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.     

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 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.     

Solubility of Muscle Proteins as a Result of Autolysis and Microbiological Growth

Bovine Longissimus dorsi samples, collected aseptically 3 hr postmortem, were used to determine natural muscle enzyme activity. Samples of this tissue were inoculated with Pseudomonas putrefaciens or Lactobacillus casei and incubated 0, 1, 7, 14, and 21 days at 2°C and 0, 1, and 3 days at 25 and 37°C. Protein solubility, pH and microbial counts were determined after appropriate periods of storage. Only small differences were noted in the solubility of muscle proteins between the muscle treatments after 21 days of storage at 2°C compared to much larger differences noted after only 3 days of storage at higher incubation temperatures (25 and 37°C).     

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 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.     

Physicochemical and microbiological changes in postmortem crayfish (Procambarus clarkii) stored at 4 °C and 25 °C

The physicochemical and microbiological parameters of postmortem crayfish stored at 4 °C and 25 °C were evaluated in order to reduce safety risks of crayfish from temperature abuse during transportation and storage. Results showed that hepatopancreas of postmortem crayfish had significantly higher contents of total volatile basic nitrogen, biogenic amines (BAs) and higher microbial loads than tail meat, regardless of storage temperature. Enterobacteriaceae and Pseudomonas counts reached log 6.41 log₁₀ CFU g⁻¹ and 6.31 log₁₀ CFU g⁻¹ in hepatopancreas of crayfish at 25 ℃ for 6 h. Putrescine and cadaverine were the main BAs in tail meat with levels of 28.18 ± 0.73 mg kg⁻¹ and 187.32 ± 3.57 mg kg⁻¹, respectively, whilst cadaverine, spermidine and spermine were the major BAs in hepatopancreas, reaching 283.45 ± 3.95 mg kg⁻¹, 62.87 ± 9.02 mg kg⁻¹ and 155.31 ± 4.55 mg kg⁻¹, respectively, after postmortem storage at 25 °C for 12 h. With time increasing, Acinetobacter, Flavobacterium, Aeromonas and Chryseobacterium at 25 °C and Acinetobacter, Flavobacterium, Psychrobacter at 4 °C in tail meat as well as Bacteroides and Muribaculaceae at 25 °C, and Acinetobacter, Psychrobacter at 4 °C in hepatopancreas, gradually became the major genus at the end of storage. Based on the results of spoilage microorganisms and biogenic amines, crayfish stored at 4 °C and 25 °C could be edible within 24 h and 6 h, respectively.     

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     

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.     

Effect of Postmortem Storage on Cold-Shortened Bovine Muscle: Analysis by SDS-Polyacrylamide Gel Electrophoresis

Myofibrils were isolated from the longissimus (L) muscle of control (CON) and cold-shortened (CS) muscles after 0, 1, 3, 7, and 10 days of postmortem storage at 2°C. Isolated myofibrils were then examined by SDS-polyacrylamide gel electrophoresis to monitor the changes in the myofibrillar proteins during postmortem storage. The main changes in CS muscles were the gradual appearance of 110,000-, 95,000-, and 30,000d-dalton components and the disappearance of desmin and troponin-T components of myofibrils. In addition, there was a gradual increase in the intensity of a protein around 55,000-daltons. CON samples showed similar changes to those of CS samples. It appears that myofibrillar proteins of cold-shortened muscles are affected by postmortem aging in a manner similar to that of the normally chilled muscles.     

Effects of Postmortem Storage and Temperature on Muscle Protein Degradation: Analysis by SDS Gel Electrophoresis

The proteolytic breakdown of the major contractile proteins of bovine longissimus muscle was examined during postmortem storage by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Samples of muscle stored at 4°C for 14 days exhibited little proteolysis of the major contractile proteins; however, samples stored at 37°C for 1 day showed significant degradation of myosin heavy chains and almost complete proteolysis of this protein by day 14. Major degradation products of the myosin heavy chains included a series of polypeptides having molecular weights between 145,000 and 125,000. These experiments demonstrate that substantial degradation of the myosin heavy chain and other muscle proteins can occur during the storage of meat, and this phenomenon was highly temperature dependent.     

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.     

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.     

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.     

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.     

Effect of Electrical Stimulation on Postmortem Property Change of Myofibrillar Proteins

Changes in biochemical properties of myofibrillar proteins of rabbit muscle, which had been subjected to electrical stimulation soon after slaughter, during postmortem storage at 0°C were investigated. Myofibrillar ATPase activity and the ATPase activity of acto-heavy meromyosin (HMM) complex, reconstituted from actin and HMM which had been prepared from at-death and postmortem muscles, decreased at first and then increased slightly during 7 days storage. In addition, the change of the dissociation constant of acto-HMM complex of electrically stimulated muscle during postmortem storage was quite small, i.e., 1.59 ± 10^?4M for at-death muscle, 1.70 ± 10^?4M for muscle stored for 1 day and 1.49 ± 10^?4M for muscle stored for 7 days. This indicates that electrical stimulation treatment minimized the postmortem change of actin-myosin interaction.     

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     

Stability of pigments and oil in pistachio kernels during storage

This work tested the stability of pigments (chlorophyll [chl] a and chlorophyll [chl] b, and lutein] and oil in pistachio kernels stored up to 14 months at three different temperatures: 10, 25 and 37 °C. The samples were hermetically packaged using two films (nylon and ethylene vinyl alcohol) with and without oxygen scavengers. For each temperature, reference samples were packaged in open bags. For both the oil and pigments, no differences were observed during storage, irrespective of packaging or oxygen scavengers. After 14 months, the oil showed very few changes: a slight increase in acidity and peroxide value (PV) irrespective of storage temperature; the spectrophotometric indices K₂₃₂ and K₂₆₈ remained the same. As for pigment stability, the lowest concentrations were observed at 37 °C with a degradation of about 62% for chl a, 44% for chl b and 57.5% for lutein. At 10 and 25 °C, the samples showed slight differences, the pigments degradations were about 46% for chl a, 33% for chl b and 37% for lutein. The degradation rate constants for the three pigments fitted a pseudo‐zero‐order kinetic in which Ea was 11.7 kJ mol^?1, 12.1 kJ mol^?1 and 18.2 kJ mol^?1 for chl a, chl b and lutein respectively.     

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.     

Histamine Formation and Bacterial Spoilage of Albacore Harvested off the U.S. Northwest Coast

Iced and previously frozen albacore were monitored for histamine formation and bacterial growth during storage at 0–37°C. The optimum temperature for histamine formation in albacore tuna (Thunnus alalunga) was 25°C, and whole fish were more susceptible to histamine formation than dressed fish at that temperature. Storage at 25°C resulted in the highest histamine level, 60.4 mg/100g in whole fish stored for 7 days. When albacore were frozen prior to storage, reduced amount of histamine was found at 7.14 mg/100 g after 7 day storage at 25°C, only after decomposition became obvious. No histamine was found in any of the albacore samples stored in ice for 18 days.