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.     

Effect of Postmortem Storage on Titin and Nebulin in Pork and Poultry Light and Dark Muscles

Purified myofibrils were prepared from samples of chicken breast and thigh muscles and from light and dark portions of pork semitendinosus muscle at death and after storage at 4° and 22°C for 1, 3, and 7 days postmortem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the effect of postmortem storage of muscle on titin and nebulin. Results indicated that titin and nebulin were more rapidly degraded in light than in dark chicken muscle. In contrast, titin and nebulin were more rapidly degraded in dark than in light pork muscle.     

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.     

Changes in Titin and Nebulin in Postmortem Bovine Muscle Revealed by Gel Electrophoresis, Western Blotting and Immunofluorescence Microscopy

Postmortem storage of bovine psoas major muscle resulted in almost complete degradation of nebulin by 48 hr; however, some intact titin was still observed after 2 wk storage at 4°C. The percentage of myofibrils having four anti-titin bands per sarcomere (immunofluorescence microscopy) was less than 1% at 45 min but increased to 65% at 48 hr after death and remained stable thereafter. A similar four band pattern was seen in frozen sections of whole muscle at 48 hr or more postmortem. The time course of nebulin degradation appeared to correlate with the titin two to four band transition.     

EFFECTS OF MUSCLE PROTEASES, ENDOGENOUS PROTEASE INHIBITORS AND MYOFIBRIL FRAGMENTATION ON POSTMORTEM AGING OF GOAT MEAT

The present study was conducted to evaluate the extent of postmortem proteolysis in longissimus dorsi, biceps femoris, semimembranosus and semitendinosus goat muscles on postmortem aging at an ambient (27C) temperature. The activities of calpains and calpastatin were determined after separation on a (diethylamino)ethyl–Sephacel column (Sigma, St. Louis, MO) and cathepsin (B, B + L and H) by carboxymethyl–Sepharose column (Sigma). The results showed that the decrease in calpain I and calpastatin activities was significantly higher than that of calpain II. Cathepsin B, B + L, H and cystatin were found to fall by 30–80% after 12 h, whereas cathepsin D decreased significantly in all the muscles. The disappearance of titin 1 and nebulin, and the appearance of a 30‐kDa component were confirmed by Western blot analysis. The appearance of the 30‐kDa component reported here explains the time‐induced structural changes of myofibrils. The Z‐line degradation had occurred by 6 h postmortem. Cathepsins are not stable compared to calpains during postmortem aging, and both enzymes may play a significant role in the proteolysis of myofibrillar proteins at ambient temperature.     

Combination of low voltage electrical stimulation and early postmortem temperature conditioning on degradation of myofibrillar proteins in Korean native cattle (Hanwoo)

The combination effect of low voltage electrical stimulation (LVES) and early postmortem (PM) temperature conditioning (2, 16, and 30°C until 3 h PM) on degradation of myofibrillar proteins were determined from Korean native cattle (Hanwoo). Myofibrils were removed at 1, 2, 3, 7, and 14 days of PM storage (2°C) and analyzed for titin, nebulin, desmin, and troponin-T by SDS-PAGE and by Western blot analysis. Degradation rate of myofibrillar proteins was affected by the combination of LVES and temperature conditioning. LVES-30°C treatment resulted in faster degradation of titin, nebulin, desmin, and troponin-T during PM storage than the other treatments. Degradation of titin took place more slowly than nebulin, desmin or troponin-T.     

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.     

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.     

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.     

Effect of lactic or acetic acid on degradation of myofibrillar proteins in post‐mortem goose (Anser anser) breast muscle

The effects of lactic acid (LA) and acetic acid (AA) on changes in myofibrillar proteins of post‐mortem goose breast muscle marinated for 24 h at 5 °C were studied. Purified myofibrils were prepared from 0.1 M LA or AA samples and controls (non‐marinated samples) after 0, 1, 3, 7 or 14 days of storage at 5 °C. The changes in myofibrillar proteins of goose muscle were examined by SDS‐PAGE. Goose breast muscle marinated in LA and AA exhibited degradation of myosin heavy chains. The appearance of ∼95 and ∼27 kDa components and the disappearance of titin and nebulin were also more rapid than for control muscle. These results suggest that acid marination enhanced the post‐mortem proteolysis of goose breast muscle. © 2000 Society of Chemical Industry     

Effect of heat shock protein 27 on the in vitro degradation of myofibrils by caspase‐3 and μ‐calpain

This study was designed to investigate the effect of heat shock protein 27 (HSP27) on the in vitro degradation of myofibrils induced by caspase‐3 or μ‐calpain. Myofibrillar proteins were prepared from at‐death beef muscles and incubated with caspase‐3 or μ‐calpain with and without HSP27, or with HSP27 alone, at 30 °C for 2 h, and protein degradation was assessed. Results showed that caspase‐3 promoted the degradation of titin, nebulin and troponin‐T, and μ‐calpain promoted the degradation of nebulin, desmin and troponin‐T, observed during normal PM ageing. Moreover, the addition of HSP27 restricted the degradation of troponin‐T in μ‐calpain‐ and caspase‐3‐treated myofilaments, and restricted the degradation of desmin in μ‐calpain‐treated myofilaments. Therefore, HSP27 may indirectly or directly interact with caspase‐3 and μ‐calpain, reducing their activity and mediating PM proteolysis of muscle proteins to affect meat tenderisation.     

Change in Titin Position in Postmortem Bovine Muscle

Myofibrils were prepared from bovine psoas muscles removed from the carcass at 3 and 48 hr postmortem and subsequently stained with a monoclonal antibody against titin. The antibody stained 2 bands per sarcomere (perpendicular to the fiber direction) in myofibrils from 3-hr muscle but often revealed 4 bands per sarcomere in the 48-hr samples. The results suggested that (1) the titin shape might be altered within the first 2 days postmortem, or (2) proteolysis of titin or a protein to which it was attached occurred.     

The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging

The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during post-mortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle.     

Survey of conditioning indicators for pork loins: changes in myofibrils, proteins and peptides during postmortem conditioning of vacuum-packed pork loins for 30 days

This study was performed to examine the changes in myofibrils, proteins and peptides during postmortem conditioning of vacuum-packed pork loins at 4 °C for 30 days. The fragmentation of myofibrils has been observed during postmortem aging for 20 days and its ratio increased until 20 days. The 32 kDa component derived from troponin T increased during storage for 20 days, while a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) among sarcoplasmic proteins was significantly degraded during storage for 15 days. Some oligopeptides increased during such storage, two peptides (peptides P1 and P2) significantly increasing during storage for 20 days. Their sequences were APPPPAEVHVHEEVH (P1) and VPTPNVSVVDLT (P2). Homology analysis showed that peptides P1 and P2 were derived from troponin T and GAPDH, respectively. It was concluded that the fragmentation of myofibrils, the increases in the 32 kDa component and peptides P1 and P2, and the decrease in GAPDH were useful as conditioning indicators of progressive degree during the storage of pork loins stored at 4 °C.     

The postmortem μ‐calpain activity,protein degradation and tenderness of sheep meat from Duolang and Hu breeds

This study aimed to investigate the differences in meat tenderisation between two Chinese native sheep breeds, Duolang and Hu. The tenderness of longissimus thoracis (LT) muscle, μ‐calpain autolysis and proteolysis of myofibrillar protein was measured at 1‐ and 7‐days postmortem storage at 4 °C. At 7‐days postmortem ageing, meat from Duolang sheep was more tender compared to that from Hu sheep. The Warner–Bratzler shear force of Duolang and Hu sheep was reduced by 55.20% and 41.51% at 7 days compared with 1 day, respectively. In Duolang LT, a higher proportion of 80‐kDa μ‐calpain was autolyzed than in the Hu samples at 1‐day postmortem ageing, but they did not differ significantly at 7 days. More titin, desmin and troponin‐T were degraded in Duolang sheep compared to Hu sheep. Additionally, pH values at the different ageing time were not significantly different, indicating that pH may be not a determinant factor contributing to the tenderness difference between the two breeds. Our data suggest the earlier activation of μ‐calpain and a larger extent of proteolysis of key structural proteins may contribute to the higher rate of tenderisation of Duolang compared to Hu sheep during postmortem ageing.     

Structural Changes in Titin and Nebulin Filaments Specific to Calcium Ions at 0.1 mM: Factors of Meat Tenderization During Postmortem Aging

ABSTRACT Postmortem structural changes in titin and nebulin filaments were investigated by incubating isolated myofibrils in a solution containing 0.1 mM calcium ions and various concentrations of a protease inhibitor. The inhibition curves showed 2 abnormal steps with increases in the concentration of leupeptin or calpastatin domain I. While the amounts of unchanged titin and nebulin were constant in the 1st step, the 2nd occurred at higher protease inhibitor concentrations. These facts indicated that excess amounts of leupeptin and calpastatin domain I caused deterioration in titin and nebulin properties, thus interfering with the binding of calcium ions. We concluded that the severance of titin and nebulin filaments in the 1st step were induced by calcium ions at 0.1 mM.     

Does the newly discovered calpain 10 play a role in meat tenderization during post-mortem storage?

The objective was to study calpain 10 during meat tenderization. Western assays were developed and changes in calpain 10, tenderization level and desmin were determined in Longissimui (LTL) during post-mortem storage. A comparison between some characteristics of calpains 1, 2 and 10 indicated differences in the pI and sub-cellular distribution. Tenderness improved gradually during post-mortem storage and was accompanied by a decline in intact desmin level. Western analysis indicated that calpain 10 is present in sheep LTL probably as two proteins (MW 73.6 and 70.7 kDa). Post-mortem storage caused a decline in the level of the 73.6 and 70.7 kDa proteins in the sarcoplasmic fraction and a concomitant increase in these proteins in the myofibrillar fraction. Changes in the myofibrillar calpain 10 proteins were strongly correlated with the rate of tenderization. To conclude, calpain 10 proteins are present in sheep LTL and the sub-cellular distribution is dynamic during post-mortem storage.     

极限pH对羊肉宰后成熟过程中肌原纤维蛋白特型的影响

为了研究羊肉宰后成熟过程中极限pH对肌原纤维蛋白特型即肌联蛋白、伴肌动蛋白、肌间线蛋白和肌钙蛋白-T降解及肌原纤维小片化指数的影响。本文选取50只羊的右侧背最长肌,贮存于4 ℃条件下,在宰后时间点分别为1 h、1、2、3、5 d和7 d时,测定其pH。按照宰后2 d的pH将肉样分成三组:高极限pH组(5.72±0.03),中极限pH组(5.54±0.01)和低极限pH组(5.40±0.02)。在每个宰后时间点,测定肌联蛋白、伴肌动蛋白、肌间线蛋白、肌钙蛋白-T降解程度和肌原纤维小片化指数(MFI)。结果表明:肌联蛋白在高极限pH组中宰后1 d开始降解;在宰后1 d时,高极限pH组肌间线蛋白相对灰度值显著低于中极限pH组和低极限pH组(p<0.05);肌钙蛋白-T在高极限pH组中,宰后1 d已出现降解条带。而伴肌动蛋白在中极限pH组中降解较快,在宰后1 d开始降解。另外在宰后1、2、3、5、7 d时,高极限pH组和中极限pH组的肌原纤维小片化指数显著高于低极限pH组的肌原纤维小片化指数(p<0.05)。极限pH通过影响这些肌原纤维蛋白降解来促进宰后肌肉成熟过程并且肌联蛋白、肌间线蛋白和肌钙蛋白-T的降解,加快了宰后前期嫩化过程。这为揭示宰后肉嫩度形成机理提供理论基础。