Liquid Loss as Effected byPost mortem Ultrastructural Changes in Fish Muscle: Cod (Gadus morhua L)...

J Sci Food Agric 1996,71,301-312

Liquid Loss as Effected by Post mortem Ultrastructural Changes in Fish Muscle : Cod (Gadus morhua L) and Salmon (Salmo salar) Ragni Ofstad,"* Bj$rg Egelandsdal,b Siw Kidrnaqc Reidar Myklebust,d Ragnar L Olsen" and Anne-Marie Hermanssonc

Norwegian Institute of Fkheries and Aquaculture, PO Box 2511, N-9002, Tromser, Norway Matforsk, Norwegian Food Research Institute, Oslov 1, 1430 A, Norway SIK, The Swedish Institute for Food Research, PO Box 5401, S40229 Gsteborg, Sweden Dept of Electron Microscopy, Institute of Medical Biology, University of Tromser, N-9002, Tromser,

Norway

(Received 22 May 1995; revised version received 2 November 1995; accepted 26 January 1996)

Abstract: This study was performed in order to assess the effect of early post mortem structural changes in the muscle upon the liquid-holding capacity of wild cod, net-pen-fed cod (fed cod) and farmed salmon. The liquid-holding capacity was measured by a low speed centrifugation test. Transmission electron microscopy was used to discover ultrastructural changes both in the connective tissue and in the myofibrils. Differential scanning calorimetric thermograms of the muscle proteins were recorded to elucidate whether fundamental differences did exist between the proteins of the raw material tested. Multivariate statistics were used to explicate the main tendencies of variations in the thermograms. The salmon muscle possessed much better liquid-holding properties than the cod muscle, and wild cod better than fed cod regardless of the storage time. Both fed cod and farmed salmon, underwent the most severe structural alterations, probably caused by the low muscle pH values. The higher liquid-holding capacity of the salmon muscle was related to species specific structural features and better stability of the muscle proteins. The myofibrils of the salmon muscle were denser and intr.a- and extracellular spaces were filled by fat and a granulated material. The differences in thermograms of muscle from wild and fed cod were largely explained by the variations in pH. The severe liquid loss of fed cod is due to a low pH induced denaturation and shrinkage of the myofibrils. Post mortem degradation of the endomysial layer and the sarcolemma may have further facilitated the: release of liquid.

Key words: salmon, wild cod, fed cod, season, post mortem changes, liquid loss, ultrastructure, scanning calorimetry.

INTRODUCTION

Lipid and water together make up about 80% of fish muscle. Depending on the properties of the flesh and how it is treated, it may gain or lose water. This is important economically since fish is sold by weight. The

* To whom correspondence should be addressed at: Fiskeri- forskning, PO Box 251 1,9002-Tromsa, Norway.

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

content of water and its distribution within the flesh also affects the quality. In spite of this, surprisingly little work has been carried out on the mechanism of inter- and intraspecies variations in liquid-holding properties.

The free water in muscle is held by capillary and surface tension forces mainly within the intracellular locations. Hence, liquid-holding capacity of muscle is highly influenced by fibril swelling/contraction and the distribution of fluid between intra- and extracellular

301

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locations (Offer and Trinick 1983). Temperature, ionic strength and pH of the muscle will strongly influence its structure and thus its liquid-holding capacity (Hermansson 1986; Wilding et a1 1986; Offer et al 1989). We have previously shown that there is a relationship between liquid-holding capacity and structural changes in coarsely chopped post-rigor muscle of both cod and salmon upon heating (Ofstad et a1 1993). In addition to pH, temperature, salt concentration and interaction effects between these factors, the choice of raw material occasioned considerable structural differences in a finely comminuted model product, thus affecting the liquid-holding properties. Both species, and whether the fish had been fed or not, affected the liquid- holding properties (Ofstad et al 1995, 1996).

It has previously been pointed out that seasonal variations associated with the nutritional status and maturation of the fish may affect characteristics such as chemical composition, post mortem pH and texture (Bilinski et a1 1984; Aksnes et a1 1986; Love 1988; Yamashita and Konagaya 1991, 1992; Haard 1992). Ang and Haard (1985) suggested that the soft muscle texture and poor water-holding capacity of the post- spawned and heavily re-fed cod was due to the low and stable ultimate pH of this fish. Similar flesh characteristics are observed in cod that are caught live and fed for long periods of time before being killed (Mohr 1986; Rustad 1992).

The high drip loss of pale, soft and exudative (PSE) porcine meat is associated with extensive post mortem ultrastructural changes both in the subcellular fractions and in the fine structure of the muscle (Cassens et a1 1963; Greaser et a1 1969; Dutson et a1 1974). PSE meat is caused by the denaturation before rigor of the proteins that takes place at low pH and/or high temperature (Bendall and Wismer-Pedersen 1962, Stabursvik et a1 1984; Offer et a1 1989; Offer 1991; Fernadez et al 1994). A similar phenomenon is reported to occur in yellowfin tuna. Burnt tuna meat which is a pale, exudative and soft meat, appears to undergo the same degradative ultrastructural changes as normal tuna muscle, except that the rate of degradation is faster (Davie and Sparksman 1986; Watson et a1 1992). Recently Tachibana et a1 (1993) have reported that the degradation of Z-discs of ordinary muscle was faster in cultured red sea bream than in the wild counterpart. Although the period immediately post mortem is crucial for the subsequent quality of fish (Brarresen 1992), there are few ultrastructural studies of early post mortem changes in fish muscle. The general post rnortem degradation in the muscle structure of goldfish and of cod, as observed after 3-4 days on ice, is similar to that observed in mammalian or avian muscle (Liljemark 1969; Bello et a1 1982). It is well established that the myofibrillar proteins differ in stability depending on the habitat temperature of the species (Hastings et a1 1985). Changes in thermal characteristics of myosin subunits

during iced and frozen storage suggest more rapid deterioration in cold water than in warm water fish (Howell et a1 1991; Davies et a1 1994).

Marked differences in both structural and chemical composition can be seen when comparing the muscle of salmon, a fatty fish species with that of cod, a lean fish species. Furthermore, structural and chemical composition vary due to the nutritional status of the fish. These may influence fish quality at the time of capture as well as influencing its deterioration after death. Indeed, it is possible that the previously reported inter- and intraspecies differences in liquid loss are a function of differing rates of deterioration (Ofstad et a1 1993, 1995, 1996). This study was therefore carried out to investigate the early post mortem changes in cod and salmon muscle. To elucidate the effects of nutritional status, wild cod harvested in different seasons were compared with net- pen-fed cod. Comparison of liquid loss, severeness of the observed ultrastructural changes, and differtial scanning calorimetric (DSC) thermograms of the muscle proteins were performed. The use of DSC in the study of protein stability has the advantage that a protein can be studied in its normal intracellular environment. Multivariate statistics, principal component analysis (PCA) and principal component regression (PCR) were used to elucidate the main tendencies of variations in the thermograms among the raw materials tested (Martens and Naes 1989). In this way we hoped to estab- lish whether fundamental differences in muscle proteins denaturation characteristics did exist between salmon and cod and between wild and fed cod and whether any such variation affected post mortem changes during ice storage.

MATERIALS AND METHODS

Raw materials

Three different groups of raw material were studied; wild cod (WC), fed cod (FC), ie cod caught live and fed in a net pen for more than 1 year before being killed, and farmed Atlantic salmon (S). WC were caught alive and kept in a tank for 1 week before being slaughtered. The fed fish, both cod and salmon, were collected from a farm located near Tromsra. The wild and fed cod used were harvested three times a year: migrating, mature condition (WMC and FMC, respectively), summer (WSC and FSC, respectively) and autumn (WAC and FAC, respectively). The salmon were harvested twice : spring being silver-bright (SO), autumn being moder- ately mature (SM). In each trial four to six fish were immediately degutted and kept in ice. The temperature of the fish muscle at slaughter was 4-7°C. Half the fish were filleted and analysed within 3 h, ie before appearance of rigor mortis. The others were kept in ice for 48 h

Liquid loss and post mortem ultrastructural changes in fish muscle 303

until the resolution of rigor m40rtis and the muscle had softened, and then filleted before being analysed. All operations were carried out at 4°C. The temperature of the fish muscle stored for 3 h in ice was 5-6°C after filleting while that stored for 48 h was 3-4°C. The proximate analyses were performed on homogenised white muscle of each fish according to Ofstad et a1 (1996). The average body index parameters, proximate composition and pH are given in Table 1.

Liquid-holding capacity (LHC)

The liquid loss was measured Ion coarsely chopped filets pooled from two to three 16sh in each trial. LHC (expressed as percent) was of the weight of liquid released per 15 g of a sample immediately centrifuged at 5°C according to the net test described by Hermansson (1986) and Ofstad et a1 (1993). Mean values were calculated from two trials by using; both filets and six repli- cates in each trial.

Transmission electron microscopy (TEM)

Thin strips of muscle were excised from the fillets near the dorsal fin of each three fish at 3 h and 48 h post mortem, respectively. The 3 11 samples were carefully stretched and pinned to dental wax plates (Allmo, Sweden) prior to fixation. Thle muscle specimens were fixed in precooled glutaralclehyde/formaldehyde (25/ 10 g kg- ') with 2 g kg- glucose in Ringer's buffer (pH 6.8) for 24 h at 4°C. Small piieces of muscle fibre were cut with a razor blade under a dissecting microscope and rinsed in pre-cooled buffer. The samples were post- fixed in osmium tetraoxide, dehydrated and embedded in Epon/Araldite, sectioned ,and stained according to Ofstad et al (1996). Sections were viewed and pho- tographed using a TEM, lOOCXII (Jeol Ltd, Tokyo, Japan). Ten blocks were prepaired from each sample and at least two blocks were sectioned either longitudinally or transversally to the fibre direction. On each occasion of testing at least two fish were examined.

Differential scanning calorimetry (DSC)

The instrument used was a Setaram Micro DSC-batch and flow calorimeter (Setaram, Lyon, France). Approx- imately 1 g of fish flesh was put into the calorimetric cells. Water was used in the reference cell. The samples were heated from 15 to 85°C at a heating rate of 1°C min-'. The baseline was subtracted and the heat- flow signal adjusted in accordance with the protein content of the fish flesh (Harbitz et al 1984). Two thermograms devoid of any visible abnormal shift, which may occur in the baseline, were used for calculations. The fish were examined on the first and second day post mortem. There was no systematic effect on the thermograms due to storage.

Statistical analysis

The thermograms (32-85"C), corrected for protein concentration variations, were subjected to a principal component analysis (PCA), principal component regression (PCR) using Unscrambler (Camo AS, Trondheim, Norway), and classification (Bayers classification) using the proc discrim of the SAS (SAS institute Inc, Cary, NC, USA) software. The purpose of PCA is to reduce the DSC-data (X-data) to a few factors which express the source of variation in the X-data. These factors are used as independent variables in a linear regression (PCR) to predict the effect upon the response variables (Y-values) (Martens and Naes 1989). Cross-validation was used to validate the calibration model, ie each object was removed from the calibration and estimated using the model calculated from the remaining samples.

RESULTS A N D DISCUSSION

Liquid-holding capacity (LHC)

Released liquid was measured in coarsely chopped fillets by the net test 3 h and 48 h post mortem. The average

TABLE 1 Average body index parameters, proximate composition per kg muscle tissue and pH (mean f standard deviation of nine fed cod,

eight wild cod and six salmon) ~

Raw Post mortem Degutted Body length Protein Water Fat FH material storage (h) weight (9) (4 (9 k g - ' ) (9 k g - 7 (9 k g - ' )

~ ~

Wild cod 3 2384 f 1954 0.67 f 0.14 171.0 f 15.8 818.0 11.1 6.75 f 0.22 48 1530 & 1157 0.60 & 0.10 175.9 f 8.4 816.1 f 9.2 6.77 f 0.13

3 4822 f 2068 0.80 f 0.09 179.2 f 15.0 812.1 f 14.2 6.23 & 0.20 48 3722 f 1187 0.79 f 0.07 176.3 f 12.4 807.8 f 9.1 6.28 f 0.10

Salmon 3 3271 f 1133 0.67 f 0.06 186.5 f 19.0 672.9 f 21.0 134.9 f 35.1 6.12 f 0.13 48 3036 f 1436 0.65 f 0.09 192.2 f 15.2 684.6 k 22.9 117.3 & 18.8 6.24 & 0.13

Fed cod

304 R Ofstad et a1

TABLE 2 Average liquid loss (YO) from coarsely chopped muscle of wild cod, fed cod and salmon according to the net test (mean & standard deviation of nine fed cod, eight wild cod

and six salmon)

Post mortem storage (h) W i l d cod Fed cod Salmon

~

3 19.70 k 9.42 31.40 f 7.00 9.53 f 2.97 48 10.61 2.09 18.79 & 2.09 1.85 k 0.86

liquid loss of each of the three groups of fish is given in Table 2. The levels of liquid loss are in accordance with previous findings (Ofstad et a1 1993). Independent of the storage time the salmon muscle possesses much better liquid-holding properties than the cod muscle and the liquid loss is higher in the fed cod than in the wild cod. As shown in Table 1, chemical composition varied between the fish types. However, correction for the different protein contents does not essentially affect the above result. In the fed fish, both cod and salmon, the muscles pH were very low both at 3 h and 48 h post mortem. The pH of the different muscle samples affects the volume of the myofibrils and this may partly explain the different water-holding capacity in fed and wild cod (Wilding et a1 1986; Offer et a1 1989).

Somewhat unexpectedly, all 3 h samples released more liquid than did 48 h samples. Even though, the flesh of the 3 h ice-stored fish appeared soft, mechanical treatment during preparation of the flesh for liquid loss measurements may have induced rigor onset. The higher liquid loss of the 3 h than the 48 h muscle is then probably caused by shrinkage of the myofibrils due to rigor contractions as described by Offer et a1 (1989). Examination of the sectioned muscle by TEM (micrographs not shown) showed that the muscle fibres fixed within 3 h had partly contracted during cutting and fixation.

Values for liquid loss showed variations due to season. Wild cod, which differed more in size and nutritional status than did the fed fish, exhibited the greatest seasonal variation (Table 3). Some of these differences in liquid loss may be explained by the different pH values; 6.67, 6.91 and 6.64 of mature (WSC), summer (WSC)

TABLE 3 Liquid loss (YO) as affected by the season of coarsely chopped muscle of wild cod according to the net test, mature (WMC); summer (WSC); autumn (WAC) (mean f standard deviation

of two WMC, three WSC and three WAC)

Post mortem storage ( h ) WMC wsc WAC

3 4.94 & 1.60 24.30 k 1.70 27.55 f 1.83 48 12.32 f 2.90 7.10 k 0.60 12.91 1.90

and autumn cod (WAC), respectively. WMC were large, migrating fish caught at the spawning grounds. The WSC, having somewhat higher liquid-holding capacity than the WMC, were small but well-fed and were visually judged to be in good condition, although post- spawn. The WAC, which possessed the poorest liquid- holding capacity, were of the same size as WSC and were caught at the same ground. However, these fish had starved and the flesh appeared soft. Love and Robertson (1967) reported a similar phenomenon in autumn caught cod; the amount of inextractable proteins was larger, when the fish were starved in the autumn and the gonads had started to mature, than otherwise.

Post mortem changes in muscle ultrastructure

Light microscopy has previously been used to reveal structural differences upon heating of fed cod, wild cod and salmon (Ofstad et a1 1993, 1996). By this technique it was not possible to explain the differences in liquid loss between cod and salmon. Electron microscopy makes it possible to obtain more detailed information on the various structural changes in relation to pre- and post-rigor condition, species and nutritional status. As shown below in Figs 1 ,2 and 4, the general post mortem degradation of the examined muscle proceeds as previously described (Liljemark 1969; Bello et al 1982; Hallet and Bremner 1988): the sarcolemma becomes progressively looser and more or less separated from the fibre, and loses its collagenous surround. The sarcoplasmic reticulum (SR) surrounding the myofibrils becomes swollen and membrane vacuoles more or less float in the sarcoplasm, both in intra- and extracellular locations. The mitochondria swell and the nuclei become more shrunken with denser chromatin. The inter-fibrillar space widens, the Z-discs of the myofila- ments disintegrate and the thin filaments of the I-bands split up. Although the post mortem degradation fol- lowed a general pattern, a qualitative observation of the micrographs indicated that there were differences between the wild and the fed fish muscle, and between cod and salmon muscle.

The ultrastructural changes were focal in nature, to the extent that changes were dramatic in one area of muscle while other areas of the same muscle appeared unaffected. There is, however, an overall pattern which can be discerned and which is described below. The micrographs presented were chosen out of many and represent the most typical structural features of the muscle samples.

Wild cod Within 3 h after death, the cell envelope, as observed between two adjacent cross-sectioned muscle cells (Fig 1 a), appears well preserved and is internally composed of sarcolemma coated by an amorphous structure (basement membrane). Outermost is the endomysial

Liquid loss and post mortem uli-rastructural changes in fish muscle 305

Fig 1. Wild cod; sections of the dorsal muscle fixed within 3 h (a,b) and 48 h after death (c,d). Transverse sections (a,c) show successive layers of sarcolemma (SI), basement membrane (BM) and collagen fibres (C) of the endomysium (E). Mitochondria (Mi) are situated beneath the sarcolemma. Myofibrils are surrounded by the sarcoplasmic reticulum (SR). Longitudinal sections (b,d) show a compact fibril structure with a distinct sarcomere pattern of A-, I-bands and H-zones, M- and Z-discs. At the Z-disc in (b) two terminal cisternae (Tc) of sarcoplasmic reticulum embrace the T-tubule (Tt). SR have partly degraded into unconnected vesicles

and membranous material in (c,d).

layer of fine collagen fibres. ]Hexagonal arrangements derived from thick and thin filaments are observable in this cross-sectional view of the myofibres. The longitudinal section of the muscle fibre looks intact, with the typical sarcomere pattern and its A-, I- and H-zones arising from the partly overlapping thick and thin filaments (Fig lb). The thin filaments are attached to the Z-discs and adjacent thick filaments are connected at the M-lines. In each sarcomere, at the Z-disc, two terminal cisternae (seen as an irregular sac of the SR) embrace the transverse tubule seen as a narrow channel.

In Fig lc, after 48 h, the integrity of the sarcolemma is maintained, but shows a tendency to separate from the myofibrils, and the outer c'ollagenous layer has split off. The mitochondria are swollen compared with those observed in Fig la, and the SR has swollen and become more vesiculated particularly in the periphery of the cell. The salient feature of this wild cod muscle is the intact myofibrillar structure with narrow intermyofibrillar spaces even after 48 h storage (Fig Id).

Fed cod Compared with the wild cod, fed cod exhibited both faster and more severe structural changes in the muscle,

as shown in Fig. 2. Notable alterations were observed already 3 h after death. In Fig 2a, the sarcolemma is separated from the myofibrils and the SR is swollen. However, small invaginations of sarcolemma, pinocytotic vesicles, show that this muscle is still in the pre-rigor condition (Liljemark 1969). Differing from the wild cod (Fig la), large amounts of granulated glycogen-like par- ticles can be seen beneath the sarcolemma (Fig 2a) (Bello et a1 1982). Rustad (1992) reported that the glycogen content in the muscle of fed cod immediately after death was about twice that in the muscle of wild cod.

The myofibrillar structure of the longitudinal sectioned muscle looks intact, but intermyofibrillar spaces are wider in the fed (Fig 2b) than in the wild cod (Fig lb). At 48 h post rnortern (Fig 2c), the mitochondria are ruptured and the nuclei have shrunken, with the chromatin clumped along the nuclear membrane. Further- more, the sarcolemma is ruptured (indicated with arrowheads in Fig 2c) and its surrounding collagenous layer has broken down. The intermyofibrillar spaces have increased and in contrast to the wild cod the Z-discs are partly disrupted, and in some places split- ting of the I-bands has occurred as indicated with arrowheads in Fig 2d.

306 R Ofstad et a1

Fig 2. Fed cod; sections of the dorsal muscle fixed within 3 h (a,b) and 48 h after death (c,d). Transverse sections (a&) show fibre base region; collagen (C), pinocytotic vesicles (P) and glycogen granules (G) are indicated. Mitochondria (Mi) is swollen in (a) and ruptured in (c). Nucleus (N) is oval in (a) and shrunken in (c). Ruptures in the sarcolemma are arrowheaded in (c). Longitudinal sections (b,d) show muscle fibre with wide intramyofibrillar spaces. The banding is indistinct and splits are visible in the I-bands as

arrowheaded in (d). Inset is enlarged view of area indicated with arrowhead.

The average muscle pH of wild and fed cod were 6.8 and 6.3, respectively. A pH closer to the iso-electric point of the myofibrillar proteins will increase the protein-protein attraction due to a smaller net charge (Wilding et a1 1986; Offer et a1 1989). This is in accordance with the wider intermyofibrillar spaces observed in the fed (Fig 2) than in the wild cod (Fig 1). The loss of water occurs by its expulsion from the myofibrils as they shrink laterally when the filaments get closer together. This shrinkage, increasing both the intermyofibrillar and extracellular spaces, reduces the capillary forces and thus liquid-holding ability (Table 2). The Z-discs link adjacent thin filaments and have a preferred lattice dimensions. These will act as a constraint on the separation/shrinkage of the thin filaments. The more the Z-disc is disrupted the weaker will be its influence on the attached thin filament lattice. So it is possible that the observed disruption of the Z-discs in the fed cod muscle (Fig 2d) could further favour the observed pH- induced shrinkage of the samples stored for 48 h, due to the reduced constraint of the thin filaments and hence the myofibrils. Moreover, the observed disruption (Fig 2c) of the sarcolemma and the extracellular collagenous layer which represents a physical barrier to release of

fluid, may increase the leakage of liquid expelled from the myofibrils.

As shown in Fig 3, ultrastructural signs of muscle myopathy were observed as central migrated nuclei in some of the cross-sectioned muscle fibres (Roberts and Bullock 1989). The effect of such an ante mortem condition upon post mortem properties has, to our know- ledge, not been elucidated. It is, however, likely that a myopathic condition of the muscle may explain some of the extended post mortem degradation observed in the fed cod, thus partly explaining the much higher liquid loss from this fish compared with the wild counterpart.

Salmon The structure of salmon muscle, known as mosaic muscle, differs markedly from cod muscle (Fig 4). The endomysial sheath which seems wider, comprises distinct bundles of collagen fibres and a clearly visible, fine flocculate material. Lipid droplets are seen both in intra- and extracellular locations. The myofibrils generally look denser and the Z-discs appear as much darker lines than in the cod. At 3 h post mortem, the muscle is still well preserved (Figs 4a and b). By 48 h after slaughter, the sarcolemma has separated from the myofibrils


structural degradation observed in salmon muscle appears less pronounced than in fed cod, but more severe than in wild cod. The better liquid-holding capacity of the salmon muscle is thus probably more related to intrinsic structural features such as the denser impression of the myofibrils, intra- and extracellular lipid droplets, and the amorphous material filling intracellular spaces. As shown at higher magnification in Fig 5a, only small aggregates of degraded myofibrillar proteins and remnants of SR may be seen in the intracellular spaces of wild cod. On the other hand, in salmon muscle these spaces are completely filled with SR remnants and an amorphous material which might be small aggregates of sarcoplasmic proteins (Fig 5b).

Seasonal variations No large structural inequalities were observed amongst fish caught during the year. We have previously shown that a more severe structural breakdown occurs in

Fig 3. Cross-section of the doraal muscle of fed cod with muscle of heavily fed, wild cod compared with the non- central migrated nuclei (N). feeding mature fish (Ofstad et a1 1996). Signs of myo-

fibrillar structural breakdown have been observed in and is partly ruptured (indicated with arrowheads in fully mature Atlantic salmon (unpublished observation) Fig 4c). In addition, disruption of the Z-discs and split- similar to those observed in mature chum salmon (Reid ting of the I-bands have occurred in some places et a1 1993). Since the intramuscular structural damage is (indicated with arrowheads in Fig 4d). The degree of focal in nature and the variations between the fish are

Fig 4. Salmon; sections of the dorsal muscle fixed within 3 h (a,b) and 48 h after death (c,d). Transverse sections (a,c) show fibre base region; endomysium (E) and collagen (C) are indicated. Ruptures in the sarcolemma are arrowheaded in (c). Longitudinal sections (b,d) show muscle fibre with dense myofibrils, dark Z-discs and intracellular lipid (L) in (b). The banding is less distinct and

splits in the I-bands are arrowheaded in (d). Inset is enlarged view of area indicated with arrowhead.

308

Fig 5. Transverse section of the dorsal muscle of wild cod (a) and salmon (b) stored 48 h in ice. Note the granulated material filling the intermyofibrillar space in salmon. Mito-

chondria (Mi) and membrane remnants (Mr) are indicated.

large, the combined use of a larger number of individ- uals and statistical sampling and morphometry tech- niques is probably necessary to disclose minor structural differences probably related to the differences in liquid loss as shown in Table 3.

Differential scanning calorimetry (DSC)

Arithmetic mean thermograms obtained for the seven different groups of fish examined are shown in Figs 6 a x . The salmon had higher enthalpies of denaturation than the cod, and wild cod had higher enthalpies of denaturation than the fed cod (t-test, P < 0.01). This indicates that different degrees of denaturation of the muscle proteins had occurred prior to the thermal denaturation in the calorimeter. Stabursvik et a1 (1984) reported a similar decrease in the enthalpies of myofibrils prepared from PSE porcine meat. The myosin peak in the DSC thermogram of the PSE meat was markedly reduced compared with the normal pork meat.

According to the most common identification of fish thermogram peaks, the major peaks at about 44 and at 54°C are due to myosin denaturation, although other proteins like collagen denature in this region as well (Hastings et al 1985; Howell et al 1991). For that reason a thorough interpretation of changes in the thermogram

20 30 40 50 60 70

R Ofstad et a1

Temperature ( ‘C)

Fig 6. Arithmetic mean thermograms for fed cod (a), wild cod (b), and salmon (c).

in this region is difficult. The last peak at 73-74°C is due to actin denaturation, while the remaining peaks, between ‘myosin’ and ‘actin’, are attributed to denaturation of sarcoplasmic proteins. In Fig 6c, one myosin subunit denatures at almost the same temperature as that at which sarcoplasmic protein denatures, this resulting in a broad peak at 56-57°C. The resolution of the corresponding peaks is better in Figs 6a and 6b. Bearing in mind the above identification, it seems that differences among the fish were present in the myosin as well as in the sarcoplasmic protein region.

Multivariate statistics The DSC results obtained from 20 fish were subjected to a principal component analysis (PCA). The scores for the first two factors, principal component (PC) 1 vs PC 2, which express the main relationships between the samples, are presented in Fig 7. Cod and salmon are clearly grouped by PC1. The major difference in the thermograms is due to the species variation.


I / W A C 1 WMC1 ! I 2 -

N x -2 -

FAG

-4 - I I I 1 I I I I -6 -3 0 3 6 9

PC 1 (52%)

Fig 7. A score plot of the thermograms of the 20 fish studied as express by the main two factors, PC1 and PC2, explaining 76% of the variation between the thermograms. FC, fed cod; WC, wild cod; S, salmon, see Materials and Methods for the

symbols of the fish samples.

The loading plot in Fig 8 corresponds to the score plot (Fig 7) in such a way that the peaks with high positive loadings relate to peaks with high positive scores (salmon-thermograms) and peaks with negative loading correspond to peaks with hiigh negative scores (cod thermograms). The loading plot (Fig 8) reveals that the most prominent differences be1 ween salmon and cod are that salmon has higher heat absorption at 44-45"C, sarcoplasmic protein which denaturates at 65°C and, finally, increased actin stability in the muscles.

A statistical approach was made using cross- validated discriminant analysis, which is a method for evaluating the differences among groups by use of a parameter integrated from the data of many variables. The two principal components, from the PCA on all fish (Fig 7) were used in an attempt to classify the thermograms into three groups; salmon, wild and fed cod (Table 4). The thermograms of salmon and cod were correctly

0.25 7-

-0.10 tI , I , 1 I I

20 30 40 50 60 70 80 90 Temperature ('C)

Fig 8. A loading plot of the two principal components, PC 1 and PC 2, of the thermograms obtained from the PCA

analysis of tb: 20 fish.

TABLE 4 Cross-validated classification summary using discriminant

analysis (Bayers classification)

Class Wild cod Fed cod Salmon ~

Wild cod 5 0 0 Fed cod 2 7 0 Salmon 0 0 6

classified which confirms that the dissimilarities between the thermograms of cod and salmon were statistically significant. This indicates species-specific differences in the stability properties of the myosin/actomyosin between those two fishes; salmon myosin and actin were less easily denatured.

The second largest cause of variation among the thermograms (PC 2, Fig 7) mainly reflects the difference between wild and fed cod caught in the autumn (scoring highest in Fig 7). In the loading plot for PC 2 (Fig 8) peaks with high positive loadings relate to peaks with high positive scores, ie wild cod thermograms in Fig 7 and vice versa fed cod thermograms. The loading plot for PC 2 reveals that the wild cod had higher heat absorption at 42°C and 47"C, but generally lower at 58°C and 75°C than the fed cod. This reflects differences in the amount of both heat denatured myosin, sarcoplasmic proteins and actin between the wild and the fed cod (Fig 6). According to Stabursvik et a1 (1984) this indicates that the myofibrillar proteins in the fed cod muscle are already partial denatured during ice storage compared with the wild cod muscle. The difference in the thermograms between wild and fed cod was significant among the autumn caught fish (Table 4). Only two cod samples with rather high pH, one fed mature (FMC) and one fed summer cod (FSC), were not correctly classified; being classified as wild cod. This confirms that there is a significant variation in the thermograms among cod.

PCR was performed using the PCA scores of 14 cod thermograms in a multiple linear regression against the measured pH values which were significantly different ( P < 0.01). Much of the difference between wild and fed cod was explained by pH, as pH could roughly be pre- dicted from the thermograms ( R = 0.77, data not shown). Six percent of the variation in the thermograms was not related to the pH variation among the cod or differences between groups of fish, but rather reflected individual variations. Hence, the major cause of variation is due to the pH differences.

The difference between ordinary and maturing salmon, being clearly grouped in Fig 7, was due to a lower and higher heat absorption at 45"C, and at 50- 70°C respectively (Fig 6). This suggests that ordinary salmon contains larger amounts of a particular sarcoplasmic protein than maturing salmon. This protein

3 10 R Ofstad et a1

source may be used as an energy supply in the non- feeding maturing fish (Love 1988).

DSC thermograms related to post mortem changes and liquid loss

Regardless of the post rnortem degradation (Fig 4), salmon possessed much better liquid-holding properties than cod (Table 2). A regression analysis (PCR) was performed by using the scoreplot in Fig 7 as X-values and liquid loss as Y-values. The variation in liquid loss was mainly explained by PC 1 (80%, R = 0.95, data not shown). According to Fig 8 this means that the peaks below 42"C, lipid, collagen and myosin head, are posi- tively correlated to (increase) the liquid loss. On the other hand, a narrow myosin peak (44-45"C), a sarcoplasmic protein denaturing at 65°C and high actin stability are negatively correlated to (decrease) the liquid loss. The narrow denaturation peak of salmon myosin, the higher actin stability (Fig 6c) and the denser impression of the myofibrils, as observed in Fig 4, indi- cate that salmon myosin and actin differ from that of cod. Thus, the better liquid-holding properties of the salmon muscle are mainly related to species-specific structural features and better stability of myosin and/or of the acto-myosin complex. This is in accordance with our previous observations. Addition of salt to salmon muscle resulted in a much more homogenous protein matrix with few intact fibres and a subsequent much better liquid-holding capacity than when salt was added to cod muscle (Ofstad et a1 1996). According to Howell et a1 (1991) the variation in myosin stability between fish species is probably related to the composition and sequences of amino acid. residues enabling a greater or lesser number of stabilising interactions to take place.

The intracellular amorphous material observed in salmon, but not in cod (Fig 5), may be small aggregates of the sarcoplasmic protein with the peak at 65°C. We have previously suggested that aggregated sarcoplasmic proteins may influence the liquid released upon heating (Ofstad et a1 1993). The connective tissue as well as non- protein components of the muscle may also be important. For example, it has been found that some of the lipids present in fish muscle bind to and .consequently stabilise actomyosin (Hamada et al 1982).

As for the muscle from PSE meat, fed cod muscle holds less water than normal or wild cod muscle (Bendall and Wismer-Pedersen 1962). This phenomenon can occur if the ultimate pH is particularly low and the muscle proteins are exposed to pH lower than they normally experience post mortem (Offer et a1 1989; Offer 1991; Fernandez et al 1994). The pH of wild cod were close to 6.8 most of the year, whereas the fed fish had pH values of about 6.25 (Table 1). Ang and Haard (1985) proposed that the soft texture and poor water- holding capacity of the extremely well fed wild cod was

due to the low ultimate pH of this fish. This decline was thought to be due to a period of heavy feeding immediately after starvation. In accordance with this we have previously observed that a more severe degradation had occurred in the re-fed post-spawn cod than in the maturing fish after storage (Ofstad et a1 1996). The higher liquid loss of fed cod compared with that of wild cod is caused mainly by the low pH induced denaturation and shrinkage of the myofibres and thus widening of the intermyofibrillar spaces as observed in Fig 2. Dis- ruption of the Z-discs and swelling of the sarcoplasmic reticulum may have reinforced this process. As previously proposed, rupture of the sarcolemma and the extracellular collagenous layer may have further facilitated the outward movement of intracellular constituents (Ofstad et a1 1993).

The present study shows that the degree and time course of post mortem structural degradation are most pronounced in the muscle of fed fish, both cod and salmon, compared with wild cod (Figs 1, 2 and 4). The reason for this is not known, but may be related to the apparently faster and more severe swelling and rupture of the cellular membranous systems and the mitochondria observed in these low-pH muscles. It has been suggested that cathepsin L from lysosomes are the most probable enzymes responsible for the post mortem hydrolysing of the major muscle structure proteins, both myofibrillar and collagenous proteins (Mikami et a1 1987; Yamashita and Konagaya 1991, 1992). These authors also suggested that muscle autolysis could be enhanced if the muscle protein were subjected to denaturation resulting from exposure of the post rnortem muscle to low pHs and/or high temperatures. The lower muscle pH in the fed fish muscle are due to a rapid and/or extended post rnortem breakdown of a larger amount of glycogen in these muscles (Rustad 1992). The nutritional status of the fish at capture may thus be an important factor influencing post mortem deterioration and thus fish quality. Fish is normally consumed after heating. The relationship between post mortem structural changes and liquid loss upon heating of fed cod, wild cod and salmon will be further discussed in a subsequent publication.

CONCLUSIONS

Both fed cod and farmed salmon underwent more severe post mortem muscle degradation than wild cod, probably caused by lower-than normal muscle pH in the fed fish. Regardless of the post mortem degradation, salmon possessed much better liquid-holding properties than cod.

The better liquid-holding capacity of salmon muscle is mainly related to species-specific structural properties of the muscle. The myofibres of the salmon muscle were


denser, and fat, membrane remnants and an amorphous material filled the intra- and extracellular spaces. The denaturation characteristics of myosin, actin and a sarcoplasmic protein differed between salmon and cod, indicating species-specific diflerences, in particular in the stability of the myosin-actomyosin complex.

The severe liquid loss in coarsely chopped muscle of fed cod is mainly related to pH-induced denaturation and shrinkage of the myofibres. Disruption of the myofibrils and swelling of SR may have reinforced this process, as well as rupture of sarcolemma and the extracellular collagenous layers which may have further facilitated the outward movement of the intracellular constituents. A myopathic condition was observed in some of the muscle samples of fed cod. The differences in thermograms between wild and fed cod were also largely explained by the variations in pH.

The liquid-holding capacity of the raw fish seems to be dependent on two main factors: (i) genetic differences in the muscle protein; and (ii) on the post mortem muscle pH and the subsequent time-dependent muscle degradation.

ACKNOWLEDGEMENT

One of the authors (RO) particularly wishes to express her gratitude to Inge Karstensen, Tone Jakobsen, Helga-Marie Bye, Berit K Martinsen and Ina Storm for their assistance. The Norwegian Research Council and Nordic Academy for Advanced Study are warmly thanked for their financial support.

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