J Sci Food Agric 1996,72,337-347

Ultramicroscopical Structures and Liquid Loss in Heated Cod (Gadus morhua L) and Salmon (Salmo salar) Muscle Ragni Ofstad* Norwegian Institute of Fisheries and Aquaculture, Tromss, Norway

Siw Kidman and Anne-Marie Hemansson SIK, The Swedish Institute for Food Research, Gsteborg, Sweden (Received 30 June 1995; revised version received 23 February 1996; accepted 3 June 1996)

Abstract: This study was performed in order to assess the effect of heating in pre- and post-rigor muscle of fed cod, wild cod and farmed salmon harvested at differ- ent times of the year. The structural changes in muscle samples pre-heated from 5 to 60°C were qualitatively evaluated using both light and transmission electron microscopy techniques. The microstructural changes are discussed in relation to the liquid loss measured by a low-speed centrifugation test. The heat-induced structural changes varied between the fish tested, reflecting different degrees of post mortem degradation prior to heating, the muscle-pH and species-specific structural properties. The fed fish, both cod and salmon, underwent the most severe structural degradation. This reflected both the low muscle pH and the more severe post mortem degradation observed in these fish prior to heating, compared with the wild cod. Heating caused extensive shrinkage of the myofib- rils and hence, widened intermyofibrillar and extracellular spaces in both the fed cod and the salmon muscle. In the sample of wild cod muscle, the extracellular spaces were narrow and the myofibrils were closely packed. The difference in heat-induced liquid loss of the fed compared with the wild cod muscle coincides with their different structural features, as observed both by LM and TEM. The better liquid-holding properties of the salmon muscle than the cod muscle are attributed to the species-specific ultrastructural features as observed with TEM. In addition to the denser appearance of the salmon myofibres, it is suggested that both fat droplets and aggregated sarcoplasmic proteins filling the inter- myofibrillar and extracellular spaces are important in preventing release of liquid upon heating.

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

INTRODUCTION

Quality loss in stored fish muscle may take the form of development of unpleasant odours and flavours, exces- sively soft texture, loss of liquid-holding capacity and development of a dry, tough texture upon cooking. Changes in texture and ability to interact with water are mediated primarily through structural changes of the

* To whom correspondence should be addressed at: A/S Forma, R & D Dept, Sofienberggt 19, PO Box 6779, St Olavs plass, N-0130 Oslo, Norway.

muscle proteins (Offer et al 1989). Knowledge about relationships between microstructure and liquid-holding properties can thus be used both in order to choose raw materials and to optimise processing conditions and hence, lead to enhanced quality of the end product.

A number of studies have been conducted using microscopic techniques for detecting heat-induced changes in mammalian meat. Disappearance of the fine filamentous structure, shrinkage and fragmentation of the myofibrils, appearance of granulated sarcoplasmic proteins beneath the sarcolemma and gelatinising of the collagenous tissue are the most prominent changes

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

338 R Ofstud, S Kidman, A-M Hermansson

noted during heating (Cheng and Parrish 1976; Jones et a1 1977; Voyle 1981). The response to thermal treatment differed according to mammalian species, type of muscle and the degree of post mortem ageing prior to cooking (Schmidt and Parrish 1971; Leander et al 1980). For example, Abugroun et al (1985) found that cooking of pre-rigor beef muscle produced more severe myofibrillar shortening than cooking of post mortem muscle.

Relatively few reports have dealt with heat-induced structural changes in fish muscle. Schaller and Powrie (1972) found, by scanning electron microscopy, that the visible damage to the myofibres was more apparent in heat-treated trout compared with beef and chicken. Corresponding observations were found in heat- processed tuna muscle by Lampila and Brown (1986). Using light microscopy, we have previously shown that there is a relationship between the liquid-holding capac- ity of both the coarsely chopped cod and salmon muscle and the tissue specific structural changes which occurred during heating. At low temperatures (20- 40"C), concomitantly with increasing liquid loss, the pericellular connective tissue underwent the most strik- ing change. The maximum liquid loss was attained when the muscle shrank due to denaturation of myosin at 45-50°C. At elevated temperatures, the liquid loss decreased when extracellular, granulated material became visible (Ofstad et a1 1993). Cod lost twice as much water as salmon upon heating. In addition to the nature of the raw material, salt concentration, pH, heating temperature and interaction effects between these factors gave rise to considerable structural alter- ations and consequently changes in the liquid-holding properties of both finely comminuted cod and salmon muscle (Ofstad et a1 1995, 1996a). Kanoh et a1 (1988) and Hatae et al(l990) suggested that drip loss and tex- tural differences between five fish species and the dark and the white muscle of yellowfin tuna when heated, were due to the different amount of sarcoplasmic pro- teins and/or differences in the fibre diameter.

Both cod and salmon are highly valued as food. Depleting stocks of wild cod will increase the demand for farmed cod. In a preceding paper we compared the liquid-holding properties and post rnortem ultrastruc- tural changes in cod and salmon muscle. Here we found that the higher liquid-holding capacity of the salmon muscle was related to species-specific structural features and better stability properties of the muscle proteins. The myofibrils of the salmon muscle were denser and intra- and extracellular spaces were filled by fat and a granulated material. On the other hand, the differences in liquid loss of wild and fed cod muscle were largely explained by the differences in pH. The severe liquid loss of fed cod was due to a low pH-induced shrinkage of the myofibrils as well as post mortem degradation of the muscle which may have further facilitated the release of liquid (Ofstad et al 1996b). The observed dif- ferences in muscle structures, degree of deterioration

and pH of the muscle in the raw condition, were sug- gested to have a profound effect upon the cooked product.

The present paper describes the effect of heating on the post mortem structural changes in cod and salmon muscle in more detail. As in our previous publication samples both from fed cod, wild cod and salmon har- vested in different seasons are investigated 3 h and 48 h post mortem. In the first part of this paper we describe the microstructural changes and then focus on how these contribute to the liquid loss. The structural changes in the heated muscle samples were qualitatively evaluated by using both light and transmission electron microscopical techniques.

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). The wild and the fed cod used were harvested three times a year: in migrating, mature condition (WMC, FMC, respectively); in summer (WSC, FSC, respectively) and in autumn (WAC, FAC, respectively). The salmon were harvested twice: in spring being silver-bright (SO) and in autumn being moderately mature (SM). Four to six fish were used in each trial. Half the fish were filleted within 3 h, ie before appearance of rigor mortis, and immediately analysed. The others were kept in ice for 48 h until the resolution of rigor mortis and the muscle had softened, and then filleted before being analysed. All operations were carried out at 4°C. Experimental procedures, body index parameters and proximate com- position of the raw material have previously been described in greater detail (Ofstad et a1 1996b).

Preparation of samples for microscopy (LM and TEM)

Blocks of whole muscle weighing approximately 15 g were taken from the fillets near the dorsal fin of each of the three fish at 3 h and 48 h post rnortem, respectively, and heated to 30, 45 and 60°C in parallel with the samples for liquid loss measurements as described by Ofstad et a1 (1993). After cooling (30 min, lOOC) the muscle specimens were fixed in pre-cooled (lOOC) 25/10 g litre- ' glutaraldehyde/formaldehyde with 2 g kg- ' glucose in Ringer's buffer (pH 6.8) for 24 h at 4°C. Small pieces of muscle fibre were cut with a razor blade under a dissecting microscope and rinsed in pre- cooled buffer. To assess the microscopic changes both light and transmission electron microscopy were used on all -samples. Light microscopy was performed on

Ultramicroscopical structures and liquid loss in heated fish muscle 339

plastic sections according to the procedure described by Ofstad et a1 (1993). Sections were viewed and photogra- phed using a Nikon Macrophot Fx microscope. The samples for TEM were post-fixed in osmium tetraoxide, dehydrated and embedded in Epon/Araldite, sectioned and stained according to Ofstad et a1 (1996a). Sections were viewed and photographed using a TEM, lOOCXII (JeoI Ltd, Tokyo, Japan). Ten blocks were prepared from each biopsy and at least two blocks were sectioned either longitudinally or transversally to the fibre direc- tion. On each test occasion at least two fish were exam- ined. The few micrographs presented here have been chosen out of many and illustrate the most typical heat- induced sample features.

Liquid-holding capacity (LHC)

The liquid loss was measured on coarsely chopped fillets pooled from two to three fish in each trial. The temperature of the minces after chopping was 4-5°C. The liquid loss, measured as the percent of weight rel- eased per 15 g sample, was determined according to the net test described by Hermansson (1986) with the modi- fication that a moistened filter paper was placed on the top of the net. The samples were either directly centri- fuged at 5°C or heated to 30, 45 and 60°C at a rate of 1°C min-', held at the temperature for 10 min and then cooled (WC, 30 min) prior to centrifugation (210 x g for 15 min) according to the procedure described by Ofstad et a1 (1993). Mean values were calculated from two trials of six replicates at each temperature.

RESULTS A N D DISCUSSION

Microstructure

The microstructure was characterized at different levels by using both light microscopy (LM) and transmission electron microscopy (TEM). LM, at low magnification, was used to investigate the gross structural changes, whereas TEM at high magnification was used to dis- cover ultrastructural changes both in the myofibrils and in the connective tissue. Heat-induced changes in cod and salmon muscle, as observed using LM, have pre- viously been reported (Ofstad et a1 1993). Therefore, emphasis here is placed on the TEM investigations.

Samples were examined for changes after being pre- heated to 30,45 and 60°C 3 h and 48 h post mortem and compared with the unheated muscle. The structural observations in the unheated samples have been pre- sented in a previously published paper (Ofstad et a1 1996b). However, the main traits as observed, by TEM, 3 h after death and after 48 h ice storage are listed in Table 1. The structure of salmon muscle differed from that of cod in the following manner. The endomysial sheath, which was wider in the salmon, comprised dis-

tinct bundles of collagen fibres, and the myofibrils looked generally denser and the Z-discs appeared as much darker lines than in the cod. Fat droplets and an amorphous material were seen both in intra- and extra- cellular locations. Apart from the structural differences between cod and salmon, the predominant change in the muscles during storage was a gradual swelling and disintegration of membranous structures and break- down of the endomysium. The intermyofibrillar space widened, the Z-discs of the myofilaments disintegrated and the thin filaments of the I-band split up. The degree of structural degradation was more pronounced in the fed fish than in the wild cod, fed cod more than salmon.

The ultrastructural post mortem changes were focal in nature, to the extent that changes were dramatic in one area of muscle while other areas of the same muscle looked much less affected. Correspondingly, structural changes occurring in the muscle during heating varied from one area of the muscle to another.

Light microscopy of heated muscle Structural changes in whole muscle heated 3 h and 48 h after death were studied by light microscopy as sections solidified by plastic embedding. Although, the overall pattern of the changes as a function of temperature from 5 to 60°C are as previously described (Ofstad et a1 1993), there were some notable differences between the cod and the salmon muscle, and between the wild cod and the fed fish, both cod and salmon muscle.

Figure 1 shows micrographs of transversely sectioned muscle of cod and salmon heated to 30°C. In cod, dark wavy lines are clearly visible in the 3 h post mortern heated muscle (Fig la), but not in the 48 h heated muscle (Fig lb). Similar looking bands referred to as contraction bands were observed in transverse sections of electrically stimulated meat (Will et a1 1980). Myofib- rillar contraction was more pronounced in cod than in salmon muscle. Contraction bands are hardly visible in the 3 h-heated sample of salmon (Fig lc). Abugroun et a1 (1985) suggested that the shortening of pre-rigor heated beef muscle was partly an active contraction, caused by heat stimulation or membrat 1 destruction releasing Ca2+ ions initiating rigor onset. Offer et a1 (1989) proposed that the heat-induced myofibrillar shrinkage was related to the species-specific rigor- development. Rigor onset depends on fish species, tem- perature of pre-rigor storage, handling before slaughter, slaughtering stress and the biological status of the fish (Love 1988; Azam et a1 1990). In accordance with our results it has been observed that during rigor cod fillets shrink to about 25% and salmon fillets to about 15% of their original length (Ssrensen, unpublished results). This may indicate that the myofibrillar shortening due to rigor onset proceeds at a slower rate and/or to a lesser extent in salmon muscle than in cod muscle.

Figure 2 shows light micrographs of transversally sec- tioned muscle of wild cod, fed cod and salmon heated to

TAB

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en

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serv

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from

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ptur

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split

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Ultramicroscopical structures and liquid loss in heated fish muscle

Fig 1. Transverse sections of plastic embedded muscle of wild cod heated to 30°C; (a) 3 h after death; (b) after 48 h storage in ice and (c) of salmon heated to 30T, 3 h after death. Note the arrowheaded contraction bands in the pre-rigor heated

cod muscle.

60°C. In the sample of wild cod (Fig 2a), the extracellu- lar spaces are narrow and the inner myofibrils which appear as small spots, are only slightly separated due to the transverse shrinkage. In fed cod (Fig 2b) and salmon (Fig 2c) the extracellular spaces containing granulated

341

Fig 2. Transverse sections of plastic embedded muscle of ice- stored (48 h) and heated (60°C) muscle of (a) wild cod; (b) fed cod and (c) salmon. Note the wide extracellular spaces with granulated material and the intracellular cavities in the fed

cod and salmon compared to the wild cod muscle.

material are wider, and the muscle cells look more rup- tured and have intracellular cavities in accordance with previously reported observations (Ofstad et a1 1993). The structural differences between these fed fish species and the wild cod probably reflect the lower muscle pH

342 R Ofstad, S Kidman, A-M Hermansson

value and the more severe post mortem degradation observed in both fed cod and salmon, compared with the wild cod (Table 1). A similar structural alteration was observed to occur in the post-spawn, heavily re-fed wild cod upon heating (Ofstad et a1 1996a). This empha- sises that the nutritional status of the fish at capture is a major factor influencing its rate of degradation, and hence the quality of the fish.

Transmission electron microscopy of heated muscle The higher resolution provided by TEM, compared with LM, made it possible to examine the structures of the heated samples in greater detail. TEM photomicrog- raphs presenting the most typical structural features of wild cod, fed cod and salmon ice-stored for 48 h prior to heating are shown in Figs 3-6. In muscle fibres, heating from 5 to 60°C causes progressive shrinkage and disintegration of both the myofibres and the peri- cellular connective tissue. Similar changes are described by others to occur in mammalian meat (Schmidt and Parrish 1971; Cheng and Parrish 1976; Jones et a1 1977; Leander et a1 1980; Voyle 1981). Muscle heated 3 h post mortem (not shown) sustained greater loss of structural integrity than that heated 48 h post mortem. Thick filaments were extensively fractured. Similar structural degradation has been reported to occur in beef muscle heated pre-rigor (Silva et a1 1993). Owing to the lower thermal stability of the fish muscle proteins, the structural changes occurred at lower temperatures than in the mammalian meat. At 60"C, the effect of heat on the ice-stored fish muscle is quite marked (Figs 3-5). This is in accordance with the observations made by Schaller and Powrie (1972), comparing thermally pro- cessed trout with chicken and beef muscle using scan- ning electron microscopy. Although the heat-induced changes followed a general pattern, structural differ- ences among the fish could easily be discerned.

Wild cod. Images of longitudinal sectioned muscle of maturing, summer and autumn cod are shown in Figs 3a, b and c, respectively. The myofibrils, which were tightly packed and well preserved prior to heating (Table l), are still closely packed after heating and sur- rounded by a rather well-structured sacroplasmic reti- culum (SR). The triad structure at the level of the Z-disc; ie two terminal cisternae (Tc) embracing the transverse tubule (Tt) can easily be discerned (indicated in Fig 3b). The thick filaments in the A-band appear indistinct, the H-zone is absent and the M-line material is lacking in all the samples. This is mainly due to the denaturation of myosin and probably some of the anchoring proteins as well. The main transition tem- perature of myosin of whole cod muscle, as measured by differential scanning calorimetry, is about 45"C, whereas actin denatures at approximately 73°C (Ofstad et a1 1996b).

The I-band and the Z-disc are absent in the mature and autumn cod (Figs 3a and c), but are rather distinct

in the summer cod (Fig 3b). Corresponding structural inequalities were not observed among the unheated samples (Ofstad et a1 1996b). This may indicate that the degree of sarcomere shortening upon heating varies between these samples. The average pH of summer cod was somewhat higher (6-9) than the pH of the mature (6.7) and the autumn cod (6.6). According to Fernandez et a1 (1994), the sarcomere length of pork meat is depen- dent upon the ultimate pH, even within minor pH differ- ences. Some of the observed differences of heat-induced structural changes in the wild cod samples may, thus be explained by the different pH values of these samples.

Fed cod. TEM (Fig 4) shows greater loss of structural integrity of the heated fed cod muscle compared with the wild cod muscle. This was expected since the fed cod muscle also showed a more severe post mortem structur- al degradation prior to heating (Table 1). Heating induced extensive myofibrillar shrinkage widening the intermyofibrillar spaces (Fig 4a). The shortening of the sacomeres is evidenced by the absence of both the H-zones and the I-bands with disintegration of the Z- discs, as indicated by areas of very low density in Fig 4a. Higher magnification (Fig 4b) reveals severe aggre- gation of the thick filaments, which emerge as obscure electron dense 'wisps' in the cross-sectioned muscle. The thin filaments are still visible as distinct points at 60°C, due to the higher T,,, (73°C) of this protein. A pH closer to the iso-electric point of the myofibrillar pro- teins will increase the protein-protein interaction thus increasing the transverse shrinkage upon heating (Hermansson 1986; Ofstad et a1 1996a). The average pH values for wild and fed cod muscle were 6.7 and 6.3, respectively. The observed structural differences between the wild and the fed cod when heated may therefore be explained by the different pH values of the samples.

In the fed cod, the sarcolemma was ruptured and the pericellular collagenous layer had disintegrated prior to heating (Table 1). This was even more evident after heating. In Fig 4c, a mixture of membrane remnants, collagen fibres and electron dense aggregates are seen as a string of pearls along the periphery of the cell as well as in the extracellular location. It has been suggested that the rupture of the sarcolemma and the extracellular collagenous layer, may facilitate the outward movement of intracellular constituents and enhance the degree of extracellular granulation (Lampila and Brown 1986; Ofstad et a1 1993).

Salmon. In salmon muscle (Fig 5), the fibres had shrunk transversally to the fibre axis and the intermyofibrillar spaces had widened in a way similar to that observed in the fed cod (Fig 4). This is in accordance with the low pH (6.13) of the salmon muscle. Corresponding to the structural observations on the unheated samples (Table l), the A-bands have a denser appearance and the I-bands are wider in the salmon (Fig 5a) than in the cod

Ultramicroscopical structures and liquid loss in heated fish muscle 343

Fig 4. TEM micrographs of ice-stored (48 h) fed cod muscle heated at 60°C. (a) Longitudinal section; (b) transverse section at higher magnification and (c) transverse section of the per- iphery of the cell. In Fig 4a the A-band, M-line and Z-disc are indicated. Note the absence of H-zones and I-bands and the wide intermyofibrilla spaces. In Fig 4b the single arrowheads point to aggregated thick filaments and the double arrow- heads point to thin filaments. In Fig 4c membrane remnants (Mr) and collagen fibres (C) are indicated. The arrowheads point to the electron-dense granules, originating from aggre-

gated proteins.

Fig 3. TEM micrographs of longitudinal sectioned wild cod muscle stored in ice (48 h) and heated at 60°C; (a) migrating mature cod, WMC; (b) summer cod, WSC and (c) autumn cod, WAC. Terminal cisternae (*) embracing the arrowheaded transverse tubule are indicated in Fig 3b. The I-band and the Z-disc are absent in the WMC and WAC, but are rather dis-

tinct in the WSC.

344 R Ofstad, S Kidman, A-M Hermansson

Fig 5. TEM micrographs of longitudinally sectioned salmon muscle stored in ice for 48 h and heated at 60°C (a, b). Note the fat droplet (F) between the myofibres. Note the filamen- tous material of melted membranous structures mixed with electron-dense aggregated proteins surrounding the fat droplet. At higher magnification in Fig 5b, it looks as if the

melted membranes coalesce with the fat droplet (F).

myofibres (Fig 4a). The Z-discs in salmon which had thickened upon heating, are still visible. The wide I-bands and visible Z-discs are in accordance with the lack of contraction bands observed in Fig 1. This further indicates that upon heating the sarcomere short- ening occurred to a lesser extend in salmon than in cod muscle.

The intermyofibrillar spaces of the raw salmon muscle were completely filled with SR-remnants and an amorphous material probably consisting of sarco- plasmic proteins (Table 1). At 60°C (Fig 5a) these mem- branous structures can be seen as a filamentous material mixed with electron-dense proteinaceous aggregates. The filamentous material surrounds the fat droplet in Fig 5a. At higher magnification shown in

Fig 5b, it looks as if these filaments coalesce with the fat droplet, thus probably adhering the fat droplet to the myofibrils. Howgate (1979) suggested that remnants of sarcoplasmic reticulum, as it degraded during frozen storage, were acting as a glue cementing the fibrils together thus producing a tougher product.

The micrographs in Fig 6 show structural alterations in the extracellular area of the salmon muscle as a func- tion of temperature when heating from 5 to 70°C. In the unheated sample (Fig 6a), the extracellular spaces are filled with bundles of collagen fibres and an amorphous material, probably sarcoplasmic proteins. At 45°C (Fig 6b), the collagen fibres which appear thicker, have swollen and lost their fibrillar structure. The melting and shrinkage temperature of collagen in salmon are to our knowledge not known. However, the thermal con- traction of cod collagen fibres is known to take place between 40 and 45°C (Sikorski et al 1984). Hence, the flocculent aggregates seen between the partly denatured collagen fibres are probably a mixture of gelatine and sarcoplasmic proteins. The sarcoplasma proteins of salmon denature between 45 and 70°C (Ofstad et a1 1996b). Accordingly, at 60°C (Fig 6c), the small aggre- gates have coarsened and are seen as electron dense granulates accumulated on the remaining collagen strands. By the use of specific protein staining, we have previously shown, that the granulated material prob- ably originated from sarcoplasmic and/or myofibrillar proteins (Ofstad et a1 1993). Lampila and Brown (1986) observed similar granules in the interstices between the shrunken muscle fibre and the endomysium of heated tuna muscle. They suggested it was a combination of heat coagulated sarcoplasmic proteins and gelatine. When heated to 70°C (Fig 6d), it can be seen that thin gelatine threads stabilise the total structure by forming a mesh in-between the granules. Similar heat-induced structures were observed in the extracellular location of the cod muscle (Fig 4c). However, there seems to be a lesser amount of the granulated material in the cod than in the salmon muscle (Fig 2).

.

Structure and liquid loss

Released liquid was measured from coarsely chopped fillets by the net test. Both the chopping of muscle and the centripetal force used in the net test will enhance the liquid loss. However, Hermansson and Strom (1989) have shown that the moisture losses which take place spontaneously on heat treatment and cooling of meat model systems afe smaller, but correspond to the mois- ture loss after the mild centrifugation included in the net test. Figure 7 shows the liquid loss measured by the net test 3 h and 48 h post mortem for fed cod (FC), wild cod (WC) and salmon (S) as a function of heating tem- perature. Salmon possesses much better liquid-holding properties than cod muscle, and the liquid loss is higher

Ultramicroscopical structures and liquid loss in heated fish muscle 345

Fig 6. TEM micrographs showing the endomysial layer of a transversally sectioned salmon muscle stored in ice for 48 h; (a) unheated muscle; (b) heated at 45"C, (c) heated at 60°C and (d) at higher magnification heated at 70°C. In the unheated sample (Fig 6a), sarcolemma (Sl) and collagen fibres (C) are indicated. During heating the sarcoplasmic proteins aggregate forming electron dense granules accumulating in partly denatured collagen strands and gelatine threads. The arrowheads in Fig 6b point to partly

denatured collagen fibres, in Fig 6d at the thin gelatine threads.

for the fed cod than for the wild cod, regardless of treat- ment. The liquid loss in salmon refers to both water and fat loss. Both the pattern and the levels of the heat- induced release of liquid are in accordance with earlier published results (Aitken and Conell 1979; Ofstad et al 1993).

The higher liquid loss of the 3 h samples than of the 48 h unheated samples is attributed to rigor-induced shrinkage of the myofibres probably caused by mecha- nical stress during preparation (Ofstad et al 1996b). The rigor-induced shrinkage which was reinforced during heating (Fig 1) tore the muscle fibres at each side of the contraction bands and caused extensive loss of struc- tural integrity. This may explain the substantial liquid loss of all the 3 h samples even at the low heating tem- peratures.

At 60°C, as observed with LM, the microstructure of fed cod and salmon muscle resembled each other. The extracellular spaces were wider and these muscle cells were more fractured with intracellular cavities com- pared with the wild cod (Fig 2). TEM revealed shrunk- en myofibres and wide intermyofibrillar spaces both in the fed cod (Fig 4) and in the salmon muscle (Fig 5), whereas in the wild cod muscle (Fig 3) the myofibrils were tightly packed. Offer et al (1989) explained the variation in the amount of water held by meat, by changes in the volume of the myofibrils. The loss of water occurs by expulsion from the myofibrils as they shrink laterally. When the filaments get closer together a compensating movement of water from intra- to extracellular location occurs. The capillary forces are reduced and the expelled water being less firmly held

346 R Ofstad, S Kidman, A-M Hermansson

45

40

35

8 30 m 8 25 - 0 20 3 U 3 15

10

5: 0 5 30 45 60

Temperature "C Fig 7. Average liquid loss (% by weight) as a function of heating temperature of coarsely chopped fish muscle of: +, fed cod; ., wild cod and; 0 , salmon measured by the net test 3 h (dotted lines, open symbols) and 48 h (whole lines, filled

symbols) after death.

can be pressed out more easily upon centrifugation. The higher heat-induced liquid loss in the fed than in the wild cod muscle (Fig 7) coincides with the wider inter- myofibrillar and extracellular spaces observed in the former when heated. Although heated salmon muscle showed structural features similar to heated fed cod muscle, the liquid-holding capacity of salmon was much better than that of both the fed and the wild cod. In the raw condition, species-specific structural features and better stability of the muscle proteins may explain the higher liquid-holding capacity of the salmon than of the cod muscle. Similar structural differences such as a denser appearance of the A-bands, wider I-bands and more distinct Z-discs were observed in the heated muscles as well. Such species-specific structural proper- ties may also explain some of the differences in liquid- holding properties between the salmon and cod muscle when heated. Furthermore, intramuscular fat and a higher proportion of sarcoplasmic proteins in salmon muscle, may be of importance in determining its higher liquid-holding capacity compared with the cod muscle. The fat droplets (Fig 5b) may plug the intermyofibrillar capillary spaces through interactions with the myofibril- lar proteins, and thereby prevent fat as well as water from being released during centrifugation. A similar effect is probably caused by the intra- and extracellular protein aggregates (Ofstad et a1 1993). The combination of gelatine network structures and aggregated protein structures (Figs 6c and d) may in addition to binding water prevent liquid losses by plugging both the intra- and extracellular capillary spaces.

Seasonal variation TEM of the heated wild cod muscle (Fig 3) exposed some structural differences between the fish harvested in

different seasons. Wild summer cod with visible I-band and Z-disc had probably undergone less sarcomere shortening upon heating. The liquid loss in wild mature, summer and autumn cod heated to 60°C was approx- imately 28, 22 and 34%, respectively. Honikel et a1 (1986) found that during storage of meat the drip loss was inversely related to the length of the sarcomere. Some of the observed differences among the wild cods in their ability to hold water may therefore be explained by the different degree of sarcomere-shortening upon heating. In our preceding paper (Ofstad et al 1996b) we showed that the variations among the differential scan- ning calorimetry thermograms of these cod samples were explained by the pH. Hence, among the tested cod samples, the different degree of fibrillar shrinkage, both transversally and longitudinally to the fibre axis and the subsequent liquid loss is probably governed by the different post mortem muscle pH values.

Only minor differences in released liquid were observed in the fed cod and salmon harvested at differ- ent times. Maturing salmon released somewhat more liquid (26%) than the ordinary salmon (20%). The TEM micrographs (not shown) revealed no striking structural differences between these fish. However, different amounts of sarcoplasmic proteins together with minor structural differences may be of importance (Ofstad et a1 (1996b).

Fish and fish products exhibit a wide range of func- tional properties, even before any effects of processing are considered. The structure of fish flesh is complicated and the interrelationships between all the components present are, as yet, obscure. Undoubtedly, the described differences in structure do have some influence upon the liquid-holding properties as well as the texture. Even within a species, the functional properties may vary due to a number of biological factors. One important factor is the nutritional status of the fish at capture influencing the post mortem pH and the subsequent time-dependent muscle degradation.

CONCLUSIONS

As seen by TEM, heating from 5 to 60°C caused pro- gressive shrinkage and disintegration of the myofibres. The heat-induced changes followed a general pattern, but the micrographs indicated that the degree of shrink- age varied between the fish groups.

The low pH and the severe post mortem degradation of the fed cod muscle caused severe myofibrillar shrink- age and extensive rupture of cells, enhancing the liquid loss upon heating.

In the wild cod muscle, which had a much higher liquid-holding capacity, the myofibrils were closely packed, whereas the degree of sarcomere shortening

Ultramicroscopical structures and liquid loss in heated fish muscle 347

varied due to season and/or minor pH differences. A lesser sarcomere shortening favoured a higher liquid- holding ability.

Salmon muscle possessed better liquid-holding properties than both the fed and the wild cod muscle regardless of the treatment and the degree of heat- induced myofibrillar shrinkage. Hence, species-specific ultrastructural features may, in addition to the intramus- cular fat and higher proportion of sarcoplasmic pro- teins, contribute to its better liquid-holding capacity.

ACKNOWLEDGEMENTS

One of the authors (RO) particularly wishes to express her gratitude to Inge Karstensen, Tone Jakobsen, Helga-Marie Bye and Ina Storm for their assistance and to Ragnar L Olsen for valuable discussions during the preparation of this manuscript. The Norwegian Research Council and Nordic Academy for Advanced Study are warmly thanked for their financial support.

REFERENCES

Abugroun H A, Forrest J C, Aberle E D, Judge M D 1985 Shortening and tenderness of pre-rigor heated beef: Part 2. Effect of heating rate on muscles of electrically stimulated carcasses. Meat Sci 14 15-28.

Aitken A, Connell J J 1979 Fish. In: Effect of Heating on Foodstuffs, ed Priestley R J. Applied Science, London, UK,

Azam K, Mackie I M, Smith J 1990 Effect of stunning method on the time of onset, duration and resolution of rigor in rainbow trout (Salmo gairdneri) as measured by visual observation and analyses for lactic acid, nucleotide- degradation products and glycogen. In: Proc IIR Con- ference on Chilling and Freezing of New Fish Products (Vol 3). International Institute of Refrigeration, Paris, France, pp

Cheng C S , Parrish Jr F C 1976 Scanning electron microscopy of bovine muscle: Effect of heating on ultrastructure. J Food Sci 41 1449-1454.

Fernandez X, Forslid A, Tornberg E 1994 The effect of high post mortem temperature on the development of pale, soft and exudative pork: Interaction with ultimate pH. Meat Sci

Hatae K, Yoshimatsu F, Matsumoto J J 1990 Role of muscle fibres in contributing firmness of cooked fish. J Food Sci 55

Hermansson A M 1986 Water- and fat holding. In: Functional Properties of Food Macromolecules, ed Mitchell J R & Ledward D A. Elsevier, London, UK, pp 273-314.

Hermansson A M, Strom S 1989 Presalting of meat raw materials. In: Proc 35th Znt Congress of Meat Science and Technology, (Vol3) Copenhagen, Denmark, pp 776-780.

pp 219-254.

249-254.

37 133-147.

693-696.

Honikel K 0, Kim C J, Hamm R 1986 Sarcomere shortening of prerigor muscle and its influence on drip loss. Meat Sci

Howgate P 1979 Fish. In: Food Microscopy, ed Vaughan J. Academic Press, London, UK, pp 273-314.

Jones S B, Carroll R J, Cavanaugh J R 1977 Structural changes in heated bovine muscles : A scanning electron microscope study. J Food Sci 42 125-131.

Kanoh S, Polo J M A, Kariya Y, Kaneko T, Watabe S , Hash- imoto K 1988 Heat-induced textural and histological changes of ordinary and dark muscle of yellowfin tuna. J Food Sci 53 673-678.

Lampila L E, Brown W D 1986 Changes in the microstruc- ture of skipjack tuna during frozen storage and heat treat- ment. Food Microstr 5 25-31.

Leander R C, Hedrick H B, Brown M F, White J A 1980 Comparison of structural changes in bovine longissimus and semitendinosus muscles during cooking. J Food Sci 45

Love R M 1988 The Food Fishes: Their Intrinsic Variation and Practical Implications. Farrand Press, London, UK.

Offer G, Knight P, Jeacocke R, Almond R, Cousins T, Elsey J, Parsons N, Sharp A, Starr R, Purslow P 1989 The structur- al basis of the water-holding, appearance and toughness of meat and meat products. Food Microstruct 8 151-170.

Ofstad R, Kidman S, Myklebust R, Hermansson A M 1993 Liquid holding capacity and structural changes during heating of fish muscle; cod (Gadus morhua L.) and salmon (Salmo salur). Food Struct 12 163-174.

Ofstad R, Kidman S, Myklebust R, Olsen R L, Hermansson A M 1995 Liquid-holding capacity and structural changes in comminuted salmon (Salmo salar) muscle as influenced by pH, salt and temperature. Food Sci Techno! 28 329-339.

Ofstad R, Kidman S, Myklebust R, Olsen R L, Hermansson A M 1996a Factors influencing liquid-holding capacity and structural changes during heating of comminuted cod (Gadus morhua L.) muscle. Food Sci Technol 29 173-183.

Ofstad R, Egelandsdal B, Kidman S, Myklebust R, Olsen R L, Hermansson A M 1996b Liquid loss as effected by post mortem ultrastructural changes in fish muscle; cod (Gadus morhua L.) and salmon (Salmo salar). J Sci Food Agric 71

Schaller D K, Powrie W D 1972 Scanning electron micros- copy of heated beef, chicken and rainbow trout muscles. J Inst Can Sci Technol Aliment 5 184-190.

Schmidt J G, Parrish Jr F C 1971 Molecular properties of postmortal muscle. 10. Effect of internal temperature and carcass maturity on structure of bovine lonissimus. J Food Sci 36 110-1 19.

Sikorski Z E, Scott D N, Buisson D H 1984 The role of colla- gen in the quality and processing of fish. Crit Rev Fd Sci Nutr 20 301-343.

Silva T J P, Orcutt M W, Forrest J C, Bracker C E, Judge M D 1993 Effect of heating rate on shortening, ultrastructure and fracture behaviour of prerigor beef muscle. Meat Sci 33

Voyle C A 1981 Scanning electron microscopy in meat science. Food Microstruc 3 405-413.

Will P A, Ownby C L, Henrickson R L 1980 Ultrastructural post mortem changes in electrically stimulated bovine muscle. J Food Sci 45 21-25, 34.

16 267-282.

1-6, 12.

301-312.

1-24.