Postmortem Storage Research Articles

Quality Comparison of Pork Loin and Belly from Three-way Crossbred Pigs during Postmortem Storage.

This study was performed to determine the meat quality characteristics of pork loin and belly from 3 different three-way crossbred pigs: Yorkshire × Landrace × Duroc (YLD), Yorkshire × Chester White × Yorkshire (YCY), and Yorkshire × Berkshire × Duroc (YBD). Each of the twenty crossed pigs were randomly selected with their live weights at the range of 110-120 kg. After being slaughtered and cooled at 0℃ for 24 h in a chilling room, the parts of loin and belly on the left side of the cooled carcasses were cut and prepared for analysis. The intramuscular fat contents of the loins from YLD were higher than those of the other crossbreds (p<0.05), and the YCY bellies had the highest moisture contents (p<0.05). Water holding capacity (WHC) of the loins from YCY were higher than the other crossbreds (p<0.05). Shear force values of the YBD loins were higher than the others (p<0.05). The TBARS values of YCY loins were significantly lower than the others at 0 d, but the difference disappeared after 14 d of storage. Sensory scores of YLD were ranked higher than the YCY or YBD in both the loins and bellies (p<0.05). The relatively high sensory values of YLD crossbred pork could be explained by the better WHC, the low shear forces, and the higher fat contents. The results indicated that the meat qualities could be altered by three-way crossbreeding.

Effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animal’s death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.

Activity of Endogenous Muscle Proteases from 4 Australian Underutilized Fish Species as Affected by Ionic Strength, pH, and Temperature

Storage conditions may influence the hydrolytic activity of endogenous muscle enzymes postmortem, rate of autolysis of myofibrillar proteins, and biological properties of hydrolyzed end products. This study investigated the effect of ionic strength, pH, and temperature on the activity of endogenous calpain-like, cathepsins B and B+L measured in crude extract obtained from deepwater flathead, silver warehou, ribaldo, and ribbonfish muscles. Activity of calpain-like enzymes in 3 examined species was significantly higher at pH 6.5 than pH 6.0 or 5.5. Raising the reaction temperature increased (P < 0.05) calpain-like activity in ribaldo. Endogenous activity of cathepsin B in ribbonfish and silver warehou declined significantly with increasing ionic strength at pH 6.5 to 6.0. The obtained results will further expand our understanding of the impact that postmortem storage conditions have on the activity of endogenous fish proteases with respect to quality and bioactivity of fish proteins and potentially diversify utilization of underutilized fish species.

Breast meat quality of broiler chickens can be affected by managing the level of nitric oxide

The objective of this study was to investigate the effects of nitric oxide (NO) on the quality of broiler chicken meat during postmortem storage. Eighteen broiler chickens were slaughtered and breast meat was randomly assigned to 1 of 3 treatments including the control group, nitric oxide synthase (NOS) inhibitor group, and NO enhancer group. The breast samples were incubated with water, NO enhancer, and NO inhibitor for 1 d and then stored at 4°C under atmospheric conditions for 4 and 7 d. Left side of breast meat was used to determine protein oxidation, lipid oxidation, water-holding capacity, and pH, whereas the right side was used for the measurement of color and drip loss. Breast meat from NO enhancer group showed the lowest water-holding capacity during refrigerated storage, whereas drip losses were not significantly (P > 0.05) different among 3 treatments. Lipid oxidation showed a significant difference (P < 0.05) only at 7 d in which NOS inhibitor group showed the highest, whereas the NO enhancer group showed the lowest levels of lipid oxidation. Carbonyl content in NO enhancer group was significantly higher than the control and NOS inhibitor treatment at 1 and 4 d of refrigerated storage. Lightness of breast meat in the NOS inhibitor group was greater, whereas redness was lower than the control and NO enhancer group at 4 and 7 d of refrigerated storage. The ultimate pH of 3 treatments was not significantly (P > 0.05) different from one another. These data indicate that NO could play a significant role in modulating the quality of fresh broiler breast meat during refrigerated storage.

Effects of tea polyphenols on the post-mortem integrity of large yellow croaker (Pseudosciaena crocea) fillet proteins

Tea polyphenols (TP) are known to be important for the post-mortem deterioration of fish muscle and can enhance food quality. To shed light on the influence of TP on the status of large yellow croaker muscle proteins, control and treated fillets (0.1% TP, 0.2% TP and 0.3% TP, w/v) were analysed periodically for myofibrillar protein functional properties (Ca2+-ATPase activity, surface hydrophobicity, total sulfhydryl content, emulsion stability index and rheological behaviour). Degradation of the myofibrillar protein myosin could be clearly observed; several proteins were also observed to vary in abundance following post-mortem storage for 25days. The present study offers new evidence that TP have an effective impact on muscle protein integrity post-mortem.

Chemical and Thermal Properties of Freshwater Prawn (Macrobrachium rosenbergii) Meat

Chemical compositions and thermal properties of cultured freshwater prawn meat (FPM) were studied. FPM contained 83.2% protein (dry basis), 62.7% of which was myofibrillar protein. Pepsin-soluble collagen (PSC) and insoluble collagen (ISC) contents were 0.63 and 0.32%, respectively. Both collagens were similar to type V collagen from porcine placenta. Glutamic acid/glutamine, arginine, aspartic acid/ asparagine, and lysine were abundant amino acids in FPM. Glycine, proline, hydroxyproline, and aspartic acid/ asparagine were predominant in both collagens. FPM exhibited thermal transition temperatures (Tmax) of 48.3 and 64.7°C, whereas Tmax of PSC and ISC were 43.0 and 46.0°C, respectively. Textural changes in FPM during post-mortem storage on ice are plausibly dependent upon its compositional and thermal properties.

Proteolytic activities in fillets of selected underutilized Australian fish species

The hydrolytic activity of major endogenous proteases, responsible for proteolysis of myofibrillar proteins during post-mortem storage, may be an indicator of the textural quality of fish which influences consumer purchasing behaviour and thus market value of the final product. Furthermore, it may also influence the type and bioactive properties of the peptides released during post-mortem proteolysis of myofibrillar proteins. This study compared the activities of cathepsins B, B+L, D, H and calpain-like enzymes in crude muscle extracted from 16 Australian underutilized fish species. Fish species had a significant effect on the activity of these enzymes with barracouta showing the highest cathepsins B, B+L, D and H activities. Activities of cathepsins B and B+L were higher than cathepsin H for all studied species. The more commercially important rock ling and tiger flathead demonstrated higher cathepsin B+L activity, whereas gemfish and eastern school whiting showed higher activity towards cathepsin B. Underutilized fish species showing higher endogenous protease activities may be suitable for fish sauce production, whereas those with lower protease activities for surimi processing.

μ-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle

Postmortem changes in proteins that have been implicated in affecting muscle integrity were examined in goose (GG) and duck (DG) gizzard smooth muscle stored at 5°C. GG and DG smooth muscles were sampled at 0, 1, 3 and 7day of storage. The pH was approximately 7 in both GG and DG samples during postmortem storage. Casein zymograms showed that 0-day μ-calpain activity was higher (p<0.05) in GG than in DG samples. As postmortem time progressed, μ-calpain was activated and autolyzed more extensively in GG than in DG samples. However, μ/m-calpain remained relatively stable in both samples. Western blots indicated that postmortem desmin degradation was more rapid in GG than in DG samples. In contrast, α-actinin remained nearly unchanged in both samples. Therefore, our results suggest that μ-calpain has an important role in the postmortem proteolysis of gizzard smooth muscle.

Proteomic Analysis of Duck Fatty Liver during Post-Mortem Storage Related to the Variability of Fat Loss during Cooking of “Foie Gras”

Fat loss during cooking of duck "foie gras" is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of postslaughter processing on fat loss during cooking, this study characterizes for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose the proteins were separated according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). Two-dimensional electrophoresis was used to analyze the S fraction and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups, respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. The shot-gun strategy was used to analyze the IS fraction, with the identification of all the proteins by mass spectrometry. This allowed identification of 554 and 562 proteins in the low-fat-loss and high-fat-loss groups, respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 × 10(-3)) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, these results suggest that the variability of technological yield observed in processing plants could be explained by different aging states of fatty livers during chilling, most likely associated with different proteolytic patterns.

Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca2+-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

Single Nucleotide Polymorphisms in Caprine Calpastatin Gene

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases-micro-calpain and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: 1) that have been previously reported to be polymorphic, intron 5 and 12 and 3'UTR; 2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SN Ps located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3'UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3'UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.

Correlation Between Electrical Conductivity of the Gutted Fish Body and the Quality of Bighead Carp (Aristichthys nobilis) Heads Stored at 0 and 3 °C

The changes in impedance change ratio (Q value), pH value, texture, K value, total volatile basic nitrogen (TVB-N), total aerobic count (TAC), drip loss, and sensory assessment (SA) of bighead carp (Aristichthys nobilis) during 0 and 3 °C storage were investigated. Simultaneously, correlation models were developed by analyzing the correlation between the measured values. The results indicate that during postmortem storage, Q value is significantly (p < 0.05) related to pH value and texture indexes (hardness, adhesiveness, springiness, cohesiveness, and resilience); during long-term storage, SA exceeds the acceptable limit on the 15th day at 0 °C and the 12th day at 3 °C, while K value, TVB-N, TAC, and drip loss increase to 72.4 %, 18.9 mg/100 g, 4.82 log10 cfu/g, and 7.09 % at 0 °C, and 78.5 %, 14.0 mg/100 g, 4.97 log10 cfu/g, and 5.08 % at 3 °C, respectively. Meanwhile, Q value declines to 18.9 % at 0 °C and 12.7 % at 3 °C. The correlation coefficients between Q and K values, TVB-N, TAC, drip loss, and SA are 0.955, 0.964, 0.966, 0.965, and 0.994 at 0 °C, and 0.969, 0.967, 0.979, 0.960, and 0.996 at 3 °C, respectively, indicating significant linear relationships (p < 0.05). Therefore, Q value can be used as a fast nondestructive method to estimate the quality of bighead carp heads during storage at 0 and 3 °C.

Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature

Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85% explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67%) and 6 days (13.3%) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.

Evaluation of pre-rigor injection of beef with proteases on cooked meat volatile profile after 1 day and 21 days post-mortem storage

This research was carried out to determine the effects of pre-rigor injection of beef semimembranosus muscle with nine proteases from plant and microbial sources, on the volatile profile of cooked beef after 1 day and 21 days post-mortem (PM) storage using Solid-phase microextraction gas chromatography mass spectrometry analysis. A total of 23 aldehydes, 5 ketones, 3 furans, 8 nitrogen and sulphur compounds, 4 alkanes, 7 alcohols and 6 terpenes were detected. Eleven volatile compounds characteristic of ginger flavour were detected in zingibain-treated meat. Benzaldehyde significantly increased (p<0.05) only in kiwifruit juice (KJ), fungal 31 protease and Asparagus protease (ASP) treated samples from 1day to 21days PM storage. A significant increase (p<0.05) in 3-methylbutanal was observed in KJ, bacterial and fungal protease treated samples at 21days PM storage. Treatments with bromelain, papain, ASP, actinidin, and KJ (except KJ 21days) proteases resulted in flavour profiles closer to that of the control beef sample.

The roles of the actin-myosin interaction and proteolysis in tenderization during the aging of chicken muscle

The objective of this study was to investigate the contribution of the changes in the actin-myosin interaction and proteolysis on meat tenderization during postmortem storage. Following slaughter, chicken breast muscles were removed and stored at 4°C. Changes in the actin-myosin interaction over 48 h of aging were determined by monitoring the Mg(2+)- and Ca(2+)-ATPase activities. Shear force values, pH, protein degradation, calpain activities, and myofibrillar ultrastructures were also investigated. Results showed that the initial weak actin-myosin interaction strengthened at 12 h postmortem followed by a gradual weakening, which was supported by a decrease in Mg(2+)-ATPase activities and a lengthening of the sarcomeres. According to SDS-PAGE and Western blotting analyses, the 30-kDa troponin-T fragment could not be readily detected until 12 h, whereas, at the same time, desmin had been rapidly degraded. However, there was a gradual decline in μ-calpain activity, commencing after about 6 h. Meanwhile, the largest decline in shear force was observed between 12 and 24 h postmortem. These findings suggest that weakening of the strong actin-myosin interaction formed at rigor may play a large role in meat tenderization during the early period of storage. It is proposed that weakening of the actin-myosin interaction results in lengthening of the sarcomeres, and then activated calpains are more able to reach their targeted sites, enabling proteolysis. These 2 factors may be involved in the conversion of muscle to tender meat during postmortem storage.

Effect of cold storage period on the quality of ram cauda epididymal spermatozoa recovered postmortem

Postmortem spermatozoa recovery is an important tool for harvesting germplasm from genetically meritorious animals and endangered species. The principal motive of this study was to find out whether ram spermatozoa within epididymides kept at 4 °C for 48 h retain their quality in terms of progressive motility, viability, and acrosomal status. Twenty-four testicles with attached epididymides were collected with 8 samples each processed at 0, 24, and 48 h of storage. A good number of motile spermatozoa could be recovered from epididymides by incision method with up to 48 h of storage, although their quality declined significantly (P < 0.05) as postmortem storage time increased (motility: 83.33 ± 1.05%, 63.33 ± 2.47%, and 44.17 ± 1.53%; and epididymal sperm output (millions): 3500.00 ± 73.97, 1220.00 ± 1.01, and 666.67 ± 19.78 for 0 (control), 24, and 48 h, respectively). Viability and intact acrosomes (%) were also acceptable, although they decreased significantly with up to 24 h of storage. In conclusion, ram epididymides could be stored at 4 °C for 48 h when epididymal spermatozoa cannot be immediately harvested and cryopreserved.

The effect of calcium chloride injection on shear force and caspase activities in bovine longissimus muscles during postmortem conditioning

Tenderness is considered as the most important quality determinant of meat. Calcium chloride application has been shown to improve tenderness by regulating endogenous proteinases. This study was designed to determine the effect of 300 mM calcium chloride injection on myofibrillar structures, caspase activities and shear force in longissimus muscles of bulls during postmortem storage of 7 days. Myofibrillar fragmentation index was determined as an index of proteolysis occurring in muscle fibers and associated proteins. Maximum tenderness was observed at days 4 and 7 in both treated and control samples. The injection of calcium chloride significantly increased myofibrillar proteolysis and improved tenderness at postmortem days 4 and 7. The treatment reduced caspase-9 activity at 4 h and day 4, whereas those of caspase-8 and -3 activities at days 1 and 4 with respect to control. The improved tenderness and increased myofibril fragmentation with decreased caspase activities suggested that the proteolytic systems activated with calcium chloride injection possibly behave independent of the caspase system.

The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30°C and sampling times were at 2, 6, 24, 48 and 120h post-mortem. After 120h at 30°C the free calcium concentration increased to 530μM from 440μM at 2°C. Incubation at temperatures higher than 2°C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.

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.

Effects of postmortem storage temperature on sea bass (Dicentrarchus labrax) muscle protein degradation: Analysis by 2‐D DIGE and MS

Storage conditions are known to be important for postmortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. In order to shed light on the influence of temperature on the status of sea bass (Dicentrarchus labrax) muscle proteins during postmortem storage, a 2-D DIGE and mass spectrometry study was performed on fish kept at either 1 or 18°C for 5 days. As expected, the greatest alterations in sea bass filet protein composition were observed upon postmortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during postmortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions.