Mild protein oxidation enhanced hydration and myofibril swelling capacity of fresh ground pork muscle packaged in high oxygen atmosphere

Abstract: This study investigated the influence of an oxygen‐enriched modified atmosphere packaging (HiOx: 80% O₂/20% CO₂) in comparison with air‐permeable polyvinylchloride (PVC) wrapping and partial vacuum (VP: 60%) packaging on the ability of myofibrils to imbibe water during retail display of fresh ground pork at 2 to 4 °C. Both HiOx and PVC muscles after 4 d showed maximum myofibril swelling and A‐band dissolution when isolated myofibrils were subjected to a graded series of salt solutions (0.2→0.4 M NaCl with 10‐mM sodium pyrophosphate, pH 6.2), while VP samples exhibited no remarkable change. Protein carbonyl content increased substantially from day 0 to 4 in all muscle samples. For HiOx, muscle hydration capacity increased linearly (P < 0.05) during storage up to 14 d, corresponding to protein carbonyl production. No significant changes in hydration properties were noted in VP muscle samples, which also maintained lower levels of protein oxidation, during storage up to 21 d. These results indicated that packaging in modified atmosphere with high levels of oxygen could result in increased protein oxidation but enhanced hydration capacity of fresh meat. Practical Application: Packaging of fresh meat, including ground pork, under an oxygen‐enriched atmosphere condition is widely used in the industry to preserve red color of meat. Results from the present study indicate that high oxygen packaging has a discordant effect on fresh ground pork: it enhances hydration but decreases water‐binding, and this seems to be caused by increased swelling of muscle fibers due to mild protein oxidation. Hence, it is advisable to employ a water‐binding agent in this type of packaging system so as to retain the moisture in stored fresh pork products while maintaining the color stability.     

Ultrastructure of Fresh, Airblast and Sodium Chloride Brine Frozen Uncooked Blue Crab Muscle

The ultrastructural changes of 0°C blast-frozen and brine-immersed (23% NaCl at 0°C) blue crab (Callinectes sapidus) levator muscle were studied by transmission electron microscopy in comparison to unfrozen, prerigor muscle. Characteristic, striated skeletal muscle features were seen in fresh muscle, including double M-lines and Z-tubules seen in some Crustacea. Changes in structural organization caused by blast freezing (slow method) included decreased density and change in shape of myofibrils and Z-Lines, and disappearance of the sacroplasmic matrix. Disintegration of Z-lines, myofibrils, and sarcoplasm was less severe due to brine-immersion freezing (rapid method). Distances between myofibrils were compacted in brine-immersed blue crab muscle samples, related to firmer objective texture measurements.     

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.     

Comparative effects of post-mortem storage and low-calcium-requiring neutral proteinase on bovine and rabbit myofibrillar proteins

The effects of post-mortem storage and of a low-calcium-requiring neutral proteinase on the myofibrils of beef Longissimus dorsi and Rectus abdominis muscles and rabbit Longissimus dorsi muscle were studied by measuring the Mg-Ca-enhanced myofibrillar ATPase activity and the changes in banding patterns on electrophoresis in sodium dodecyl sulphate. During ageing, the changes in the ATPase activity and myofibrillar proteins were qualitatively different between rabbit and bovine muscles, whilst differences in intensity were only observed between the two bovine muscles. The most prevalent component appearing upon conditioning had a mol. wt of 27 000 in rabbit muscle and of 30 000 in bovine muscles. In both species, the incubation of myofibrils with the low-calcium-requiring neutral proteinase mimicked the post-mortem changes. This would suggest that this enzyme has a qualitatively different effect on rabbit and bovine myofibrils.     

Ca(2+)-activated protease in stored muscle

To establish the rôle of Ca²⁺-activated protease (CAF) in meat tenderness, the properties of CAF extracted from stored muscle, and the effects of CAF on the ultrastructure of myofibrils under the conditions obtaining in stored muscle were studied.

The yield and activity of crude CAF extracted from stored muscle were higher than that from muscle immediately post mortem, but no difference of the enzymic properties (time course of activity, pH dependence and Ca²⁺ dependence) was observed between the two preparations. An electron microscopic study of the effects of CAF on myofibril structure indicated that CAF was active under the conditions actually obtaining in stored muscle.

From the results obtained in this paper, it was confirmed that the CAF had an important rôle in meat tenderness.     

Functional Properties of Myofibrillar Proteins from Cold-Shortened and Thaw-Rigor Bovine Muscles

Prerigor bovine sternomandibularis muscles were stored at 15, 0 and ?29°C to examine cold-shortening and thaw-rigor effects on myofibrillar protein extractability and gelation properties of myofibrils and salt-soluble protein (SSP). Frozen muscle that underwent severe contraction at thawing showed greater protein extractability (35%) than muscles stored at 0 and 15°C (27% extractability). Of the three tempered muscles, thaw-rigor muscle produced the strongest myofibril gel and cold-shortened muscle formed the most elastic SSP gel as determined by dynamic shear and penetration measurements. However, thermally induced changes in gel viscoelastic moduli for all protein samples followed similar patterns. Results indicated that physicochemical changes accompanying muscle contraction affected protein network formation during gelation.     

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.     

Treatment and Post-Mortem Aging Effects on the Z-line of Myofibrils from Chicken Pectoral Muscle

SUMMARY: Changes in the morphology of myofibrils prepared from chicken pectoral muscle during post-mortem storage at 5°C were examined by light and electron microscopy. When the 24-hr stored samples were blendorized, electron micrographs showed two types of destruction in the Z-lines of sarcomeres and myofibrillar fragments: (1) The degradation and/or disappearance of Z-lines. (2) The breakdown of the junction of Z-line and I-filaments. A change in the state of the Z-line and the junction of the Z-line and I-filaments appeared to be indispensable for the fragmentation of the myofibrils. It was also shown through phase contrast microscopic observations that sarcoplasmic proteins, participating in the glycolytic cycle, may play a role in the fragmentation of the myofibrils. Evidence has not been obtained, to date, on the participation of proteolytic enzymes in the fragmentation phenomenon.     

Ultrastructure of mechanically tenderised pork muscle

Porcine biceps femoris muscles were mechanically tenderised by the use of a meat activator. The kind and degree of damage of muscle tissue were then examined under an electron microscope. It was observed that several changes, known from the studies of the post mortem muscles, were much more frequent in tenderised than in intact muscles. Additional changes were found as: disruption of the contractile system or its expansion till the A- and I-bands disconnected. Thus we suggest that mechanical tenderisation, by destroying several linkages between muscle fibres, between myofibrils and within myofibrils, may be responsible for lattice expansion and increase of brine uptake and overcoming the myofibrillar and connective tissue toughness of porcine meat.     

Microstructural changes in Teruel dry-cured ham during processing

Dry-cured ham, "Protected Designation of Origin (PDO)" Teruel, is a high quality meat product processed using a traditional dry-curing procedure. The objective of this paper was to study the microstructure of the Biceps femoris and Semimembranosus muscles during "PDO Teruel" dry-cured ham processing using electron microscopy techniques (Cryo-SEM, SEM and TEM). The Semimembranosus and Biceps femoris muscles from raw ham show the typical structure of muscle tissue in meat. The muscle fibres appear firmly attached to one another by the endomysial connective tissue; the myofibrils inside the cells are strongly attached to each other and to the sarcolemma. In raw ham, the typical structural elements that constitute the sarcomere can be observed by TEM. Important microstructural changes are observed following salting; reflected in the fact that the Z-disks inside the myofibrils are no longer in line and there is important degradation of the cell membranes in this stage. At the end of the process, an accumulation of solutes, which could be products from proteolysis occurring mainly in the last stage of ripening, could be observed in both muscles. Thus the proteolysis and dehydratation phenomena that take place during "PDO Teruel" dry-cured ham processing occur at an ultrastructural level, and this is the basis to produce a product with the sensory features appreciated by consumers.     

A "Cold Shortening" Effect in Avian Muscle

SUMMARY— Experiments were conducted to determine the effect of post-mortem temperatures between 0 and 20°C on the degree of shortening in isolated pectoralis major muscles of chickens and turkeys. A "cold shortening" effect in these muscles is described and compared to post-mortem pH, average sarcomere length of isolated myofibrils, and relative solubility of myofibrillar and sarcoplasmic proteins.
The degree of muscle shortening at each temperature after various periods post-mortem indicated that shortening was essentially complete after 3 hr in chickens and 5 hr in turkeys. Shortening in muscles stored at 0°C was significantly greater (P < .01) than in the 12–18°C temperature range. Shortening was greatest in muscles stored at 20°C. The degree of gross shortening observed was directly related to the average sarcomere length of isolated myofibrils. Post-mortem decline in pH was not significantly correlated (P > .05) with shortening. Extractability of myofibrillar and sarcoplasmic proteins after 5 hr at either 0 or 16°C was determined and found to be unrelated to the degree of post-mortem shortening.     

SDS-PAGE Conditions for Detection of Titin and Nebulin in Tender and Tough Bovine Muscles

Purified myofibrils were prepared from infraspinatus (tender) and rhomboideus (tough) muscles at 7 days postmortem and examined for myofibrillar/cytoskeleta1 protein degradation by using sodium dodecyl sulfate polyactylamide gel electrophoresis (SDS-PAGE). Four acrylamide/bisacrylamide ratios (37:1, 50:1, 75:l and 100:1) and two SDS-PAGE gel buffers (Tris-HCl, pH 8.0 and 8.9) were used to determine the optimum conditions for detection of titin and nebulin. Titin was degraded to a greater extent in myofibrils from the infraspinatus than in myofibrils from the rhomboideus. Very little nebulin was detected in either muscle. Use of acrylamide/bisacrylamide ratio of 37:1 and a gel buffer of pH 8.0 provided the most optimum conditions for detecting differences in the resolution of titin, nebulin and their apparent degradation products.     

Thermal Gelation of Brown Trout Myofibrils: Effect of Muscle Type, Heating Rate and Protein Concentration

The thermal gelation properties of myofibril solutions (KCl 0.6M; pH 6.0) from reared brown trout white and red muscles were analyzed by thermal scanning rheometry. With a heating rate of 1°C/min, red muscle myofibrils exhibited a lower gelation capacity than white muscle myofibrils at low temperatures. No difference was observed above 60°C where solid gels were formed from the two myofibril types. Increasing protein concentration or reducing heating rate increased the values of the rheological parameters at 80°C for the two muscle type myofibrils. With a low heating rate (0.25°C/min), white muscle myofibrils formed stronger gels whatever the temperature.     

Storage of Yellowtail (Seriola quinqueradiata) White and Dark Muscles in Ice: Changes in Content of Adenine Nucleotides and Related Compounds

Adenine nucleotides and their related compounds in white and dark muscles of yellowtail Seriola quinqueradiata were measured and compared during ice storage. In white muscle, adenosine 5′-triphosphate was degraded rapidly to inosine 5′-monophosphate, which then underwent gradual build-up of inosine (HxR). In dark muscle, HxR accumulated very rapidly and the K value, a freshness index, thus increased at a greater initial rate than in the white muscle (62 vs 3%/day). Urate occurred in a small amount only in the white muscle stored for over 40 days (0.2 μmole/g), while little was found in the dark muscle during the entire storage period. Nicotinamide adenine dinucleotide, which existed in both the fresh white and dark muscles (0.8 vs 0.4 μmole/g), decreased rapidly in the early stage of storage.     

Glycolysis and associated changes in beef carcasses

Post-mortem glycolysis was investigated at several locations in intact sides of beef. The time required for the pH to fall to 6.0 in six major hindquarter muscles ranged from 2.2 to 13.6 h, varying with muscle and depth in the carcass. A good correlation (r=0.97) was observed between the rate of ATP turnover and muscle temperature. In the muscles in the round the rate of glycolysis increased with depth, as did the extent of denaturation of myofibrils, creatine phosphokinase and also drip loss.     

Investigation of the activity of cathepsin B in red shrimp (Solenocera crassicornis) and its relation to the quality of muscle proteins during chilled and frozen storage

Cathepsin B is a cysteine protease that has important effects on the quality of muscle products. In this study, the changes of cathepsin B activity and its relation to muscle proteins were investigated in intact and beheaded shrimp during chilled and frozen storage. The obtained results indicated that the water holding capacity (WHC), shear force, hardness, and myofibrillar protein (MP) content all significantly decreased in both the intact and beheaded shrimp samples with increasing storage period (p < 0.05). Specifically, beheading shrimp exhibited much more stable characteristics than intact shrimp samples during both chilled and frozen storage. The enzyme activity results suggested that cold temperature and storage induced the release of cathepsin B from the lysosomes to the mitochondria, sarcoplasm, and myofibrils in the muscle tissues. Furthermore, SDS-PAGE and transmission electron microscopy (TEM) analysis revealed that beheading the shrimp greatly inhibited the dissociation of shrimp muscle proteins during storage. The current findings suggest that cathepsin B located in the head of shrimp was likely transferred to the muscle through the first abdominal segment during storage, accelerating the dissociation of the muscle proteins. Therefore, beheading the shrimp was conducive to prolonging the shelf-life of stored shrimp products.     

Post-mortem changes of connectin in chicken skeletal muscle

To investigate the influence of connectin (titin) on meat tenderization during conditioning, changes in the content and the electrophoretic pattern of the connectin in chicken myofibrils isolated by gel permeation chromatography were studied. Significant difference of the content and the electrophoretic pattern of the connectin isolated was not observed between the preparations from fresh and stored muscle. Therefore, the connectin is unlikely to be responsible for the meat tenderization caused by conditioning.     

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.     

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0–0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6–7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3–9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.     

Liquid Loss as Effected by Post mortem Ultrastructural Changes in Fish Muscle: Cod (Gadus morhuaL) and Salmon (Salmo salar)

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 intra- 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.