Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes

Unacceptable water-holding capacity costs the meat industry millions of dollars annually. However, limited progress has been made toward understanding the mechanisms that underlie the development of drip or purge. It is clear that early postmortem events including rate and extent of pH decline, proteolysis and even protein oxidation are key in influencing the ability of meat to retain moisture. Much of the water in the muscle is entrapped in structures of the cell, including the intra- and extramyofibrillar spaces; therefore, key changes in the intracellular architecture of the cell influence the ability of muscle cells to retain water. As rigor progresses, the space for water to be held in the myofibrils is reduced and fluid can be forced into the extramyofibrillar spaces where it is more easily lost as drip. Lateral shrinkage of the myofibrils occurring during rigor can be transmitted to the entire cell if proteins that link myofibrils together and myofibrils to the cell membrane (such as desmin) are not degraded. Limited degradation of cytoskeletal proteins may result in increased shrinking of the overall muscle cell, which is ultimately translated into drip loss. Recent evidence suggests that degradation of key cytoskeletal proteins by calpain proteinases has a role to play in determining water-holding capacity. This review will focus on key events in muscle that influence structural changes that are associated with water-holding capacity.     

Microstructural changes in rabbit meat wrapped with Pteridium aquilinum fern during postmortem storage

This work studied the microstructural degradation of rabbit semimembranosus muscle wrapped with Pteridium aquilinum fern fronds, stored at 4 °C during the first 72 h postmortem. At the microstructural level, practically all the perimysial and endomysial connective tissue was destroyed and a small degradation of the myofibrils could be observed after the first 24 h postmortem; in addition, a degradation of the sarcolemma and the generation of numerous gaps were seen. After 32 h postmortem, tissue fibres were broken, cell membranes were more strongly degraded, and the connections between the sarcolemma and the myofibrils had disappeared. After 72 h postmortem, intercellular connections became degraded inside the muscle bundles and intercellular gaps became larger. The proteolytic activity is attributed to fern endogenous enzymatic activity in addition to the typical endogenous enzymatic activity of meat postmortem.     

Effect of Postmortem Storage on Degradation of the Myofibrillar Protein Titin in Bovine Longissimus Muscle

Purified bovine longissimus muscle myofibrils were prepared from muscle at death and from muscle samples stored at 2°, 25°, or 37°C for 1, 3, and 7 days postmortem. Tbe myofibrils were analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Titin migrated as a closely spaced doublet of very high molecular weight (M_r～ 1 × 10⁶) in myofibrils from at-death muscle samples. With increased storage time and temperature, the top band of the titin doublet gradually disappeared. the lower doublet band (putative breakdown product of upper band) remained after 7 days storage at 2° or 25°C, but disappeared by 3 days of postmortem storage at 37°C. Thus, titin is degraded in postmortem muscle, and the rate of degradation is enhanced by increases in storage time and temperature.     

Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.     

Gel Electrophoretic Analysis of the Protein Changes in Ground Beef Stored at 2°C

Purified myofibrils and sarcoplasmic proteins were prepared from ground (GR) and intact (CON) beef semitendinosus muscle samples after 0, 1, 3, 6, and 10 days of storage at 2°C. SDS-polyacrylamide gel electrophoresis analysis revealed the following major postmortem changes in GR samples: the gradual disappearance of nebulin and desmin, appearance of 110,000-, 95,000- and 30,000-dalton polypeptides, and an increased content of myosin light chain-3 and 55,000-dalton component in myofibrils. Also noted was emergence of 100,000- and ?500,000-dalton polypeptides and diminution of 300,000-dalton protein in the sarcoplasmic fraction. Since GR samples showed proteolytic changes similar to those of CON samples, it was concluded that grinding had little effect on postmortem muscle protein degradation.     

Postmortem Degradation of Titin and Nebulin of Beef Steaks Varying in Tenderness

Purified myofibrils were isolated from “tender” and “less-tender” bovine longissimus muscle at death and at 1, 3, 7, and 14 days of postmortem storage (4^oC). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect changes in the myofibrillar/cytoskeletal proteins, titin and nebulin. Titin and nebulin bands were observed to be less intense on gels from “tender” than from “less-tender” steaks. These results suggest that titin and nebulin were more rapidly degraded in “tender” than in “less-tender” steaks, and that the extent of beef loin steak tenderness may be dependent upon the postmortem degradation of titin and nebulin.     

Effect of Cathepsin D on Bovine Myofibrils under Different Conditions of pH and Temperature

Purified cathepsin D was incubated with bovine skeletal muscle myofibrils under in virro conditions resembling those found in postmortem muscle. SDS-PAGE analysis of myofibrils treated at pH 5.5 and 37°C and the sedimented, showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. The cathepsin D treated myofibrils were not fragmented to any greater extend than untreated myofibrils. Raising the pH and/or lowering the temperature greatly reduced the effectiveness of cathepsin D suggesting that the enzyme does not play a principal role in the tenderization process occurring in muscle postmortem.     

Ultramicroscopical Structures and Liquid Loss in Heated Cod ( Gadus morhuaL) and Salmon ( Salmo salar) Muscle

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 different 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 myofibrils 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 intermyofibrillar and extracellular spaces are important in preventing release of liquid upon heating.     

Effects of Postmortem pH and Temperature Muscle Structure and Meat Tenderness

Bovine longissimus muscles with postmortem pH in the range 5.5 - 7.0 were subjected to different postmortem temperatures of 1°, 4°, 25° and 37°C. Intact beef sides with different postmortem pH were also subjected to two different environmental temperatures of 1° and 25°C. High pH muscles exhibited an extensive degradation of Z-lines, whereas low pH muscles showed a preferential degradation of M-lines and myosin heavy chains. Intermediate pH muscles did not show much degradation of muscle proteins, resulting in tougher meat than either low or high pH muscles. High postmortem temperatures enhanced the degradation of muscle proteins in excised and incubated muscle strips, but the delayed chilling of intact beef sides at 25°C for 8-hr did not affect either the structural changes or meat tenderness.     

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.     

Comparison of fresh beef and camel meat proteolysis during cold storage

The objective of this research was to determine the difference in myofibrillar fragmentation of camel meat and beef during postmortem aging. Semitendinosus muscle was excised at slaughter and muscle pH was measured at 6, 12, 24, 48, and 72h postmortem. Myofibril fragmentation index was measured on 1, 3, 5, and 7 days postmortem. Also, myofibrils isolated from semitendinosus muscles of camel and cattle at 1, 3, 5 and 7 days postmortem storage were analyzed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results showed that the camel semitendinosus muscle had significantly higher myofibril degradation values compared to that in beef which was supported by a difference in troponin-T degradation and appearance of a 30kDa band. Postmortem pH decline of camel meat was significantly slower than that of beef. This study demonstrated that the semitendinosus protease activity of camel meat was superior to that of beef, which may have been due to the difference in pH decline.     

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.     

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.     

Biochemistry of postmortem muscle — Lessons on mechanisms of meat tenderization

It is certain that meat tenderness is a highly valued consumer trait and thus definition of the multiple processes that influence meat tenderness will provide clues toward improving meat quality and value. The natural process by which meat becomes tender is complex. Tenderness development is dependent on the architecture and the integrity of the skeletal muscle cell and on events that modify those proteins and their interaction. Specifically protein degradation and protein oxidation have been identified as processes that modify proteins as well as the tenderness of meat. The intracellular environment is a major factor that controls these events. Ultimately, the interplay between these events determines the rate and extent of tenderization. Given the intricacy of the structure of the muscle cell, coupled with the complexity of the regulation of protein modification and the ever-changing intracellular environment it is not surprising that this area of research is a very dynamic field. Just as the overall integrity and function of muscle cells does not depend on a single protein, but rather on the coordinated interaction of several proteins, the structural weakening of muscle cells during postmortem aging also must not depend on the degradation of a single myofibrillar or other cytoskeletal protein. The proteins mentioned in this review are located in different regions of the muscle cell, and most have been implicated in some manner as being important in maintaining the structure and function of the muscle cell. Oxidation of myosin heavy chain, a predominant protein in the myofibril, is known to promote aggregation and toughening of meat. Degradation of proteins such as desmin, filamin, dystrophin, and talin (all located at the periphery of the Z-line) may disrupt the lateral register and integrity of the myofibril themselves as well as the attachments of the peripheral layer of myofibril to the sarcolemma. Degradation of the proteins within the myofibril that are associated with the thick and thin filaments may allow lateral movement or breaks to occur within the sarcomeres of postmortem aged samples. Titin, nebulin, and troponin-T, by their ability to directly interact with, or modulate the interaction between, major proteins of the thick and thin filaments and (or) the Z-line, play key roles in muscle cell integrity. Disruption of these proteins, especially titin and nebulin, could initiate further physicochemical and structural changes that result in myofibril fragmentation and loss of muscle cell integrity, and ultimately in tenderization of the muscle. In order to make real progress in this area, the scientific community must have a global appreciation of how both the structural proteins and the key proteases are influenced by the vast changes that occur during the conversion of muscle to meat.     

Comparison of postmortem changes in goose cardiac and breast muscles at 5 °C

BACKGROUD: The tenderness of goose heart is an important consideration in its utilization as a popular meat product. It is generally thought that postmortem degradation of myofibrillar proteins may improved meat tenderness. Little information, however, is available regarding the postmortem changes in goose cardiac muscle. Therefore, the postmortem proteolysis between goose cardiac and breast muscles at 5 °C is compared. RESULTS: The pH is higher (P < 0.05) in cardiac samples than in breast samples. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots results indicate that postmortem degradation of titin and desmin and the appearance of the 28 and 30 kDa components are faster in breast muscle than in cardiac muscle. CONCLUSION: Our results may suggest that goose postmortem proteolysis occurs more rapidly in breast muscle than in cardiac muscle at 5 °C. Copyright © 2008 Society of Chemical Industry     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

Skeletal muscle fiber type and myofibrillar proteins in relation to meat quality

Although numerous studies have reported the relationships among muscle fiber characteristics, lean meat content and meat quality, controversial perspectives still remain. Conventional histochemical classifications may be involved in a high level of error, subjectivity and it could not clearly explain variety of myofibrillar protein isoforms. Therefore, more information is needed on how different factors, such as species, breeds, gender, nutrient conditions, physiological state of animals, and environment factors, affect ultimate meat quality in order to evaluate these uncertainness. Unfortunately, there is little information that completely covers with relationship among the muscle fiber types, myofibrillar proteins and enzymatic proteolysis. In addition to the perspective of postmortem metabolism, protein quality control in skeletal muscle and proteolytic degradation of muscle proteins during postmortem period could help to clarify this relationship. Therefore, the present review will focus on muscle fiber types, typing methods, muscle proteins and meat quality, and will summarize aspects of enzymatic view of proteasome.     

The effect of proteasome on myofibrillar structures in bovine skeletal muscle

When bovine myofibrils are incubated with the 20S proteasome their structure is rapidly damaged with loss of material, particularly from the Z discs and I bands. After 24 hr of incubation the myofibrils rupture and debris appears. Certain myofibrillar proteins, including nebulin, myosin, actin and tropomyosin, are hydrolysed during the incubation; others are solubilised (α-actinin). The 20S proteasome completely and rapidly hydrolyses purified myofibrillar proteins in an energy-independent manner. This shows that the 20S proteasome probably plays a role in the postmortem transformation of muscle and more generally in the hydrolysis of cellular proteins.(1).     

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.