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.     

Postmortem Changes in Mule Duck Muscle Marinated in Red Wine

Postmortem changes in duck breast muscle as affected by red wine marination at 5 °C were studied. Myofibrils were purified from the control (CON) and the red wine marinated muscle (RW) after 0, 1, 3, 7, and 14 d storage. The results showed that myofibril fragmentation index (MFI) was significantly (p < 0.05) higher in RW than in CON samples. Myosin heavy chain, α‐actinin, troponin‐T, titin, and nebulin were degraded more rapidly as seen on SDS‐PAGE in RW than in CON samples. Our results suggested that red wine marination can cause postmortem changes in mule duck breast muscle.     

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.     

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.     

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。     

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.     

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.     

骨架蛋白降解对冰鲜草鱼质构的影响

利用免疫组化技术检测草鱼背肌白肉中骨架蛋白在冰藏条件下的变化,同时通过超微结构、剪切力、滴水损失率分析草鱼背肌组织精微结构和质构特性的变化,以期阐明草鱼骨架蛋白降解与鱼肉质构劣化之间的关系,从而为调控冰鲜淡水鱼品质提供理论依据。结果显示：冰藏21 d内伴肌球蛋白、伴肌动蛋白、抗肌营养不良蛋白分别降解了76.10%、78.21%、71.14%;冰藏14 d后草鱼肌纤维明暗带模糊,Z带、M带被破坏,肌纤维结构出现严重断裂松散现象。冰藏10 d内剪切力由81 N快速下降至32 N,滴水损失率由1.6%增加至7.8%;将骨架蛋白的灰度与剪切力及滴水损失率进行相关性分析发现,剪切力与伴肌球蛋白、抗肌营养不良蛋白的灰度呈极显著正相关（P＜0.01）;滴水损失率与伴肌球蛋白、抗肌营养不良蛋白的灰度值呈极显著负相关（P＜0.01）。综上所述,骨架蛋白降解可能是草鱼在冰藏期间肌纤维结构破坏以及质构劣化的重要原因。     

CATHEPSIN D AND ITS EFFECTS ON MYOFIBRILLAR PROTEINS: A REVIEW1

The molecular and enzymatic properties of the extensively studied enzyme cathepsin D are reviewed and additional information concerning its activity presented. Cathepsin D at pH 5.5 (37°C) degraded several myofibrillar proteins. The most rapidly hydrolyzed included titin and perhaps nebulin, myosin heavy chain, and M and C-proteins. The effects of cathepsin D on myofibrillar structure under these conditions included reduction in A band width, cleared central region in the A band, and dislocation of the Z line. Temperature was found to exert a strong influence on activity of cathepsin D and maximum activity was observed at 45°C with both muscle and hemoglobin substrates. Activity was evident at even higher temperatures and approximately 49% remained at 55°C (hemoglobin assay). Low temperature (i.e., < 15°C) however, has been observed to result in almost complete inactivity of the enzyme. The implications of this information for involvement of cathepsin D in postmortem proteolysis and tenderization were discussed.     

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.     

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     

Degradation of structural proteins and their relationship with the quality of Mandarin fish (Siniperca chuatsi) during post-mortem storage and cooking

This study was carried out to investigate the changes in the microstructure and structural proteins (titin, nebulin, desmin and tropomyosin (TPM)) of mandarin fish muscle as well as the relationship between the structural proteins and the quality characteristics of fish muscle during post-mortem storage and after cooking. During post-mortem storage, titin was degraded initially, accompanied with an increase of the I-band length. Subsequently, the degradation of desmin induced a gradual breakage of the myofibrils. The following degradation of nebulin accelerated the destruction of N₂-lines, which occurred with a slightly fuzzy Z-disc. Finally, TPM began to degrade, and the remaining desmin was also degraded further. Principal component analysis (PCA) and correlation analysis indicated that the changes in the quality characteristics of fish muscle were intimately related to the degradation of structural proteins. Hence, these structural proteins could be the biomarkers to monitor the quality characteristics of fish muscle.     

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     

Post mortem changes in porcine longissimus muscle incubated with 0.1 M calcium lactate

The effects of calcium lactate incubation at 5 °C on post mortem changes in porcine longissimus muscle were studied. Myofibrils were purified from control (CON) and calcium lactate‐incubated (CL) muscles. Samples were taken at 0, 1, 3, 7 and 14 days post mortem. SDS‐PAGE results showed that the disappearance of titin, nebulin, tropomyosin and troponin‐T and the appearance of a ～30 kDa component were more pronounced in CL samples than in CON samples. Western blots labelled with a monoclonal antibody to desmin also demonstrated that desmin degraded more quickly in CL samples. Our data suggested that calcium lactate incubation might accelerate the post mortem changes in porcine longissimus muscle via activation of calpains. © 2001 Society of Chemical Industry     

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.     

Lipid Oxidation Potential of Beef, Chicken, and Pork

Beef and pork longissimus dorsi and semimembranosus muscles and chicken breast and thigh muscles were excised 24 hr postmortem from carcasses of marketweight grain-finished feedlot beef cattle, marke-tweight hogs on a typical finishing diet, and broilers on a commercial grain diet. Muscle samples were immediately ground and formed into patties and stored raw or after cooking, at 4°C (cooked) or ?20°C (raw and cooked). TBA values (on sample weight basis) of frozen raw samples were higher for beef and pork than for chicken, as was heme iron content. However, TBA values of cooked samples were highest for chicken thigh muscles, which contained the most polyunsaturated fatty acids, at all storage temperatures.     

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.     

THE DEGRADATION OF MYOFIBRILLAR PROTEINS IN BEEF AND LAMB USING DENATURING ELECTROPHORESIS - AN OVERVIEW

Degradation of specific myofibrillar proteins has been followed in many studies using SDS-PAGE (denaturing) electrophoresis and these have shown that proteins such as titin, nebulin, troponin-T, desmin, filamin and vinculin are degraded at different rates during postmortem storage of meat. Although informative, electrophoresis is usually restricted to qualitative interpretation of the gels and there have been few studies that have quantitatively linked the degradation of specific proteins to changes in toughness. Improved quantitative methods (including scanning densitometry, image analysis, and modeling approaches) will be necessary to determine the protein and/or protein alterations that cause postmortem tenderization. This approach is complementary to other measurements of proteolysis where the objective is to more adequately explain the variation in toughness.     

Postmortem changes in myofibrillar-bound calpain 3 revealed by immunofluorescence microscopy

An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.     

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.