Structural changes in marinated fillets of gilthead sea bream (Sparus aurata L.) during vacuum storage at refrigerated temperature

Fillets of Sparus aurata were marinated in a mixture of salt (5.5%) and citric acid (48.9 g/kg) for 1 h, then vacuum packed and refrigerated for 1, 5, 16, and 21 d. Structural and ultrastructural parameters were evaluated in 6 specimens per time period. At day 1, the preservative solution appeared among the muscle fibers, thus increasing the interfibrillar spaces; cytoplasmic organelles were swollen and the sarcolemma-endomysium appeared very altered and electron-dense. At 5 to 10 d, the marinade solution had penetrated into the muscle fibers. The subsarcolemmal and intermyofibrillar spaces were dilated and granular lines of electron-dense material appeared among the fibers. From 16 d, broad interfibrillar spaces were occupied by granular material derived from denatured sarcolemma-endomysium and denatured myofibrils. Sarcomeres were already altered from initial stages, mainly at the I-band level. The Z-line also appeared disrupted. Such alterations were more severe from 10 d on, such that most of the sarcomeres showed disintegration of myofilaments.     

Muscle tissue structure and flesh texture in gilthead sea bream, Sparus aurata L., fillets preserved by refrigeration and by vacuum packaging

Fillets were analysed from 66 gilthead sea bream specimens (Sparus aurata L). Thirty-six fillets were refrigerated (2 °C). The rest of the fillets were vacuum-packed and refrigerated. Muscle and sensorial parameters were evaluated for 22 days post-mortem.Textural parameters were higher in early stages. The refrigerated fillets had the lowest values. Most of textural parameters showed a negative correlation with the detachments among fibres in both groups.Ultrastructural results in vacuum-packed fillets showed that sarcolemma-endomysium was gradually disrupted, with almost a complete loss at 22 days. Initially, the detachments of myofibrils from the sarcolemma-endomysium were scarce. Mitochondria and sarcoplasmic reticulum were swollen from the first stages onwards. From 16 days onwards, the intra-cytoplasmic organelles were significantly altered and the smooth reticulum appeared hypertrophied, indicating an increase of the autophagic mechanisms. Sarcomeres were gradually altered, mainly at the I-band level, which showed a loss of actin filaments and Z-line disruptions from 12 to 16 days onwards.In non-vacuum fillets the muscle tissue was already markedly altered from the early stages. From five days onwards, the detachments of myofibrils from the sarcolemma were frequent and the hypertrophied smooth reticulum and the degraded lysosomes were already observed.     

Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels

The structure of muscle injected with specific cysteine protease inhibitors was examined to determine whether inhibitors cause denaturation and the degradation post-mortem of myofibrillar proteins was followed using SDS electrophoresis. Given the central role of calcium in theories of tenderisation the level of free calcium was measured during the early post-mortem period. The protease enzyme inhibitor E-64 was injected into the m. longissimus et thoracis lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). Muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). Muscle samples were selected from eight portions of the LTL (1-day post-mortem, from six different carcasses) for examination by transmission electron microscopy. Matching light images of myofibrils were obtained after determination of myofibrillar fragmentation. Free calcium concentration was determined for all samples (n=191) using an ion selective electrode excluding those 'at death'. Light images of myofibrils from treated samples showed normal striations and no evidence of denaturation or aggregation compared to control samples. This also applied to the samples processed for examination by electron microscopy. Appearance of the 30-kDa subunit increased with time (P<0.001) post-mortem. The interaction between ageing and stimulation had an effect (P<0.001) on the amount of a protein designated M1. The amount of M1 measured pre-rigor was greater for stimulated muscle, but the rate of decline was also greater through to day 2 post-mortem. Proteolysis was very rapid in the first 24 h post-mortem in ovine muscle. Ageing had an effect (P<0.001) on the free calcium concentration, which increased as muscle aged. As a covariate pH also had an effect (P< 0.05). Based on a non-linear model when the concentration of free calcium reached a plateau (～110 μM) the predicted pH was 5.5 (ultimate). From the qualitative observation of images and the levels of free calcium in injected muscle there is no support for the view that the inhibitors bind to sarcomere proteins, occupying sites to which calcium might bind. The levels of free calcium do not provide support for the view that m-calpain has a role in post-mortem tenderisation, but do suggest along with results of protein degradation that activation of μ-calpain is likely to occur before the pH drops to 6.2-6.1.     

Structural and ultrastructural changes of full-cycle cultured Pacific bluefin tuna (Thunnus orientalis) muscle slices during chilled storage

Background: This study examined the structural and ultrastructural changes of dorsal and ventral muscle tissues of full‐cycle cultured Pacific bluefin tuna (PBT), Thunnus orientalis Temminck & Schlegel 1844, cut into slices simulating sashimi and placed in chilled storage for varying periods. Structural and ultrastructural changes were determined in order to understand the physical texture by breaking strength measurement. Results: Progressive deterioration of myofibril structure was observed during chilled storage (4 °C) of PBT muscle slices over 5 days post mortem . Muscle degradation included detachment between myofibres, detachment of the plasmalemma, disruption of mitochondria, loss of Z‐line density and alignment, cementation of myofibrils, loss of the hexagonal arrangement of thick versus thin myofilaments and migration of subsarcolemmal nuclei to intermyofibrillar spaces. Conclusion: Loss of myofibre‐myofibre adhesion, detachment of the plasmalemma and disruption of other components did not lower the breaking strength of PBT muscle. This provides evidence that the muscle breaking strength of PBT is not only associated with the detachment of myofibres or detachment of the plasmalemma. Other factors that produce cement‐like substances, such as cementation of the myofibrillar components and degradation of the sarcoplasmic reticulum, may also increase breaking strength. Copyright © 2011 Society of Chemical Industry     

Post-exsanguination Infusion of Ovine Carcasses: Effect on Tenderness Indicators and Muscle Microstructure

Lambs were assigned to 3 treatment groups: control (Ctr), infused with 10% volume (by wt) of a tenderizing blend (NCa), and NCa plus 0.015M CaCl₂ (WCa). Compared to Ctr and WCa, NCa-treated samples had lower shear force values (P < 0.05) and higher percent change in myofibrillar fragmentation index (P < 0.05). SDS-PAGE of infused samples revealed the appearance at 24 hr postmortem of 22-30 kd protein components. Scanning electron micrographs of NCa myofibrils showed they were more fragmented, fractured, or split and had wider interfibrillar spaces compared to Ctr and WCa. The fracture plane of muscles immediately postmortem was along the endomysial-sarcolemmal sheath, while at 24 hr postmortem the sheath was weakened enough for the fracture to occur along the surface of the myofibrils.     

Pelvic suspension and fast post-mortem chilling: Effects on technological and sensory quality of pork - A combined NMR and sensory study

The effects of pre-rigor excision, which results in a fast post-mortem chilling; pelvic suspension, which results in a muscular stretching; control treatment of m. longissimus dorsi on water distribution measured by (1)H NMR relaxometry and on sensory properties were investigated in two sub-studies including a total of 16 pigs. Determination of sarcomere length revealed significant effects of the treatments on the degree of contraction, as pre-rigor excision and pelvic suspension resulted in shorter and longer sarcomere length, respectively. In addition, an effect of treatment on pH measured at 24h post-mortem was found, as pre-rigor excision was associated with a higher ultimate pH. NMR measurements revealed that water distribution in the meat was affected to a minor degree by the various treatments. However, an interaction between treatment and ageing period was observed, as the data demonstrated an effect of pre-rigor excision on water distribution in the cooked samples when meat was only aged for 2 days, but this effect was eliminated when meat was aged for 5 days. The effect of pre-rigor excision on water distribution in cooked meat after 2 days of ageing is suggested to reflect structural constraints in the meat that are eliminated during ageing. Sensory analyses demonstrated strong interactions between treatment and position on the muscle and between treatment and ageing period. However, in general pre-rigor-excised meat was sensed as significantly juicier compared with the other two treatments. Accordingly, the study demonstrates that under the present conditions the ultimate pH is more important for juiciness than the sarcomere length.     

Post-mortem Changes in Breast Muscles of Mule Duck

Post-mortem changes in myofibril fragmentation index and degradation of myofibrillar proteins of duck breast muscles at 5°C was investigated. Muscle samples were collected immediately after killing and from the stored carcasses at 5°C for 1, 3, 7, and 14 days post mortem . The results showed that the MFI of the breast muscles increased with time post mortem . SDS-PAGE of myofibrils indicated that the disappearance of troponin-T accompanied concurrently with the appearance of 32–30 kDa components. After 7 days of storage, α-actinin was degraded, and a 98 kDa component appeared. Titin 1 and nebulin also disappeared after post-mortem storage for 3 days.     

Manipulation of the pre-rigor glycolytic behaviour of bovine M. longissimus dorsi in order to identify causes of inconsistencies in tenderness

The aim of this study was to monitor the effects of the alteration of the pre-rigor environment of the bovine LD muscle using controlled temperature regimes in order to gain an insight into the early post-mortem pH/temperature/time interactions which are important from the point of view of tenderness and to identify possible reasons for inconsistencies in beef tenderness. LD muscles (n=12) were hot-boned within 90min post-slaughter, cut into three pieces which were randomly placed in polyethylene bags and submerged in water baths pre-set at the following temperatures; 0, 5, 10, 15, 20 and 25°C for 8 h post-mortem then stored at 2°C for up to 14-days post-mortem. The rate of pH decline increased with increasing temperature. Muscles incubated at 0 and 5°C were cold shortened however not all of these muscles were tough as indicated by Warner Bratzler shear force values (WBSF). A pH range of 5.9-6.2 at 3 h post-mortem (pH(3)) produced consistently tender beef where cold-shortening was avoided. Cold shortened muscles showed the greatest variation in tenderness at 14 days post-mortem and underwent the greatest amount of tenderisation (ΔWBSF) and proteolysis between days 2 and 14 post-mortem. Proteolysis of cold shortened muscle may induce variation in tenderness in these muscles.     

Changes in structure of psoas major and minor and semitendinosus muscles of calves, heifers and cows during post-mortem ageing

Changes occurring during post-mortem ageing in structure of psoas major and minor (PM) and semitendinosus (ST) muscles of calves, heifers and cows were observed. Samples from muscles taken 3 h after slaughter and after 6 and 12 days of storage at the temperature of 4?°C were analysed using light and transmission electron microscopy. Three hours after slaughter muscle fibres were close to each other and sarcomeres showed normal structure of A- and I-bands, M-lines and Z-disks. The space between myofibrils and sarcolemma and also between single myofibrils in muscle fibres increased during storage. Structure of sarcomeres underwent continuous degradation, length of sarcomeres increased due to enlargement of I-bands accompanied by Z-disk degradation. During ageing structural changes in myofibrils took place faster, were more intensive in muscles of younger animals and were more extensive and faster in PM than in ST muscles.     

Effect of heating rate on shortening, ultrastructure, and fracture behavior of prerigor beef muscle

Rapidly heated prerigor beef is tender because of incompletely described myofibrillar disruption and tissue fracture. This study was designed to evaluate the effects of heating rate on heat-induced myofibrillar shortening, ultrastructural changes, and fracture behavior in prerigor triceps brachii muscle. Rapid heating (2°C/2 min) to 53°C caused (P < 0·05) more severe myofibrillar shortening in a shorter time and at higher muscle pH and temperature, less muscle weight loss, and shorter sarcomeres than slow heating (2°C/12 min) to 47 or 53°C. Rapid heating caused more extensive degradation of A and I bands, greater loss of the tridimensional pattern of myofibrils, more fragmentation and melting of myofibrils, widened intermyofibrillar spaces, and maximum separation of fiber bundles as compared to slow heating. Slow heating caused extensive shortening but not extensive degradation and disruption of myofibrils. Muscles slowly heated to 53°C sustained greater loss of structural integrity than those slowly heated to 47°C, but fracture behavior was similar. Separation and fracture occurred near the perimysial/endomysial junction in all heated samples, but the perimysium remained affixed to the endomysium at one side of the interface in many rapidly heated samples. Longitudinal fractures showed a granular endomysium and large numbers of supercontraction nodes alternating with areas of sarcolemmal membrane fragmentation and fiber tearing in rapidly heated samples. Alterations of myofibrillar ultrastructure and fiber structure, and separation of bundles, may account for enhanced tenderness of rapidly heated prerigor muscle.     

Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef

Changes in proteoglycans (PGs) during post-mortem ageing of bovine m. semitendinosus were studied. Electron microscopic observations made it clear that there were two types of PGs in bovine m. semitendinosus immediately post-mortem: PGs were arranged regularly in the basement membrane and PGs associated with collagen fibrils in the perimysium. After 28 days ageing at 4 °C, no PG was observed in the basement membrane, and the greater part of PGs in the perimysium had disappeared. The total amount of PGs decreased with time post-mortem. SDS-polyacrylamide gel electrophoresis indicated that PGs with a high molecular weight disappeared within 7 days post mortem. These results suggest that PGs are degraded during post-mortem ageing of beef. The degradation of PGs seems likely to be the main factor in the weakening of intramuscular connective tissues, i.e., separation of collagen fibrils and fibres from the endomysium and the perimysium, which results in the partial tenderization of beef during post-mortem ageing.     

Studies on Bovine Natural Actomyosin 2. Physico-Chemical Properties and Tenderness of Muscle

SUMMARY: Studies were made of physicochemical characteristics of natural actomyosin from bovine longissimus of different post-mortem ages and tenderness classifications. Reduced viscosity, ATP sensitivity, and "actin" content (polyethylene sulfonate treatment) were higher for natural actomyosin prepared from muscle 12-24 hr post-mortem than from pre-rigor muscle, which confirms previous reports for rabbit natural actomyosin. A higher actin to myosin ratio in actomyosin from muscle 12-24 hr was therefore postulated. A stronger interaction of actin and myosin in actomyosin from muscle 12-24 hr post-mortem than from pre-rigor or aged muscle was also suggested by reduced viscosity and ultracentrifugation data. Reduced viscosity differences between actomyosins from tough and tender muscle suggested a higher gel character in actomyosin from tough muscle. This possibly indicated a higher content of α-actinin. No consistent differences in ATP sensitivity, myosin and actin content of natural actomyosin of tough and tender muscle were found. Natural actomyosin from muscle aged post-mortem showed the appearance during analytical ultracentrifugation of an additional component which sedimented at about 11S to 12S. This component appeared in the actomyosin prepared from tender muscle after 24 hr but did not appear until 10 days in the actomyosin from tough muscle.     

Post-Mortem Changes in Subcelllular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 2. Electron Microscopy.

SUMMARY— Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates of normal and pale, soft, exudative (PSE) porcine muscle at 0 and 24 hr post-mortem and examined by electron microscopy. No differences were observed between normal and PSE myofibrils obtained at death. PSE myofibrils prepared at 24 hr post-mortem had more granular appearing filaments and wider Z lines than normal myofibrils at 24 hr. The PSE heavy sarcoplasmic reticulum fraction obtained at death had a higher proportion of granular material than the same fraction from normal muscle. Several structural differences between the other PSE and normal fractions were also observed, especially at 24 hr postmortem. This study indicated that the composition of the subcellular fractions changed with time post-mortem and that this change should be considered when analyzing biochemical data from these fractions. However, the differences observed could not explain the large changes in calcium accumulating ability that have been shown to occur post-mortem.     

POSTMORTEM CHANGES IN MYOFIBRILLAR PROTEINS OF GOAT SKELETAL MUSCLES

Postmortem changes at 5C in myofibrillar proteins of longissimus dorsi (LD), biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles and myofibrillar structure of LD muscle of goat were investigated. Muscle samples were immediately collected after killing, and from carcasses stored at 5C for 3, 6, 9, 12 and 20 days. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of myofibrils indicated the appearance of a 30 kDa component, depending on the type of the muscles. A new 55 kDa component appeared in BF and SM muscles during postmortem. Titin I and nebulin also disappeared during storage. The disappearance of titin 1 and nebulin and the appearance of a 30 kDa component were confirmed by Western blot analysis. The Transmission Electron Microscopy studies showed that after 3 days postmortem, Z‐disks stayed unaltered. After 6 days postmortem, a little ultrastructural alteration was observed, and at 12 days postmortem a considerable degradation of Z‐disk ultrastructure was shown. The Z‐disk degradation, which results in the fragmentation of myofibrils and the appearance of 30 kDa components, is the major change observed in goat skeletal muscles during postmortem.     

Protein extractability and thermal gel formability of myofibrils isolated from skeletal and cardiac muscles at different post-mortem periods

The effects of the post-mortem ageing period on the extractability of myofibrillar proteins from pork cardiac and rabbit skeletal muscles under various conditions of pH and ionic strength were studied with particular reference to the changes in the solubility of individual myofibrillar proteins and their denaturation characteristics. The ultimate influence of these changes on the heat-induced gel forming ability of myofibrils isolated from cardiac and skeletal muscles at different post-mortem stages was also investigated. Results showed that pork cardiac myofibrils always exhibited lower solubility than those from rabbit skeletal muscles under identical conditions of pH, ionic strength and temperature. SDS-PAGE profiles indicated several quantitative differences in the relative proportion of individual protein species present in cardiac and skeletal myofibrils. The solubility of various proteins present in myofibrils was also affected differently on heating in 0-1 and 0-6 _M NaCl solution at various pH values. Thermal denaturation of cardiac myofibrils occurred at about 10°C higher than that of skeletal myofibrils as revealed by differential scanning calorimetry. Cardiac myofibrils formed much weaker heat-induced gels than those produced by skeletal myofibrils under identical conditions of temperature, pH, ionic strength and protein content.     

Ultrastructural changes in pre- and post-rigor beef muscle caused by conventional and microwave cookery

Examination of cooked pre-rigor muscle by TEM (transmission electron microscopy) and SEM (scanning electron microscopy) revealed that all cookery methods resulted in the development of supercontraction bands alternating with areas showing tissue fragmentation and tearing. Microwave cookery produced smaller and less dense supercontraction nodes in pre-rigor muscle with less tearing and fragmentation but more fibre separation. Although cold-shortened pre-rigor muscle cooked by all methods also exhibited supercontraction bands with some tearing and fragmentation in adjacent sarcomeres, the samples cooked by microwaves showed a more uniform repeating pattern of small stretched areas alternating with dense contracted areas. Cooking of muscle in full rigor resulted in myofibrillar protein coagulation and shrinkage but supercontraction nodes were absent. Cold-shortened bicarbonate treated muscle was relatively intact after cooking, exhibiting fusion of the myofibrils and an absence of intermyofibrillar spaces. Results are discussed in relation to possible effects upon tenderness.     

Manipulation of the pre-rigor phase to investigate the significance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi

The objective of this study was to evaluate the importance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi (LD) muscle over a 21-day period. This was done by altering the pre-rigor glycolytic behaviour of hot-boned LD muscles using different early post-mortem temperature regimes. Hot-boned LD muscles (n=8) were cut in two, randomised, placed in polythene bags and submerged in a water bath set at 5°C (rapidly chilled) and 15°C (slowly chilled) for 8h post-mortem. All muscles were then stored at 2°C for up to 21 days post-mortem. The temperature regimes altered the glycolytic behaviour of the muscles in the pre-rigor period. The slowly chilled muscles exhibited a faster (P<0.01) pH fall than rapidly chilled muscles. Cold shortening was induced in rapidly chilled muscles as they had shorter (P<0.01) sarcomere lengths than slowly chilled muscles up to day 21 post-mortem. Warner Bratzler shear force values (WBSF) deemed cold-shortened muscles as tougher than noncold shortened up to day 14 post-mortem. Both cold-shortened and noncold-shortened muscles tenderised over time to an extent where there was no significant difference in WBSF values by day 21 post-mortem. SDS-PAGE protein profiles indicated that the rate of proteolysis was faster in slowly chilled muscles when compared to rapidly chilled muscles. However by day 21 post-mortem it appeared that rapidly and slowly chilled muscles underwent proteolysis to the same extent.     

Effect of pre-rigor stretch and various constant temperatures on the rate of post-mortem pH fall, rigor mortis and some quality traits of excised porcine biceps femoris muscle strips

Porcine biceps femoris strips of 10 cm original length were stretched by 50% and fixed within 1 hr post mortem then subjected to temperatures of 4 °, 15 ° or 36 °C until they attained their ultimate pH. Unrestrained control muscle strips, which were left to shorten freely, were similarly treated. Post-mortem metabolism (pH, R-value) and shortening were recorded; thereafter ultimate meat quality traits (pH, lightness, extraction and swelling of myofibrils) were determined. The rate of pH fall at 36 °C, as well as ATP breakdown at 36 and 4 °C, were significantly reduced by pre-rigor stretch. The relationship between R-value and pH indicated cold shortening at 4 °C. Myofibrils isolated from pre-rigor stretched muscle strips kept at 36 °C showed the most severe reduction of hydration capacity, while paleness remained below extreme values. However, pre-rigor stretched myofibrils - when stored at 4 °C - proved to be superior to shortened ones in their extractability and swelling.     

Temperature induced denaturation of myosin: Evidence of structural alterations of myosin subfragment-1

Denaturation of myofibrillar proteins in porcine longissimus thoracis et lumborum muscle was investigated after pre-rigor temperature incubation at 20, 30 and 40 °C. At 24 h myofibrils were isolated and myosin was further cleaved by chymotrypsin. High temperature pre-rigor induced release of myosin S1 (subfragment-1), less (P < 0.05) Ca²⁺-ATPase activity and structural alterations of the region of the myosin molecule that harbors S1. Surface hydrophobicity of myofibrils from the 40 °C group increased (P < 0.001), suggesting a temperature-induced structural rearrangement exposing hydrophobic groups on the surface of myofibrils which in turn may explain the reduced water-holding of PSE meat.     

Injection of marinade with actinidin increases tenderness of porcine M. biceps femoris and affects myofibrils and connective tissue

BACKGROUND: Marination of beef muscles with brine solutions containing proteolytic enzymes from fruit extracts has been shown to tenderize meat. However, the effect of marination with actinidin on tenderness of pork muscles has not been investigated. Tenderness and eating quality of porcine M. biceps femoris was investigated by Warner–Bratzler (WB) shear test and sensory evaluation after injection of brine containing up to 11 g L^?1 actinidin‐containing kiwi fruit powder and 2, 5 or 9 days of storage. RESULTS: Actinidin decreased WB shear force, increased tenderness and did not affect flavour and juiciness. Injection of 2.8 g L^?1 actinidin powder and storage for 2 days resulted in WB shear force values similar to control samples stored for 5 or 9 days. In samples injected with 10 g L^?1 actinidin powder, degradation of desmin and percentage of heat‐soluble collagen (P < 0.05) increased compared to control samples. Myofibrillar particle size tended to decrease (P < 0.1) with increasing actinidin concentration. No major changes were observed by proteome analysis. Atomic force microscopy showed actinidin‐induced damage of endomysium surrounding isolated single muscle fibres. CONCLUSION: Our results indicate that actinidin tenderizes pork M. biceps femoris by affecting both the myofibrils and connective tissue. Copyright © 2009 Society of Chemical Industry