Postmortem Storage Research Articles

The calpain system in three muscles of normal and callipyge sheep.

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibition of the calpains in postmortem muscle is modulated in some as yet unknown manner.

Myofibrillar Structural Weakening and Tenderization of Uncooked Cold Shortened Bovine Muscle during Postmortem Storage

It is known that bovine muscles stored before the rigor mortis in low temperature cause the cold shortening. This study was carried out in order to clarify the relation of weakening of myofibrillar structure during storage on meat tenderization of cold shortened bovine semimembranous muscle. The muscle was excised immediately after slaughter from the dressed carcass and divided into muscle blocks, and then stored antiseptically at 4°C. The muscle restrained for two days on the carcass was excised on day 3 postmortem and similarly treated. The value of breaking strength and elasticity of both muscles gradually decreased with the postmortem time. However, the values of both breaking strength and elasticity in the excised muscle decreased but less than the restrained muscle, showing that the cold shortening developed in the immediately excised muscle. Measurement on the weakening of the myofibrillar structures of the cold shortened muscle showed that the weakening of the Z-disks progressed linearly after 3 days postmortem, agreeing with decline of the stiffness of the muscle. The weakening of the interaction between actin and myosin that affects sarcomere length hardly occurred throughout the storage time. And, the degradation of α-connectin rapidly progressed after 4 days postmortem. These results suggest that the weakening of the Z-disks and the degradation of connectin filaments were related to the tenderization of uncooked cold shortened bovine muscle during postmortem storage.

Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.

Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep.

Properties of the calpain bound to myofibrils in longissimus muscle from callipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of postmortem storage at 4 degrees C. Western analysis has shown that most of this calpain is mu-calpain, although the sensitivity of the antibodies used in the earlier studies could not eliminate the possibility that up to 10% of the calpain was m-calpain. The calpain is bound tightly, and very little is removed by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total mu-calpain was bound to myofibrils from at-death muscle, and this increased to approximately 40% after 1 d postmortem. The amount of myofibril-bound mu-calpain increased only slightly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu-calpain increases with time postmortem until after 10 d postmortem, when all myofibril-bound mu-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the specific activity of extractable mu-calpain from the same muscle. It is unclear whether this low specific activity is the result of unavailability of the active site of the myofibril-bound calpain to exogenous substrate. The myofibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha-actinin and undergoes additional autolysis when incubated with Ca2+; all these activities occurred slowly considering the amount of myofibril-bound calpain. Activity of the myofibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibitors, E-64 and iodoacetate; was more effectively inhibited by a broader-based protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 to 45%) by calpastatin. Release of undegraded alpha-actinin and autolysis are properties specific to the calpains, and it is unclear whether some of the myofibril-bound proteolytic activity originates from proteases other than the calpains or whether the active site of myofibril-bound calpain is shielded from the inhibitors. Activities and properties of the myofibril-bound calpain were identical in longissimus muscle from callipyge and normal sheep, although previous studies had indicated that the "normal" longissimus was much more tender than the callipyge longissimus. Hence, it seems unlikely that the myofibril-bound calpain has a significant role in postmortem tenderization of ovine longissimus.

Histochemical characterization of cytotoxic brain edema. Potassium concentrations after cerebral ischemia and during the postmortem interval

Cytotoxic edema is a phenomenon of the ischemically damaged brain. In the present study we tested a histochemical method that detects this phenomenon based on potassium (K+) levels in the brain. In a first series focal cerebral ischemia was induced by arterial occlusion in 23 gerbils (Meriones unguiculatus). After survival times of 30, 60 and 120 min, the animals were killed and brain section histochemically stained for potassium and quantitatively evaluated with a morphometric method. The results were compared with those using physicochemical techniques. A distinct K+ depletion could be demonstrated in the area of the focal ischemia within a survival time of 30 min, the depletion growing thereafter with increasing survival time. In a second series histochemical and chemical methods were used to study the stability of K+ levels in undamaged brains of 15 healthy rats during postmortem intervals of 2.5 and 5 h. Within these intervals K+ levels were clearly depleted, apparently as a result of cerebral spinal fluid (CSF) diffusion. Even if neuronal injury can be demonstrated histochemically after very brief survival times of about 30 min, postmortem storage of the cadavers rendered detection impossible due to electrolyte and water diffusion. In autoptic human cases, therefore, this technique is of no practical utility in detecting cytotoxic brain edema in postmortem tissue.

Ionic strength-induced inactivation of mu-calpain in postmortem muscle.

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.

Rigor temperature and meat quality characteristics of lamb longissimus muscle.

The present experiment was conducted to determine the effect of muscle temperature during the prerigor and early postrigor period on meat tenderness, postmortem proteolysis, calpain system activity, water-holding capacity, and color. Lamb longissimus muscle (n = 14) from the right and left carcass sides was excised immediately after dressing, divided into an anterior and posterior sample, vacuum-packaged, and stored overnight at 5 to 35 degrees C. Further storage, up to 14 d postmortem, was at 2 degrees C. Tenderness at 1 d postmortem, tenderization during further storage, and postmortem proteolysis were negatively affected by overnight incubation above 25 degrees C. This effect could be explained by an effect of temperature on muscle contraction and activity of the calpain system. Muscle contraction was at a minimum after incubation at 15 degrees C. Water-holding capacity was negatively affected by incubation above 25 degrees C. Color scores improved with increasing incubation temperature at 1 d postmortem. However, after 14 d of postmortem storage, no differences in color scores were observed. Based on the present results and results of other groups, a temperature around 15 degrees C at the onset of rigor seems optimal to maximize tenderness without having detrimental effects on water-holding capacity or color.

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.

Desmin Degradation in Postmortem Fish Muscle

ABSTRACTThe degradation of desmin was studied in postmortem white dorsal muscle from sea bass, brown trout, turbot and sardine, stored at 0–4°C. Based on desmin specific antibody tests, results showed that the extent and rate of desmin degradation in postmortem fish muscle varied considerably among species. There was no degradation of desmin during the first 4 days postmortem storage of sea bass and brown trout, whereas 10 to 20% of muscle desmin was degraded during the first 4 days of postmortem storage of turbot. Sardine muscle presented a very complex pattern of desmin degradation products and aggregated desmin fragments detected within the first 24h. Most polypeptides were larger than the nondenatured desmin. Desmin degradation would not be a suitable marker for evaluation of postmortem changes of stored fish.

Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.

Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle.

Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (P<.05) in A than in F1 B x A and B carcasses. Calpastatin activity was higher (P<.05) in muscle from B than in muscle from A and F1 B x A steers, and postmortem storage (O vs 48 h) and CaCl2 injection reduced (P<.05) the activity of the calpains and calpastatin. Strip loin and top sirloin steaks from A and F1 B x A steers were more tender (P<.05) than steaks from B steers; however, top round steak tenderness did not differ (P>.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts.

Application of labeling techniques to archival temporal bone sections.

Pathology of the human inner ear has traditionally been studied in celloidin-embedded, hematoxylin and eosin-stained sections of the temporal bone. Although the traditional histologic approach has yielded valuable information, it is now possible to extend these studies to include analysis of molecules using immunohistochemical and histochemical staining techniques. Fourteen antibodies and 6 lectins have been applied to 420 archival, celloidin-embedded human temporal bone sections. Tissues had been fixed in 10% formalin, embedded in celloidin, sectioned, and stored for as many as 40 years. The staining intensities varied among sections, so they were ranked from 'no label" to "dense label." To investigate the relationships between the extent of postmortem changes (PMCs), storage time, and staining intensity for each antibody, the sections were graded according to their PMCs, which ranged from good preservation of the temporal bone histologic structure to severe postmortem autolysis. Although statistical analysis indicated that both extent of PMCs and storage time in general decrease the staining intensity, both poorly fixed tissue and sections stored for a long time can yield good immunostaining results with some antibodies.

The extent of proteolysis is independent of sarcomere length in lamb longissimus and psoas major.

The objective of this experiment was to determine the effect of sarcomere length on postmortem proteolysis and meat tenderness. Eighteen Dorset market-weight sheep were slaughtered conventionally. The longissimus thoracis et lumborum and psoas major from each carcass were either left intact on the carcass (control), which was chilled at 0 degrees C, or excised from the carcass and chilled in an ice slurry (0 degrees C). At 24 h, control muscles were excised, and all muscles were cut into sections and assigned to 1 or 10 d of postmortem storage at 2 degrees C. Sarcomere length was shorter (P < .01), as intended, in the shortened relative to the control treatment and in longissimus relative to psoas major (1.36 vs 1.69 microm, raw longissimus; 1.45 vs 3.03 microm, raw psoas major). Sarcomere length was not affected (P > .05) by aging time. Western blot analysis of troponin-T and desmin indicated no effect (P > .05) of the shortened treatment compared to the control on the extent of proteolysis. Regardless of aging time or treatment, troponin-T was more degraded (P < .01) in longissimus than in psoas major (38.1 vs 23.5%) and desmin tended to be more degraded (P = .08) in longissimus than in psoas major (50.4 vs 35.1%). Regardless of muscle or treatment, aging 10 d compared to 1 d increased degradation of troponin-T (46.3 vs 15.3%) and desmin (69.3 vs 16.1%). Warner-Bratzler shear force was greater (P < .01) in the shortened treatment than in control (6.9 vs 3.8 kg), greater (P < .01) in longissimus than in the psoas major (6.5 vs 4.2 kg), and greater (P < .01) with 1 d than with 10 d of aging time (6.1 vs 4.6 kg). A muscle x aging time interaction (P < .05) indicated shear force declined more in longissimus than in psoas major during aging. We conclude that sarcomere length did not affect the extent of proteolysis. However, sarcomere length may have an indirect effect on tenderization during aging due to its effect on initial tenderness.

Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

Changes in the calpains and calpastatin during postmortem storage of bovine muscle.

Changes in activity and protein status of micro-calpain, m-calpain, and calpastatin in bovine semimembranosus muscle during the first 7d of postmortem storage were monitored by using assays of proteolytic activity, SDS-polyacrylamide gel electrophoresis, and Western blot analysis. Extractable m-calpain activity changed slightly during the first 7d after death (decreased to 63% of at-death activity after 7d), whereas extractable calpastatin activity decreased substantially (to 60% of at-death activity after 1d and to 30% of at-death activity after 7d of postmortem storage) during this period. Extractable micro-calpain activity also decreased rapidly (to 20% of at-death activity at 1d and to less than 4% of its at-death activity at 7d after death) during postmortem storage. Western blot analysis showed that the 80-kDa subunit of m-calpain remained undegraded during the first 7d after death but that the 125- to 130-kDa calpastatin polypeptide was gone entirely at 7d after death. Hence, the calpastatin activity remaining at 7d originates from calpastatin polypeptides that are 42 kDa or smaller. The 80-kDa micro-calpain subunit was almost entirely in the 76-kDa autolyzed form at 7d after death; this form is proteolytically active in in vitro systems, and it is unclear why the postmortem, autolyzed micro-calpain is not active. Over 50% of total muscle micro-calpain is tightly bound to myofibrils 7d after death; this micro-calpain is also nearly inactive proteolytically. Unless postmortem muscle contains some factor that enables micro-calpain in this muscle to be proteolytically active, it is not clear whether micro-calpain could be responsible for any appreciable postmortem myofibrillar proteolysis.

Effects of postmortem storage on the ultrastructure of the endomysium and myofibrils in normal and callipyge longissimus.

These experiments were conducted to examine ultrastructural changes in longissimus from normal and callipyge lamb during 14 d of postmortem storage at 4 degrees C. Six crossbred ewe lambs (1/2 Dorset x 1/2 Romanov) were grain-fed and slaughtered at approximately 250 d of age. Leg conformation score was the basis for classifying carcasses into normal and callipyge. The normal and callipyge longissimus had mean Warner-Bratzler shear force of 2.8 (2.7, 2.4, and 3.4) and 9.0 (12.2, 6.9, and 7.9) kg, respectively, after 14 d of postmortem storage. The results of transmission electron microscopy demonstrated ultrastructural changes, including sarcolemma detachment, loss of myofibril lateral attachments, and I-band breaks in normal longissimus. Detachment of sarcolemma from myofibrils occurred in both phenotypes, but it was delayed by several days in callipyge longissimus. Thus, the sarcolemma detachment seems not to contribute significantly to postmortem tenderization. The endomysium of both phenotypes did not change with postmortem storage. In normal longissimus, the percentage of fractured I-bands increased from 0% at d 1 to 11% at d 3 (P<.05) and did not change between 3 and 14 d (15%) postmortem (P>.05). However, postmortem storage did not affect (0 to 3%) the frequency of the I-band breaks in the callipyge longissimus (P>.05). Therefore, the break in the I-band region in postmortem muscle is a change that is associated with postmortem tenderization. We conclude that the major factor responsible for the toughness of meat from callipyge longissimus is the postmortem stability of myofibrils.

Effect of prerigor freezing and postrigor calcium chloride injection on the tenderness of callipyge longissimus.

The effect of rapid prerigor freezing and postrigor freezing and postrigor calcium chloride injection on the tenderness of callipyge longissimus was studied. Ewe and wether lambs (n = 49; 1/2 Dorset x 1/2 Romanov) were grain-fed and slaughtered at approximately 250 d of age. Based on leg conformation scores, 23 of the carcasses had the callipyge phenotype. Within each phenotype, approximately one-half of the carcasses were chilled conventionally (24 h at -2 degrees C). At approximately 17 min postmortem, the remaining carcasses were submersed in liquid nitrogen for 15 min and then held at -2 degrees C for 4 d. At 1 d postmortem for carcasses chilled conventionally and at 4 d postmortem for carcasses frozen in liquid nitrogen, the longissimus muscles from both sides were removed. The longissimus from one side of each carcass was vacuum-packaged and aged (1 degrees C) conventionally for 7 or 14 d. The remaining muscles were injected with a 2.22% solution of food-grade calcium chloride at 5% by weight, vacuum-packaged, and aged as above. Liquid nitrogen freezing was effective in limiting sarcomere shortening (1.99 vs 1.63 microm; P < .05). Warner-Bratzler shear force values of callipyge longissimus were 222 and 232% of that of normal longissimus after 7 and 14 d postmortem, respectively (P < .001). Also, trained panel tenderness rating was decreased by 49.4% in untreated callipyge longissimus after 14 d postmortem. Liquid nitrogen, calcium chloride injection and their combination did not affect d-14 longissimus shear force and sensory tenderness for normal lambs because untreated muscles were already tender. Liquid nitrogen freezing improved the shear force and sensory tenderness rating of callipyge longissimus by 30 and 86.2% after 14 d postmortem, respectively. Calcium chloride injection improved the shear force and sensory tenderness of callipyge longissimus by 36.7 and 86.2% after 14 d postmortem, respectively (P < .001). The most effective treatment for mitigating the callipyge effect on tenderness was the combination (freezing and calcium chloride injection) treatment, which improved the shear force and sensory tenderness by 51.2 and 124.2% after 14 d postmortem, respectively (P < .001). We conclude that either treatment can effectively mitigate the negative effect of callipyge phenotype on longissimus tenderness. Callipyge lamb carcasses subjected to the combination of prerigor liquid nitrogen freezing, postrigor calcium chloride injection, and 14 d postmortem storage had tenderness similar (P > .05) to that of normal, untreated carcasses after 14 d of postmortem storage.

Effect of Freezing on Lens Capsule Mechanical Behavior

Postmortem storage of lens capsules by freezing was found to increase capsular thickness slightly (5%). The effect on mechanical properties was minor. Ultimate tensile strength and ultimate stiffness were not changed by freezing. Ultimate strain was slightly increased (2%) and the elastic modulus calculated at 40% strain was decreased (20%). Frozen capsules were found to relax to a slightly lower value than fresh ones (2%). The results show that freezing tends to soften the tissue, possibly due to an increased hydration. In spite of this effect, which has to be kept in mind, postmortem freezing seems to be an acceptable storage method when collecting tissue for biomechanical research.

The formation of non-volatile amines in relation to concentrations of free basic amino acids during postmortem storage of the muscle of scallop ( Pecten maximus), herring ( Clupea harengus) and mackerel ( Scomber scombrus)

The concentrations of the non-volatile biogenic amines that form during storage of the adductor muscle of scallop ( Pecten maximus ) and the skeletal muscles of mackerel (Scomber scombrus) and herring ( Clupea harengus ) were determined by high-performance liquid chromatography, using postcolumn derivatisation with o-phthalaldehyde. Initially, concentrations of amines were low in all muscles but, as time of storage progressed, the concentration of agmatine increased steadily in scallop muscle reaching a level of 100 mg per 100 g wet weight after 10 days of storage in ice or 3 days at 10°C. In mackerel and herring only slight increases were observed, with levels of less than 1.0 mg after 10 days in ice or 2 days at 10°C. In scallop, arginine, the precursor of agmatine, decreased steadily in concentration. In herring muscle, histidine, the precursor of histamine, decreased considerably in concentration, whereas in mackerel it changed only slightly. © 1997 Published by Elsevier Science Ltd