ablank was prepared for each type of activity by adding distilledwater instead of enzyme extract to the reaction mixture. Amountof the fluorogenic product 7-amido-4-methylcoumarin (AMC)liberated was measured fluorimetrically at 460 nm after excitationat 360 nm (10 nm slits). (Perkin Elmer 3000 Fluorescence Spec-trometer, Perkin Elmer Inc., Buckinghamshire, UK). A standardcurve of fluorogenic product was established. Activities are givenas nmol AMC liberated/g wet weight/min. The analyses were runin triplicate.

The activities of cathepsin D/E-like enzymes were measuredagainst a synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)-acetyl-glycyl-lysyl-prolyl-isoleucyl-leucyl-phenylalanyl-phenyl-alanyl-arginyl-leucyl-lysyl-N-(2,4-dinitrophenyl)-arginine-NH2) ob-tained from Calbiochem (Merck, Darmstadt, Germany) andSigma (Sigma Chemical Co., St. Louis, MO, USA) (Yasuda et al.,1999). Samples were diluted with distilled water to a protein con-centration of 5 mg/ml if the protein level of the sample exceededthis level. The concentration of this substrate (dissolved in di-methyl sulfoxide and Bis–Tris buffer pH 6.0) was 200 lM in50 mM Bis–Tris pH 6.0. The activities were determined at 4 �C,pH 6. The incubation time and stop solution types were testedand the protocol was optimized at 4 �C, 10 min incubation, with5% TCA used as stop solution. The amount of stop solution was ad-justed compared to Yasuda et al. (1999). Enzyme extract (10 ll)

1402 L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408

and buffer (10 ll) were mixed and left for 10 min before adding thesubstrate (10 ll). The reaction mixture was incubated for 10 minbefore the stop solution (2.5 ml) was added. A blank was preparedby adding distilled water instead of enzyme extract to the reactionmixture. The amount of the fluorogenic product analogue (7-meth-oxycoumarin-4-yl) acetyl (MCA) was measured fluorimetricallywith excitation at 328 nm and emission at 393 nm (10 nm slits).A standard curve of fluorogenic product analogue was established.Activity is given as nmol MCA liberated/g wet weight/min. Theanalyses were run in triplicate.

2.9. Sensory analyses

The fillet index includes assessment of appearance, texture andodour of the products. The fillets were given demerit scores of 0–2,or 0–3 points for the different attributes (based on Esaiassen, Dahl,Eilertsen, Gundersen, & Sivertsvik, 2008). The odour was evaluatedas sea fresh, neutral, fishy or ammonia/sour, giving 0, 1, 2 or 3points, respectively. The other attributes evaluated were gaping(0: none – 3: severe), colour (0: homogeneous white – 2: yellow,translucent), surface (0: dry and shiny – 2: dispersed), and texture(0: natural – 3: very soft). The fillet index is the sum of scores for allthe attributes.

A descriptive sensory analysis was performed at Nofima Marin,Tromsø, where 19 sensory properties (smell, appearance, colour,taste and texture) were analysed. A trained sensory panel of sevenpersons analysed the samples and classified each property on ascale from 1 to 9 where 1 is no intensity and 9 is clear intensity.The sensory panel was calibrated in a test experiment with twocalibration samples. In the calibration, one control and onestressed sample were presented. Afterwards, five fillets from eachgroup were used for sensory analysis. Each evaluator got one sam-ple from each of the fillets. The fillets were kept in ice until the sen-sory analysis. On the day of analysis, the fillets, without skin andbones, were cut in 2–3 cm cubes and wrapped in aluminium foil,and heated with steam from boiling water in a kettle and servedto the evaluators in randomized order at the time of testing.

2.10. Statistical analysis of results

Results are given as average ± standard error of the mean.The effect of handling stress on a given variable was statistically

analysed applying one-way t-tests assuming unequal variances.However, paired t-tests were used when comparing proteolyticactivities at different assay pH values within treatment groupsand sampling days, as well as for comparing proteolytic activitiesat the day of slaughter and sampling day.

The integer results of the fillet index method were analysed bythe Mann–Whitney U test (Mann–Whitney U Test, 2011). Due tovalues below the detection limit for blood lactate, these resultswere analysed by the Mann–Whitney U test with the normalapproximation.

The results from the sensory analysis were analysed by analysisof variance (ANOVA) for significant differences between groups foreach of the sensory properties.

The level of significance is 95% unless otherwise stated.

3. Results and discussion

3.1. Biological characteristics of the fish

The fish in the two groups were of equal size (length, weightand condition factor) and condition (hepatosomatic and gonadoso-matic indices) (Table 1). Application of short-term stress is not ex-pected to change these characteristics. The results are in

accordance with previous studies (Kristoffersen et al., 2006;Mørkøre, 2006). By chance, the body length of the control fish inour selection for further biochemical analyses tended to be lowerthan the corresponding stressed fish. However, neither the guttedweight nor the condition factor was influenced, maintaining thestatement of equal characteristics in the two groups.

3.2. Assessing stress levels

Circulatory levels of cortisol, glucose, lactate and pH are often usedmeasures of the primary (cortisol) and secondary stress response infish (Barton, 2002; Barton & Iwama, 1991; Bonga, 1997). When com-paring control fish and stressed fish immediately following slaughterin the present experiment, stressed fish displayed significantly in-creased circulatory levels of cortisol, glucose and lactate and signifi-cantly reduced blood pH, thus clearly demonstrating an elevatedlevel of stress in this group (Table 1). Similar results were obtainedin the cod selected for further biochemical analyses. No linear rela-tionships were found between cortisol, glucose or lactate level andslaughter order within treatment (results not shown). In agreementwith previous investigators of pre-slaughter stress in the Atlanticcod (e.g. Digre, 2011; Erikson et al., 2011; Misimi, Erikson, Digre,Skavhaug, & Mathiassen, 2008; Olsen, Sundell et al., 2008; Olsen,Sørensen, Larsen, Elvevoll, & Nilsen, 2008), we found that the magni-tude of the stress response parameters were low compared to otherfish species (Barton & Iwama, 1991). This observation in the presentstudy may partly be due to the fact that our cod were fed prior toslaughter, as it has been shown that feed-deprived cod display in-creased and prolonged responsiveness to stress as compared to fedcod (Olsen, Sundell et al., 2008). In the control group, the circulatorylevels of particularly cortisol and lactate seemed somewhat elevatedcompared to reported basal levels for this species (Brown et al., 2010;Hemre, Lambertsen, & Lie, 1991; Morgan, Wilson, & Crim, 1999;Olsen, Sundell et al., 2008). Accordingly, it appears that some degreeof stress was experienced also by the fish in the control group dueto handling and confinement/crowding (and possibly combined withincreased swimming activity) in the sweep net just prior to slaughter,and this may have reduced potential differences between the controland stressed group in the present experiment. Another relevant as-pect is that effects of pre-slaughter treatment may also be due to fac-tors such as environmental hypoxia and/or altered swimming activitywhich are not necessarily specific to the stress response per se (e.g.Bonga, 1997; Erikson et al., 2011; Herbert & Steffensen, 2005; Nelson,Tang, & Boutilier, 1994). This combination of factors may increase therelevance of their studies to the situation in commercial slaughtering,and a combination of stressors was therefore chosen also in the pres-ent experiment. In the present study however, a rich supply of freshseawater may have minimised stress and anaerobic metabolismdue to environmental hypoxia.

3.3. Muscle pH

Muscle pH in the stress group was significantly lower than inthe control group at slaughter (p < 0.05) (Table 1). This is in agree-ment with previous studies (Kristoffersen et al., 2006; Poli, Parisi,Scappini, & Zampacavallo, 2005; Stien et al., 2005). In addition tothe effect of stress per se, the pre-slaughter handling stress in thepresent study included periodical chasing of the fish in the holdingtank, presumably leading to increased muscular activity, reducedmuscle energy levels and thus a higher production of lactic acidand a reduced muscle pH. In addition, H+ formed by ATP degrada-tion may also have contributed to the reduction in muscle pH(Robergs, Ghiasvand, & Parker, 2004; Strasbourg, Xiong, & Chiang,2008). In a previous study in the Atlantic cod, however, Olsenand coworkers found that pre-slaughter crowding stress increasedthe amount of residual blood in the fillet but did not influence

Table 1Physical characteristics and physiological stress parameters directly after slaughter, muscle pH and properties related to muscle water and proteins after five days of iced storage(in all fish, and in fish selected for further biochemical analyses).

All fish* Fish used for biochemical analyses**

Control Stressed p Values Control Stressed p Values

At slaughter Length (cm) 74.5 ± 1.0 74.5 ± 1.0 0.950Y 72.4 ± 1.2 75.4 ± 1.2 0.093Y

Gutted weight (kg) 4.15 ± 0.16 4.06 ± 0.18 0.719Y 3.8 ± 0.2 4.03 ± 0.18 0.518Y

Fulton’s K-factor (g/cm3) 1.27 ± 0.03 1.23 ± 0.04 0.493Y 1.28 ± 0.05 1.19 ± 0.05 0.248Y

Hepatosomatic index (%) 12.9 ± 0.4 12.7 ± 0.4 0.695Y 12.5 ± 0.7 13.0 ± 0.6 0.630Y

Gonadosomatic index (%) 4.7 ± 0.5 4.5 ± 0.5 0.823Y 4.7 ± 0.8 4.2 ± 0.3 0.605Y

Blood pH 7.69 ± 0.02 7.52 ± 0.02 0.000X 7.67 ± 0.03 7.50 ± 0.03 0.001X

Cortisol (ng/ml) 19 ± 3a 44 ± 6c 0.001X 22 ± 6d 50 ± 15e 0.070X

Glucose (mmol/l) 4.4 ± 0.2b 8.3 ± 0.6 0.000X 4.3 ± 0.4d 8.4 ± 0.6 0.000X

Lactate (mmol/l) 1.4 4.4 0.000X 1.5 6.1 0.000X

Muscle pH 7.68 ± 0.04 7.29 ± 0.05 0.000X 7.66 ± 0.03 7.31 ± 0.08 0.001X

After iced storage Muscle pH – – – 6.19 ± 0.02 6.21 ± 0.02 0.584Y

Water holding capacity (%) – – – 92.3 ± 0.6 90.3 ± 0.8 0.048Y

Water content (%) – – – 78.5 ± 0.2 78.82 ± 0.11 0.225Y

Water-soluble proteins (mg/g) – – – 41.5 ± 1.3 41.7 ± 0.8 0.903Y

Salt soluble proteins (mg/g) – – – 38 ± 3 41 ± 4 0.572Y

Results are given as mean ± standard error, except for lactate where medians are given (all samples: U = 476.5, z = 5.50; selection: U = 98.0, z = 3.63). Water holding capacity isgiven as amount of water retained after centrifugation in percent of the original total water in the sample. Water content is given as % of wet weight. Soluble proteins aregiven in mg protein per g wet weight.* n = 22 unless otherwise stated.** n = 10 unless otherwise stated.

a n = 21.b n = 20.c n = 19.d n = 9.e n = 7.X p Values are one-sided.Y p Values are two-sided.

L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408 1403

muscle pH (Olsen, Sørensen et al., 2008). This lack of effect on mus-cle pH may be explained by a different level of stress in their study,possibly including a difference in muscular activity and thus lac-tate levels although this was not measured.

The muscle pH decreased from day 0 to day 5 (Table 1). The ulti-mate pH was in the same range as found in previous studies onfarmed net-pen fed cod (Ofstad et al., 1996; Rustad, 1992) andwas not affected by pre-slaughter handling stress. This is in accor-dance with other studies in this species (Kristoffersen et al., 2006;Olsen, Sørensen et al., 2008; Stien et al., 2005).

3.4. Water holding capacity, water content and protein solubility

Water holding capacity (WHC) in the control group was signif-icantly higher than in the stress group (p < 0.05) (Table 1). An in-creased drip loss due to handling stress was reported from icedstorage of intact pieces of cod muscle sampled at the day of slaugh-ter (Bjørnevik & Solbakken, 2010). This difference was not ob-served for samples excised after 3 days of iced storage. However,they did not subject the muscle to any centrifugation in the driploss assay. Erikson and coworkers found no effect of stress onwater holding capacity after 7 days of iced storage (Erikson et al.,2011). This strongly indicates that the stress effect will vary withstorage period. The rate of pH decline in the muscle post mortemis important, as a rapid pH decline may cause soft texture and poorwater holding capacity of the meat (Ang & Haard, 1985). As alreadydiscussed, a significant decrease in muscle pH due to handlingstress was observed (Table 1). It is therefore probable that the rateof pH decline was increased in the stressed group, influencing boththe muscle proteins and the endogenous proteolytic enzymes,thereby affecting the water holding capacity.

Water content and protein extractability properties did not dif-fer significantly due to handling stress (Table 1). This is in agree-ment with other studies reporting that handling stress did not

significantly affect amounts of water- and salt soluble proteins inAtlantic cod (Erikson et al., 2011) and salmon (Sigholt et al., 1997).

3.5. Protease activities

Two different homogenizing procedures were used for the sam-ples from the day of slaughter and for the samples after 5 days ofstorage. The effect of the homogenization was analysed for severalsamples that were homogenized using both procedures. No effectof the homogenization procedure on the enzyme activity wasfound (results not shown).

3.5.1. General protease activity and peptide contentHandling stress did not have significant effect on general prote-

ase activity (GPA) after 5 days of iced storage (Fig. 2A). The resultsfrom previous studies (Hultmann & Rustad, 2004, 2007) showedthat there was no significant difference in general proteolytic activ-ity (against haemoglobin) due to storage time.

When muscle proteins are degraded by proteolytic enzymes, anincrease in fragments of different lengths (from free amino acids tolarge peptides) is expected. The amount of TCA soluble material(smaller peptides and free amino acids) in the muscle was deter-mined in order to investigate the total effect of any changes in pro-teolytic activities due to handling stress. Peptide contents wereonly determined after 5 days of iced storage, not directly afterslaughter. The amount of TCA soluble peptides in the control groupwas somewhat higher than in the stress group after storage in icefor 5 days, although not significant (two-sided p = 0.090) (Fig. 2B).This indicates that the total activity may have been changed due tohandling stress. However, reporting the amount of ‘‘peptides’’, noattempts have been made to investigate the composition of thismaterial. It is known that both peptides and free amino acids willcontribute to the colour development; their impact varies greatly(Peterson, 1979). Therefore, the composition of the peptide fractionin the control and stressed groups may be different. Immediately

0.0

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1.2

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TC

A s

olub

le p

eptid

es/g

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wei

ght/h

aa

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olub

le p

eptid

es/g

wet

wei

ght

a

a

A

B

Fig. 2. (A) General protease activity in muscle of the control and stress groups after5 days of iced storage measured at 30 �C. (B) Amount of TCA soluble peptides in themuscle of control and stressed fish after 5 days of iced storage. Bars indicatestandard error of the mean (n = 10). Different letters denote significant differences(p < 0.05) due to handling (a and b).

1404 L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408

after slaughter, the muscle pH was reduced due to handling stress(Table 1). It is therefore reasonable to assume that the activities ofproteolytic enzymes differed between the stressed and control fish.However, as there are both endopeptidases and exopeptidases,measurement of increase in TCA soluble material may not beappropriate for describing changes in proteolytic activities.

3.5.2. Activity of cathepsin B-like and cathepsin B/L-like enzymesHandling stress did not significantly affect the activities of

cathepsin B or cathepsin B/L analysed at pH 6 and 7 at any sam-pling time, although the activities were reduced in the stressedgroup (Fig. 3A and B).

In this study, the release of AMC from N-carbobenzoxy-arginine-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-MCA)was always more extensive than from N-carbobenzoxy-phenylala-nyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-MCA). Theenzymes have different affinities towards different substrates, andthis would also be influenced by assay pH. We therefore cannot statethat the activity of cathepsin B is much higher than the activity ofcathepsin B/L, only that more AMC was released from the cathepsinB substrate. No attempts were made to elucidate the contribution ofcathepsin B to the degradation of Z-Phe-Arg-MCA. As discussed byGodiksen and Nielsen (2007), the reported results on degradationof these substrates by different cathepsins are not unambiguous.In herring, the activities of cathepsin B and L could be differentiatedby acidification of a crude extract. However, we have chosen todetermine the AMC liberating activity from several substrates atconditions relevant for the fish to characterize the available activi-ties in the muscle. On the contrary, a more extended release ofAMC from the cat B/L substrate than the cat B substrate has been re-

ported in extracts from salmon muscle (Bahuaud, Gaarder, Veiseth-Kent, & Thomassen, 2010; Bahuaud, Mørkøre et al., 2010; Bahuaudet al., 2009). The activities of salmon cathepsin B and B/L immedi-ately after slaughter increased due to handling stress, although thedifferences were not significant. The relative expression of cathepsinL gene was significantly increased in salmon subjected to long-termstress. Furthermore, cathepsin B and B/L activities and cathepsin Lexpression were positively correlated to detachments in the musclestructure, and negatively correlated to muscle pH and firmness(early post mortem) (Bahuaud, Mørkøre et al., 2010). A significantnegative correlation between cathepsin L activity at day 2 and sal-mon fillet firmness (at day 2 and 5) has also been reported (Bahuaud,Gaarder et al., 2010). However, in a study with different lipid sourcesin the salmon feed, no relationships were found between the totalactivities of cathepsin B and L-like enzymes or the activities leakingfrom lysosomes immediately after slaughter and the later observedmuscle degradation (myofibre–myofibre detachments), suggestingthat other proteases may have been involved (Bahuaud et al., 2009).

During storage for 5 days, activity of cat B analysed at pH 7 in-creased significantly (p < 0.05) while activity of cat B analysed atpH 6 was stable. According to Hultmann and Rustad (2007), activ-ity of cathepsin B-like enzymes in farmed cod fillets was equalafter 4 and 10 days of iced storage. This was also found for cathep-sin B-like enzymes in ice stored Atlantic salmon (Hultmann &Rustad, 2004). However, activity of cat B/L increased after 5 daysof storage. Bahuaud and coworkers observed stable total cathepsinB and L activity during iced storage for 24 h of previously super-chilled Atlantic salmon (Bahuaud et al., 2008). A stable cathepsinB and B/L activity was also found by Duun and Rustad (2008) dur-ing superchilled (�1.5 and �3.6 �C) storage of Atlantic salmon.

Activity of cat B analysed at pH 6 was significantly higher thanat pH 7 in all groups, in accordance with previous studies (Hult-mann & Rustad, 2007). Likewise, the activity of cat B/L analysedat pH 6 was significantly higher than at pH 7 in all groups.

3.5.3. Activity of collagenase-like enzymesHandling stress did not significantly affect the activity of colla-

genase-like enzymes, although the activity increased somewhatdue to handling stress immediately after slaughter (p = 0.060 forpH 6 and 0.092 for pH 7, two-sided tests). No significant differ-ences were seen after 5 days of iced storage (even though the activ-ity in the stress group was still higher than in the control group)(Fig. 3C).

During storage, activity of collagenase-like enzymes decreasedsignificantly. This is in accordance with earlier results on farmedcod (Hultmann & Rustad, 2007). The decrease in collagenase activ-ity during storage is simultaneous with the decline in muscle pH.The activity of collagenase analysed at pH 6 was significantly lowerthan at pH 7 in all groups. Some previous studies suggest that thesoftening of cod muscle observed during iced storage is causedmore by collagenase activity than by proteolysis of myofibrils(Hernandez-Herrero, Duflos, Malle, & Bouquelet, 2003; Montero& Mackie, 1992; Shigemura, Ando, Harada, & Tsukamasa, 2004).The pH may also affect the properties of the collagen molecules di-rectly. Reducing pH may lead to weakening of the connective tissue(Love, Lavety, & Garcia, 1972).

Montero and Mackie (1992) indicated that degradation of colla-gen is initiated during development of rigor mortis. In cod, handlingstress led to a more rapid initial contraction followed by a slowerrate while the unstressed fish had a virtually linear rate of contrac-tion (Stien et al., 2005). This may have induced more rapid disrup-tion of the structures. The progress of rigor mortis was not assessedin our fillets. Collagenase-like enzymes could play a role in thedegradation of collagen in cod muscle leading to texture softeningduring storage. However, as the pH will influence both the

0

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12

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aAx

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et w

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aBaB

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B D

Fig. 3. Activity of specific proteolytic enzymes against appropriate synthetic coumarin based substrates (given in Section 2.8) at 4 �C. Bars indicate standard error of the mean(n = 10). (A) Cathepsin B-like enzymes. (B) Cathepsin B/L-like enzymes. (C) Collagenase-like enzymes. (D) Cathepsin D/E-like enzymes. Different letters denote significantdifferences (p < 0.05) due to handling (within sampling day and assay pH) (a and b), storage (within treatment and assay pH) (A and B), and assay pH (within handling andsampling day) (x and y).

L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408 1405

conformation of the collagen molecules and the collagenase activ-ity this is difficult to validate.

3.5.4. Activity of cathepsin D/E-like enzymesHandling stress did not significantly affect the activity of cathep-

sin D/E-like enzymes, although the activity was decreased in thestressed group. During storage, activity of cat D/E decreased signif-icantly (p < 0.05) (Fig. 3D). Bjørnevik and Solbakken (2010) studiedcathepsin D activity at pH 2.8 (37 �C), using haemoglobin as sub-strate. No significant effects of handling stress were seen, and theactivity increased significantly during the early storage period.However, the activity was highest in the stressed group at day 0and highest in the control group after 3 and 8 days of iced storage.

Recent studies showed that cathepsin D had a local pH opti-mum in trout muscle at pH 6.0 (Godiksen, Morzel, Hyldig, & Jessen,2009). Their results indicated that cathepsin D is active post mor-tem outside the lysosomes. Ashie and Simpson (1997) showed thatcathepsin D often exists in several isomer forms with a broad opti-mal pH range, found in Tilapia mossambica. Wang, Martinez, andOlsen (2009) concluded that the degradation of cod myosin heavychain (MHC) occurred even at very low storage temperatures andthat the process was pH dependent. Moreover, cathepsin D can de-grade cod isolated myofibrils. Stored muscle with low degradationof MHC was observed at pH 6.3 (as well as at pH 7.0 and 8.0) com-pared to pH 5.5, but there were differences in the proteolytic frag-ments produced depending on the pH (Wang et al., 2009).

Proteolytic activities have been determined in muscle extractswithout subcellular fractionation. By this, it is not possible to tellwhether handling stress caused leakage of cathepsins from lyso-somes. Bahuaud and coworkers argued that cathepsin activitiesmay not be related to quality differences due to treatment, as a

measure of enzymes leaking out of the lysosomes is required (Bah-uaud et al., 2008). However, by combining the results from the pro-teolytic assays with information about muscle pH during storage, itis possible to discuss changes in proteolytic profiles due to han-dling stress.

The effect of assay pH on proteolytic activity obviously dependson the type of enzyme investigated. Cathepsin B- and L-like en-zymes seem to be more active at pH 6 than 7, most evident earlypost mortem, while the opposite result is shown for collagenase-like activity. On the day of slaughter, muscle pH was above 7 anddeclined to 6.1–6.3 during storage. Therefore, the activities of col-lagenase-like enzymes may be important in the early post mortemperiod, while the activities of cathepsin B-, D-, E-, and L-likeenzymes become more important as the pH declines.

3.6. Sensory analyses

Five fish from each group were subjected to sensory analysis asraw fillets (reported as fillet index) and as cooked pieces after5 days of iced storage. The fillet index was not significantly influ-enced by handling stress (mean 1.8 in the control and 1.0 in thestress group, Mann–Whitney U = 17.5, n1 = n2 = 5, p = 0.310, two-tailed). The results show that the cod were of good quality.

The results from sensory analysis of cooked pieces showed thatthe unstressed and stressed cod were quite similar (Fig. 4). Theonly significant differences found due to handling stress were inyellow colour and hardness 1. Generally, the differences withingroups were larger than between groups, and the reported differ-ences due to handling stress are probably of no practical impor-tance. This is in agreement with Sveinsdóttir et al., (2010), wherethe only differences due to handling stress were seen in texture

0

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9Sea smell

Sourish smell

Foddery smell

Old/stale smell

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Yellow colour *

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Bitter taste

Hardness 2

Juiciness

Fibre formation

ChewinessControl

Stressed

Hardness 1

Cohesiveness

Gloss

Sea tasteSourish taste

Foddery taste

Sweet taste

Old/stale taste

Fig. 4. Radar diagram of the average values of sensory properties for the two groups of farmed cod. ⁄ Indicates significant difference (p < 0.05) between the control and thestress groups (n = 5).

1406 L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408

attributes. They also included investigation of consumers’ liking,and those results were highly dependent on test setting.

The score of yellow colour in control fish fillets was significantlyhigher than in the stressed fish fillets, but they are still low inintensity. However, no significant differences in white colour orgloss were observed although the stressed fish were slightly morewhite and glossy than the control fish. This can be explained by thelower muscle pH in the stressed fish compared to the control fishdirectly after slaughter, which may have influenced the muscleproteins. Also, the pH of the stressed group probably declined morerapidly to the stable level during storage. It has been reported thatthe change in pH caused by stress induced more rapid changes inprotein conformation leading to lighter and more opaque raw fishflesh appearance (Stien et al., 2005). However, we did not find anydifferences in protein solubility to support this (Table 1). A signif-icant effect of pH on flesh colour is also seen in halibut, wherecathepsins may also contribute to the colour (Hagen, 2011).

Moreover, the score of hardness 1 in the control fish fillets wassignificantly higher than in the stressed fish fillets. As discussedearlier, the handling stress also reduced the water holding capac-ity. This may be explained by changes in protein properties impor-tant both for water holding capacity and textural properties in themuscle. This partly agreed with Stien et al. (2005) where the filletswere significantly softer in the stressed cod fillets compared to un-stressed fillets during the rigor period, whereas no significant dif-ference was found post rigor. An increased shear force due tohandling stress was reported in raw muscle of farmed cod,although the difference was not significant (Bjørnevik & Solbakken,2010). Similarly, cod chased to exhaustion in reduced water leveltended to have softer texture than dip-netted cod (Digre et al.,2010).

4. Conclusion

The combination of pre-slaughter stressors applied induced sig-nificant changes in blood physiology and muscle pH at the time ofslaughter, demonstrating that the treatment was a stressful event

for the fish. Despite this, except from a significant reduction inwater holding capacity, reduction of yellow colour and value ofhardness 1 after iced storage, no severe reduction of quality wasseen in the fillets from stressed fish compared to control fish. Thisis also in general agreement with other studies in this species. Nev-ertheless, fish welfare should be taken into account when slaugh-tering farmed cod. By analysing the activity of several specificproteolytic enzymes, however, we conclude that the actual prote-olytic profiles were influenced both by handling stress and storagetime. The activity of collagenase-like enzymes was found to be sig-nificantly higher at pH 7 than at pH 6, and decreased during icedstorage, whereas the opposite was found for cathepsin B- and B/L-like enzymes. Further, the muscle pH was significantly reducedduring storage. Altogether, this leads to the conclusion that the col-lagenase-like enzymes were most important in the early post mor-tem period, whereas the contribution of cathepsins increased asthe muscle pH was reduced. This may be important if the fish issubjected to further processing.

Acknowledgements

We want to thank Ronny Jakobsen (Nofima), and Diana Herbon(Lise-Meitner Schule, Germany) for skilful technical assistance, andSvein Kristian Stormo for performing the cortisol analysis. Thefinancing of the work by the Research Council of Norway is grate-fully appreciated (Grant No. 178979/S40).

References

Anderson, M. L., & Ravesi, E. M. (1968). Relation between protein extractability andfree fatty acid production in cod muscle aged in ice. Journal of the FisheriesResearch Board of Canada, 25, 2059–2069.

Ang, J. F., & Haard, N. F. (1985). Chemical composition and postmortem changes insoft textured muscle from intensely feeding Atlantic cod (Gadius morhua, L).Journal of Food Biochemistry, 9, 49–64.

Ashie, I. N. A., & Simpson, B. K. (1997). Proteolysis in food myosystems – A review.Journal of Food Biochemistry, 21, 91–123.

Bahuaud, D., Gaarder, M., Veiseth-Kent, E., & Thomassen, M. (2010). Fillet textureand protease activities in different families of farmed Atlantic salmon (Salmosalar L.). Aquaculture, 310, 213–220.

L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408 1407

Bahuaud, D., Mørkøre, T., Langsrud, Ø., Sinnes, K., Veiseth, E., Ofstad, R., &Thomassen, M. S. (2008). Effects of �1.5 �C super-chilling on quality ofAtlantic salmon (Salmo salar) pre-rigor fillets: Cathepsin activity, musclehistology, texture and liquid leakage. Food Chemistry, 111, 329–339.

Bahuaud, D., Mørkøre, T., Østbye, T. K., Veiseth-Kent, E., Thomassen, M. S., & Ofstad,R. (2010). Muscle structure responses and lysosomal cathepsins B and L infarmed Atlantic salmon (Salmo salar L.) pre- and post-rigor fillets exposed toshort and long-term crowding stress. Food Chemistry, 118, 602–615.

Bahuaud, D., Østbye, T. K., Torstensen, B. E., Rørå, M. B., Ofstad, R., Veiseth, E.,Thomassen, M. S., & Ruyter, B. (2009). Atlantic salmon (Salmo salar) musclestructure integrity and lysosomal cathepsins B and L influenced by dietary n�6and n�3 fatty acids. Food Chemistry, 114, 1421–1432.

Barrett, A. J., & Heath, M. F. (1977). Lysosomal enzymes. In J. T. Dingle (Ed.),Lysosomes – A laboratory handbook (2nd Ed., pp. 19–145). Amsterdam, TheNetherlands: North Holland.

Barrett, A. J., & Kirschke, H. (1981). Cathepsin B, cathepsin H, and cathepsin L. In L.Lorand (Ed.), Methods in enzymology. Proteolytic enzymes, Part C (pp. 535–561).New York: Academic Press.

Barton, B. A. (2002). Stress in fishes: A diversity of responses with particularreference to changes in circulating corticosteroids. Integrative and ComparativeBiology, 42, 517–525.

Barton, B. A., & Iwama, G. K. (1991). Physiological changes in fish from stress inaquaculture with emphasis on the response and effects of corticosteroids.Annual Review of Fish Diseases, 1, 3–26.

Bjørnevik, M., & Solbakken, V. (2010). Preslaughter stress and subsequent effect onflesh quality in farmed cod. Aquaculture Research, 41, e467–e474.

Bonga, S. E. W. (1997). The stress response in fish. Physiological Reviews, 77,591–625.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry, 72, 248–254.

Brown, J. A., Watson, J., Bourhill, A., & Wall, T. (2008). Evaluation and use of theLactate Pro, a portable lactate meter, in monitoring the physiological well-beingof farmed Atlantic cod (Gadus morhua). Aquaculture, 285, 135–140.

Brown, J. A., Watson, J., Bourhill, A., & Wall, T. (2010). Physiological welfare ofcommercially reared cod and effects of crowding for harvesting. Aquaculture,298, 315–324.

Damsgård, B. (2008). Fish welfare and ethical qualities in aquaculture. In T. Børresen(Ed.), Improving seafood products for the consumer (pp. 490–510). Boca Raton, FL:CRC Press.

Digre, H. (2011). Slaughter methods and processing of farmed Atlantic cod (Gadusmorhua). Welfare aspects and flesh quality (Unpublished PhD thesis) NorwegianUniversity of Science and Technology, Department of Biotechnology,Trondheim, Norway.

Digre, H., Erikson, U., Misimi, E., Lambooij, B., & van de Vis, H. (2010). Electricalstunning of farmed Atlantic cod Gadus morhua L.: A comparison of an industrialand experimental method. Aquaculture Research, 41, 1190–1202.

Duun, A. S., & Rustad, T. (2008). Quality of superchilled vacuum packed Atlanticsalmon (Salmo salar) fillets stored at �1.4 and �3.6 �C. Food Chemistry, 106,122–131.

Erikson, U., Digre, H., & Misimi, E. (2011). Effects of perimortem stress on farmedAtlantic cod product quality: A baseline study. Journal of Food Science, 76,S251–S261.

Esaiassen, M., Dahl, R., Eilertsen, G., Gundersen, B., & Sivertsvik, M. (2008). Pre-rigorfilleting and brining of farmed cod: Influence on quality and storage stability.LWT – Food Science and Technology, 41, 724–729.

Godiksen, H., Morzel, M., Hyldig, G., & Jessen, F. (2009). Contribution of cathepsins B,L and D to muscle protein profiles correlated with texture in rainbow trout(Oncorhynchus mykiss). Food Chemistry, 113, 889–896.

Godiksen, H., & Nielsen, H. H. (2007). New method to discriminate betweencathepsin B and cathepsin L in crude extracts from fish muscle based on asimple acidification procedure. International Journal of Food Science andTechnology, 42, 102–106.

Haard, N. F. (1992). Control of chemical composition and food quality attributes ofcultured fish. Food Research International, 25, 289–307.

Hagen, Ø. (2011). Protease activity impacts flesh colour during post-mortem storageof farmed Atlantic halibut (Hippoglossus hippoglossus): A condition referred to aschalky halibut. Food Chemistry, 125, 1294–1298.

Hemre, G. I., Lambertsen, G., & Lie, Ø. (1991). The effect of dietary carbohydrate onthe stress response in cod (Gadus morhua). Aquaculture, 95, 319–328.

Herbert, N. A., & Steffensen, J. F. (2005). The response of Atlantic cod, Gadus morhua,to progressive hypoxia: Fish swimming speed and physiological stress. MarineBiology, 147, 1403–1412.

Hernandez-Herrero, M. M., Duflos, G., Malle, P., & Bouquelet, S. (2003). Collagenaseactivity and protein hydrolysis as related to spoilage of iced cod (Gadusmorhua). Food Research International, 36, 141–147.

Hultmann, L., Rørå, A. M. B., Steinsland, I., Skåra, T., & Rustad, T. (2004). Proteolyticactivity and properties of proteins in smoked salmon (Salmo salar) – effects ofsmoking temperature. Food Chemistry, 85, 377–387.

Hultmann, L., & Rustad, T. (2002). Textural changes during iced storage of salmon(Salmo salar) and cod (Gadus morhua). Journal of Aquatic Food ProductTechnology, 11, 105–123.

Hultmann, L., & Rustad, T. (2004). Iced storage of Atlantic salmon (Salmo salar) –effects on endogenous enzymes and their impact on muscle proteins andtexture. Food Chemistry, 87, 31–41.

Hultmann, L., & Rustad, T. (2007). Effects of temperature abuse on texturalproperties and proteolytic activities during post mortem iced storage of farmedAtlantic cod (Gadus morhua). Food Chemistry, 104, 1687–1697.

Inubushi, T., Kakegawa, H., Kishino, Y., & Katunuma, N. (1994). Specific assaymethod for the activities of cathepsin L-type cysteine proteinases. Journal ofBiochemistry, 116, 282–284.

Kinoshita, M., Toyohara, H., Shimizu, Y., & Sakaguchi, M. (1992). Modori-inducingproteinase active at 50 �C in threadfin bream muscle. Nippon Suisan Gakkaishi,58, 715–720.

Kristoffersen, S., Tobiassen, T., Steinsund, V., & Olsen, R. L. (2006). Slaughter stress,postmortem muscle pH and rigor development in farmed Atlantic cod (Gadusmorhua L.). International Journal of Food Science and Technology, 41, 861–864.

Lefèvre, F., Bugeon, J., Aupérin, B., & Aubin, J. (2008). Rearing oxygen level andslaughter stress effects on rainbow trout flesh quality. Aquaculture, 284,81–89.

Licciardello, J. J., Ravesi, E. M., Lundstrom, R. C., Wilhelm, K. A., Correia, F. F., &Allsup, M. G. (1982). Time-temperature tolerance and physical-chemical qualitytests for frozen red hake. Journal of Food Quality, 5, 215–234.

Love, R. M., Lavety, J., & Garcia, N. G. (1972). The connective tissues of fish. VI.Mechanical studies on isolated myocommata. Journal of Food Technology, 7,291–301.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Proteinmeasurement with the Folin phenol reagent. Journal of Biological Chemistry,193, 265–275.

Lupatsch, I., Santos, G. A., Schrama, J. W., & Verreth, J. A. J. (2010). Effect of stockingdensity and feeding level on energy expenditure and stress responsiveness inEuropean sea bass Dicentrarchus labrax. Aquaculture, 298, 245–250.

Mann–Whitney U Test. (2011). Accessed September 2011 from http://elegans.swmed.edu/~leon/stats/utest.cgi.

McIlvaine, T. C. (1921). A buffer solution for colorimetric comparison. Journal ofBiological Chemistry, 49, 183–186.

Misimi, E., Erikson, U., Digre, H., Skavhaug, A., & Mathiassen, J. R. (2008). Computervision-based evaluation of pre- and postrigor changes in size and shape ofAtlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) fillets duringrigor mortis and ice storage: Effects of perimortem handling stress. Journal ofFood Science, 73, E57–E68.

Montero, P., & Mackie, I. M. (1992). Changes in intramuscular collagen of cod (Gadusmorhua) during post-mortem storage in ice. Journal of the Science of Food andAgriculture, 59, 89–96.

Morgan, M. J., Wilson, C. E., & Crim, L. W. (1999). The effect of stress on reproductionin Atlantic cod. Journal of Fish Biology, 54, 477–488.

Mørkøre, T. (2006). Relevance of dietary oil source for contraction and quality ofpre-rigor filleted Atlantic cod, Gadus morhua. Aquaculture, 251, 56–65.

Morzel, M., Chambon, C., Lefevre, F., Paboeuf, G., & Laville, E. (2006). Modificationsof trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity. Journalof Agricultural and Food Chemistry, 54, 2997–3001.

Morzel, M., Sohier, D., & Van de Vis, H. (2003). Evaluation of slaughtering methodsfor turbot with respect to animal welfare and flesh quality. Journal of the Scienceof Food and Agriculture, 83, 19–28.

Nelson, J. A., Tang, Y., & Boutilier, R. G. (1994). Differences in exercise physiologybetween two Atlantic cod (Gadus morhua) populations from differentenvironments. Physiological Zoology, 67, 330–354.

Ofstad, R., Egelandsdal, B., Kidman, S., Myklebust, R., Olsen, R. L., & Hermansson, A.M. (1996). Liquid loss as effected by post mortem ultrastructural changes in fishmuscle: Cod (Gadus morhua L.) and salmon (Salmo salar). Journal of the Science ofFood and Agriculture, 71, 301–312.

Ofstad, R., Kidman, S., Myklebust, R., & Hermansson, A. M. (1993). Liquid holdingcapacity and structural changes during heating of fish muscle: Cod (Gadusmorhua L.) and salmon (Salmo salar). Food Structure, 12, 163–174.

Olsen, S. H., Sørensen, N. K., Larsen, R., Elvevoll, E. O., & Nilsen, H. (2008). Impact ofpre-slaughter stress on residual blood in fillet portions of farmed Atlantic cod(Gadus morhua) – Measured chemically and by Visible and Near-infraredspectroscopy. Aquaculture, 284, 90–97.

Olsen, R. E., Sundell, K., Hansen, T., Hemre, G.-I., Myklebust, R., Mayhew, T. M., &Ringø, E. (2002). Acute stress alters the intestinal lining of Atlantic salmon,Salmo salar L.: An electron microscopical study. Fish Physiology and Biochemistry,26, 211–221.

Olsen, R. E., Sundell, K., Ringø, E., Myklebust, R., Hemre, G.-I., Hansen, T., & Karlsen,Ø. (2008). The acute stress response in fed and food deprived Atlantic cod,Gadus morhua L. Aquaculture, 280, 232–241.

Peterson, G. L. (1979). Review of the Folin phenol protein quantitation method ofLowry, Rosebrough, Farr and Randall. Analytical Biochemistry, 100, 201–220.

Poli, B. M., Parisi, G., Scappini, F., & Zampacavallo, G. (2005). Fish welfare and qualityas affected by pre-slaughter and slaughter management. AquacultureInternational, 13, 29–49.

Robergs, R. A., Ghiasvand, F., & Parker, D. (2004). Biochemistry of exercise-inducedmetabolic acidosis. American Journal of Physiology – Regulatory, Integrative andComparative Physiology, 287, R502–R516.

Rustad, T. (1992). Muscle chemistry and the quality of wild and farmed cod. In H. H.Huss, M. Jakobsen, & J. Liston (Eds.), Quality Assurance in the Fish Industry,proceedings of an international conference, Copenhagen, Denmark, 26–30 August1991 (pp. 19–27). Amsterdam, The Netherlands: Elsevier Science Publishers B.V.

Shigemura, Y., Ando, M., Harada, K., & Tsukamasa, Y. (2004). Possible degradation oftype I collagen in relation to yellowtail muscle softening during chilled storage.Fisheries Science, 70, 703–709.

1408 L. Hultmann et al. / Food Chemistry 134 (2012) 1399–1408

Sigholt, T., Erikson, U., Rustad, T., Johansen, S., Nordtvedt, T. S., & Seland, A. (1997).Handling stress and storage temperature affect meat quality of farmed-raisedAtlantic salmon (Salmo salar). Journal of Food Science, 62, 898–905.

Statistics Norway. (2011). Accessed September 2011 from http://www.ssb.no/english/subjects/10/05/fiskeoppdrett_en.

Stien, L. H., Hirmas, E., Bjørnevik, M., Karlsen, Ø., Nortvedt, R., Rørå, A. M. B., Sunde,J., & Kiessling, A. (2005). The effects of stress and storage temperature on thecolour and texture of pre-rigor filleted farmed cod (Gadus morhua L.).Aquaculture Research, 36, 1197–1206.

Strasbourg, G., Xiong, Y. L., & Chiang, W. (2008). Physiology and chemistry of ediblemuscle tissues. In S. Damodaran, K. L. Parkin, & O. R. Fennema (Eds.), Fennema’sfood chemistry (4th Ed., pp. 923–973). Boca Raton: Taylor & Francis.

Sveinsdóttir, K., Martinsdóttir, E., Thórsdóttir, F., Schelvis, R., Kole, A., & Thórsdóttir,I. (2010). Evaluation of farmed cod products by a trained sensory panel andconsumers in different test settings. Journal of Sensory Studies, 25, 280–293.

Thomas, P. M., Pankhurst, N. W., & Bremner, H. A. (1999). The effect of stress andexercise on post-mortem biochemistry of Atlantic salmon and rainbow trout.Journal of Fish Biology, 54, 1177–1196.

Wang, P. A., Martinez, I., & Olsen, R. L. (2009). Myosin heavy chain degradationduring post mortem storage of Atlantic cod (Gadus morhua L.). Food Chemistry,115, 1228–1233.

Yasuda, Y., Kageyama, T., Akamine, A., Shibata, M., Kominami, E., Uchiyama, Y., &Yamamoto, K. (1999). Characterization of new fluorogenic substrates for therapid and sensitive assay of cathepsin E and cathepsin D. Journal of Biochemistry,125, 1137–1143.