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.     

The Effect of µ/m‐Calpain on Protein Degradation of Chicken Breast Meat

This study was designed to determine the effects of µ/m‐calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12 hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4 °C. The samples were used to evaluate troponin‐T and desmin degradation, calpain activity, and myofibril ultrastructure at 12 hr, day 1, day 3, and day 7. Casein zymography revealed that µ‐calpain could not be detected in all samples after 12 hr postmortem. The calpain inhibitor inhibited µ/m‐calpain activity and reduced troponin‐T and desmin degradation during 7 day postmortem. The caspase inhibitor inhibited µ/m‐calpain activity and, troponin‐T and desmin degradation before day 3 postmortem. The findings suggest that, µ/m‐calpain had an effect on cytoskeletal protein degradation after 12 hr postmortem.     

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.     

Influence of muscle type on the evolution of free amino acids and sarcoplasmic and myofibrillar proteins through the manufacturing process of Celta dry-cured ham

The effect of the muscle type [semimembranosus (SM) and biceps femoris (BF)] on the evolution of free amino acids and sarcoplasmic and myofibrillar proteins throughout the manufacture of Celta dry-cured ham was studied. A total of thirty Celta hams were analyzed, 5 in the green stage, 5 after the end of the salting stage, 5 after 120 days of post-salting, 5 after the end of the drying–ripening stage, 5 after 165 days and 5 after 330 days of “bodega” step. Possibly due to its higher moisture values, the BF muscle underwent higher protein degradation than the SM muscle. The average of total FAA content increased significantly (P < 0.001) from 567.3 and 570.3 mg/100 g of dry matter in the raw pieces to 7884.1 and 4595.9 mg/100 g of dry matter at the end of the “bodega” stage for BF and SM muscles, respectively. SDS-PAGE analysis also revealed that myofibrillar proteins were more intensely degraded in BF muscle, differences being more evident for the 48.9 kDa and 33.1 kDa proteins.     

EFFECT OF FROZEN TEMPERATURE AND STORAGE TIME ON CALPAINS, CATHEPSINS (B, B + L, H AND D) AND THEIR ENDOGENOUS INHIBITORS IN GOAT MUSCLES

The effects of frozen storage on the biochemical properties of myofibrils, muscle proteinases (cathepsins and calpains) and their endogenous inhibitors were investigated. Longissimus dorsi, biceps femoris, semimembranosus and semitendinosus muscles from goat were frozen (?15C) and studied up to 120 days. The results showed that the percentage change in sarcomere length was 8.4–13.1. The calpain activity was determined after separation on a diethylaminoethyl–Sephacel column (Sigma, St. Louis, MO). Significantly greater percentage of calpain II activity was recovered when compared to calpain I. There was a 15–25% loss in calpastatin inhibitory activity, and the cystatin level fell by 11–16% after 80 days. Cathepsin B, B + L, H and D were very stable when compared to calpains. The calcium concentration may also be the factor for calpain activation. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis result showed the appearance of 55 kDa components. It was concluded that calpains, not cathepsins, play an important role in the proteolysis of myofibrillar proteins at the freezing temperature.     

Effect of ion fluid injection on beef tenderness in association with calpain activity

Sodium pyrophosphate plus sodium chloride (PPi) was injected into pre-rigor, hot boned biceps femoris (BF) and semimembranosus (SM) muscle from 12 heifer carcasses. The PPi injection caused higher pH values between 10 and 48 h post-mortem than found in the controls for both muscles (P<0.05). PPi injection resulted in faster decreases in the activities of μ-calpain and calpastatin than in the control for both muscles with time post-mortem (P<0.05). There were significant differences between treatments in both the BF and SM (P<0.05). There was evidence that PPi-injection elevated pH, and accelerated activation of calpains, resulting in improved tenderness. The rates of degradation of titin and troponin-T as well as the appearance of 95 and 30 kDa peptides were faster in the PPi-injected muscles than the controls. PPi-injection elevated muscle pH, which was attributed to acceleration of the calpain activation. It is concluded that PPi-injection improved beef tenderness by accelerating activation of calpain.     

Influence of caspase3 selective inhibitor on proteolysis of chicken skeletal muscle proteins during post mortem aging

The objective of this study was to investigate the potential contribution of apoptosis related downstream executioner caspase3 to post mortem skeletal muscle proteolysis by use of caspase3 selective inhibitor DEVD–CHO (N-acetyl–Asp–Glu–Val–Asp–CHO). After slaughter, four chicken breast muscles were removed and cut into small pieces, then marinated in treatment solution containing DEVD–CHO, or in control solution, and stored at 4 °C for 1, 3 or 7 d. Meat samples were obtained and used for detecting muscle protein degradation or calpain activity. Results showed that DEVD–CHO had inhibited the degradation of muscle skeletal proteins (titin, nebulin, desmin and troponin-T) significantly, whereas the activity of calpains had not been influenced. Therefore, the degradation of muscle proteins should not been exclusively attributed to the calpain system, and the effector caspase3 may be a new protease involved in meat post mortem tenderization.     

Broad‐spectrum inhibition of proteolytic enzymes by allicin and application in mitigating textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets

The inhibitory effect of allicin on proteolytic enzymes and textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets was investigated. The results of in vitro study showed that allicin inhibited the activity of cathepsin B, L and D, calpain and collagenase in crude extract of grass carp muscle. Among endogenous enzymes, cathepsin B, L and collagenase were the most susceptible to allicin. Proteolysis of myofibrillar proteins by either crude enzyme or cathepsin B and L was almost prevented by allicin when employed at a concentration higher than 100 mm . After storage of 21 days, shear force of fillets treated with allicin at 10–100 mm was 39–51% higher than that of control. Myofibrillar proteins of fillets during storage were well protected against degradation when allicin concentration increased to 100 mm , as evidenced by SDS‐PAGE. Therefore, allicin could be a potential broad‐spectrum inhibitor to retard softening of fish fillets via mitigating myofibrillar proteolysis by endogenous enzymes especially cathepsin B and L during ice‐storage.     

Changes in degradation and phosphorylation level of titin in three ovine muscles during postmortem

The objective of this study was to investigate the degradation of titin and its phosphorylation level in three muscles from sheep. The MFI and pH from the longissimus lumborum (LL), semimembranosus (SM) and psoas major (PM) muscles were measured at 30 min, 1, 2, 7, 14, 21 and 28 days postmortem. Myofibrillar proteins were extracted, separated by SDS‐PAGE and quantified by phosphor‐specific staining. Phosphorylation of titin was predicted by Pro‐Q Diamond‐SYPRO Ruby staining. Two days after exsanguination, the pH and MFI of the PM were higher than those of the LL and SM muscles (P < 0.05). The sarcomere length of the PM muscle was also longer than that of the LL and SM muscle (P < 0.05). PM muscles had a highest phosphorylation level (P < 0.05) at 0.5 h postmortem and showed the greatest degree of titin degradation over 28 days. This suggests that phosphorylation of titin might accelerate its degradation.     

Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb

This study is concerned with the rate of protein turnover in the hind limb muscle bed of intact lambs, the activity of calpain proteolytic system in the M. biceps femoris, and subsequent rates of myofibre breakdown and tenderisation in the M. longissimus dorsi. Feed restriction increased protein degradation in hind-limb muscle of lambs (p<0.1), with a concominant decrease in the extractable activity of calpastatin (p<0.01), the endogenous inhibitor of calpain. IGF-1 analog treatment decreased both protein degradation and assayed μ-calpain activity (p<0.05) with no effect on the activity of calpastatin. β-Agonist treatment increased hind-limb protein synthesis (p<0.01), calpastatin activity (p<0.1) and decreased (p<0.01) μ-calpain activity, but did not effect protein degradation. Significant correlations were observed between Myofibril Fragmentation Index (MFI) values during post-mortem storage and initial post-slaughter calpastatin activity at days 3 (r=-0.34, p<0.1), 5 (r=-0.58, p<0.01) and 9 (r=-0.58, p<0.1), and μ-calpain activity at days 5 (r=0.35, p<0.1) and 9 (r=0.41, p<0.05). However, stronger correlations were observed between the ratio of μ-calpain to calpastatin, an estimate of potential μ-calpain proteolytic activity, and the rate of myofibril fragmentation (r=0.75, p<0.001) and tenderisation (r=-0.64, p<0.01) during aging. These results are consistent with the calpain system being the major proteolytic system involved in myofibril fragmentation and hence aging-related tenderisation of meat.     

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.     

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.     

Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua)

Collagenase activity and changes in muscular protein of iced Atlantic cod stored for 9 days were studied. The crude fish muscle extract showed maximum collagenase-like activity against bovine insoluble tendon collagen at 48 h of incubation at 37 °C. Collagenase activity against synthetic substrate increased (P<0.05), especially for fish in initial and advanced stages of decomposition. These results suggest that endogenous collagenases and other proteases may be responsible for the destruction of fine collagenous fibrils in the skeletal muscle of cod. The content of titin 1 decreased when decomposition was advanced. Moreover, a progressive degradation of sarcoplasmic proteins with a molecular weight of 100, 94, 85 and 80 kDa was observed. Results suggest that softening of cod muscle during iced storage is caused more by collagenase activity than by proteolysis of myofibrils.     

Influence of gender and spawning on thermal stability and proteolytic degradation of proteins in Australian red claw crayfish (Cherax quadricarinatus) muscle stored at 2 °C

Thermal stability and proteolytic degradation of male (M), nonspawning female (F) and spawning female (SF) red claw crayfish (Cherax quadricarinatus) muscle proteins during refrigerated storage (2 °C) were investigated. The thermal transition temperatures (T_max) of myosin and actin remained relatively constant during storage, but their enthalpies of denaturation (ΔH) increased, especially in SF samples. SF muscle proteins were more heat‐stable (greater T_max and ΔH values, P < 0.05) than M and F muscle proteins. Protein degradation occurred in all muscle groups, more rapidly in M and F muscles than in SF muscle. The diminishments of a 69‐kDa component and troponin‐I and the appearance of a 55‐kDa polypeptide represented the most salient proteolytic changes. The results suggested that the spawning status was a more significant factor than gender in affecting the quality of red claw muscle proteins and their changes during refrigerated storage.     

Comparative study on chemical composition,thermal properties and microstructure between the muscle of hard shell and soft shell mud crabs

Chemical composition, thermal properties and microstructure of the muscle from hard shell and soft shell mud crabs were studied. Both lump and claw muscle of soft shell mud crabs contained a lower protein content but higher moisture and salt contents than those from hard shell mud crab (p < 0.05). Calcium and magnesium were the major minerals in the muscle of hard and soft shell mud crabs (240.5–699.2 ppm). Approximately one-third of calcium was observed in the muscle of soft shell mud crab, compared to that of hard shell mud crab. Copper, iron and zinc were the trace minerals with the amount less than 50 ppm. Hydroxyproline content ranged from 7.92 to 8.88 mg/g wet muscle in all samples, except claw muscle from soft shell mud crab, which contained considerably low hydroxyproline content (2.75 mg/g wet muscle). Sarcoplasmic and alkali-soluble proteins were the major constituents in all muscles, except claw muscle from soft shell mud crab, in which sarcoplasmic protein was the dominant component. T_max from myosin of lump muscle were 47.58–48.08 °C with enthalpy of 0.20–0.21 J/g, whereas myosin from claw muscle had lower T_max (45.00–47.48 °C) with lower enthalpy (0.17–0.18 J/g). Lump muscle bundles of hard and soft shell mud crabs aligned orderly, while claw muscles of both crabs had partial disintegrations and the porous structure was observed in that from soft shell crab.     

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.     

Changes on enzymatic activity and on sarcoplasmic and myofibrillar proteins of frozen-stored hake (Merluccius merluccius) pre-treated by high pressure

High-pressure (HP) pre-treatments were applied on European hake (Merluccius merluccius) followed by a frozen accelerated experiment (−10 °C). A central composite design ranging pressure levels (150–450 MPa) and frozen storage time (0–150 days) was used, being evaluated the enzymatic activities and muscle proteins. Acid phosphatase and calpain activities decreased after 150 days of frozen storage (58%/56% and 38%/56% for non-/HP-treated samples, respectively). Cathepsin B showed higher reductions (98%) for longer storage times. Furthermore, HP and frozen storage did not affect significantly cathepsin D activity, only slightly decreasing at 169 MPa. Furthermore, HP seemed to not affect myofibrillar proteins, while sarcoplasmic proteins were clearly affected by HP and frozen storage time, resulting in a reduction of about 53% or 23% for 431 or 450 MPa, respectively. Thus, HP could be used to lowering the deleterious effect of proteases on frozen European hake.     

Colour,lipid and protein stability of Rhea americana meat during air- and vacuum-packaged storage: Influence of muscle on oxidative processes

Physicochemical characteristics and oxidative stability during storage were determined in Gastrocnemius pars interna (GN) and Iliofiburalis (IF) muscles of Rhea americana. Glycolytic potential (GP) and pH decline of muscles were measured within the first 24 h post mortem. Colour, lipid and protein stability were determined during storage of meat, i.e. 5 days under air-packaging at 4 °C, or 28 days under vacuum-packaging at 4 °C. In parallel, anti-oxidant status of muscles was estimated by measuring α-tocopherol content and anti-oxidant enzyme activities (superoxide dismutase and catalase), while pro-oxidant status was evaluated by determining haeminic iron and long chain fatty acids (especially polyunsaturated fatty acids). The ultimate pH was similar in both muscles, but the GP value was significantly higher in IF than in GN muscle. Haeminic iron and alpha-tocopherol content differed between muscles, with 30% more haeminic iron (p < 0.05) and 134% more alpha-tocopherol (p < 0.001) in IF than GN muscle. The IF muscle presented higher lipid content and lower PUFA/SFA ratio (polyunsaturated fatty acids/saturated fatty acids) than GN muscle. With storage under air-packaging, lipid and protein oxidation of rhea muscles increased up to 275% and 30%, respectively. This increase was more rapidly and marked in IF muscle. The IF also showed high level of metmyoglobin accumulation after 3 days of storage (47%) and was rejected by 1 consumer out of 2 in sensorial analysis. Under vacuum-packaging, both muscles showed a high stability of colour and no oxidation of lipids and proteins.     

Calpain and cathepsin activities in post mortem fish and meat muscles

Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and this contrasts with muscle of mammalian meats. The tenderization can be partly attributed to the acid lysosomal cathepsins and cytosolic neutral calcium-activated calpains. In this study, these proteases from fish and bovine muscles were quantified and compared. The cathepsin B and L activities were in more important amounts in sea bass white muscle than in bovine muscle. On the other hand, cathepsin D activity was 1.4 times higher in meat that in fish muscle, while cathepsin H was negligible in both muscles. Calpain activities were similar in both types of muscle. Moreover, calpastatin (calpain endogenous inhibitor) level is 3.9 times higher in sea bass white muscle. These differential activities are considered in relation to their probable involvement in post mortem degradation of muscle.     

Postmortem degradation of desmin and calpain in breast and leg and thigh muscles from Taiwan black‐feathered country chickens

BACKGROUND: Several studies have reported that the postmortem changes are more rapid in breast muscles (BM) than in leg and thigh muscles (LM) of chickens. However, the reasons for the differences in postmortem proteolysis of BM and LM are still uncertain. The purpose of this study was therefore to compare the postmortem degradation of desmin and calpains in BM and LM from Taiwan black‐feathered country chickens at 5 °C. RESULTS: The pH was lower (P < 0.05) in BM than in LM. Western blot indicated that postmortem desmin degradation was more rapid in BM than in LM. Casein zymograms showed that at‐death µ‐calpain activity was higher in BM than in LM. As postmortem time proceeded, µ‐calpain was activated and autolyzed more extensively in BM than in LM. However, the µ/m‐calpain activity remained stable during postmortem storage in both BM and LM. CONCLUSION: Our results suggest that the more rapid postmortem proteolysis found in BM than in LM at 5 °C similar with the previous study could be mainly explained by both greater amounts and faster activation and autolysis of µ‐calpain in BM. Copyright © 2010 Society of Chemical Industry