Postmortem Storage Research Articles

Physicochemical and quality characteristics of New Zealand goat meat and its ultrastructural features

This study investigated the quality characteristics, physicochemical indexes and ultrastructure of New Zealand goat meat. The goat meat samples were sourced from New Zealand feral goats and Boer crossbreeds. Rawlongissimus thoracis(LT) andsemimembranosus(SM) muscles were evaluated after 48 hr of post-mortem storage at 4 °C. The cooked meat characteristics were studied after sous vide cooking at 60 °C for 6 h. Meat samples from both goat breeds exhibited intermediate pH values in the range of 5.76 and 5.98. Feral goat meat had higher collagen with lower solubility compared to Boer crosses. All the samples had high peak shear force from 62.1 to 87.3 N, and the variations were highly influenced by pH (p < 0.01) and collagen content (p < 0.05) and are dependent on muscle type (p < 0.01). For color, meat from feral and Boer varies significantly for redness (p < 0.01). Transmission electron micrographs from all samples revealed short sarcomeres ranging from 1.4 to 1.8 µm. Some super contracted muscle fibers were also observed, suggesting that muscles have undergone severe cold-shortening. Our findings indicate that regardless of genotype, the challenge for goat meat quality is its inherently high pH and its susceptibility to cold-shortening that impacts its technological properties.

Label-free quantification proteomics reveals potential proteins associated with the freshness status of crayfish (Procambarus clarkii) as affected by cooking

In order to characterize the freshness status of raw material of cooked crayfish products, protein and physicochemical changes were investigated in raw and cooked crayfish from fresh-live state to the early postmortem using Label-free quantification proteomics, total volatile basic nitrogen (TVB-N) and biogenic amines (BAs) analysis. Results showed that the TVB-N and BAs contents in both raw and cooked crayfish remained low levels and no obvious changes were observed within 24 h of postmortem storage at 4 °C. Altogether, six differentially expressed proteins (DEPs) were detected in both C6/C0 and C24/C0 groups by proteomics. Furthermore, five among the six DEPs were verified both in cooked and raw crayfish samples by parallel reaction monitoring. Among the six DEPs, “Unigene990_S_0_Gene.8754” changed most significantly with the increasing of postmortem time and not obviously influenced by heating, indicating that “Unigene990_S_0_Gene.8754” may be highly associated to the freshness status of crayfish.

Muscle tissue structural changes in bombay-duck (Harpodon nehereus) induced by formaldehyde solution during post-mortem storage

Formaldehyde is a harmful compound that is usually used as an illegal preservative agent in fisheries products to prevent spoilage and extend shelf life. In several fishery products, formaldehyde can be formed endogenously during the post mortem process through degradation of trimethylamine oxide (TMAO) by enzymatic process. Exogenous and endogenous formaldehyde can affect the textural and structural change in fish muscle tissue significantly influencing flesh quality. This study was aimed to characterize the structural change of the muscle tissue in Bombay duck (Harpodon nehereus) that is immersed with formaldehyde solution on chilling storage temperature. The Scanning Electron Microscope (SEM) analysis was used to identify and characterize the structural change in muscle tissue samples, which were immersed with formaldehyde solution 0%, 1%, 3%, and 5%. SEM analysis identified that in transversal section samples immersed with formaldehyde solution 0% (control) intermyofibrillar space was increased significantly during post mortem storage. This was in line with the increased of myofibril, connective tissue and sarcolemma lost on samples. Furthermore, on samples that were immersed with formaldehyde solution 1%,3%, and 5% the degradation of myofibril and intermyofibrillar was can be held and the connectivity tissue muscle still showed strong aggregation during post mortem storage.

Oxidative Stress Contributes to Cytoskeletal Protein Degradation of Esox lucius through Activation of Mitochondrial Apoptosis during Postmortem Storage.

This study investigated the role of oxidative stress in the mitochondrial apoptotic pathways and structural protein degradation of fish during postmortem storage by measuring oxidative stress levels, mitochondrial antioxidant enzyme activity, mitochondrial dysfunction, apoptotic factors, and structural protein degradation (n = 3). The results revealed that reactive oxygen species (ROS) increased gradually within the first 12 h and then decreased (p < 0.05) in mitochondria. Lipid peroxidation was increased, and superoxide dismutase, catalase, and glutathione peroxidase activities were decreased in mitochondria (p < 0.05). Furthermore, oxidative stress induced mitochondrial membrane opening, mitochondrial swelling, as well as the depolarization of mitochondrial potential. This led to an increase in the release of cytochrome c from mitochondria and caspase-3 activation. Ultimately, oxidative stress promoted small protein degradation (troponin-T and desmin) and induced myofibril susceptibility to proteolysis. These observations confirmed that oxidative stress mediated the activation of mitochondrial apoptotic factors-promoted protein degradation, initiating the deterioration of fish muscle through the mitochondrial apoptotic pathway.

Estimation of the proteome affecting changes in tenderness of yak meat during storage by label-free mass spectrometry.

BackgroundTenderness is the main quality of meat products. However, the meat tenderness formation is a complex biological process, and pathways and proteins that affect the tenderness of yak meat are unknown.MethodsLabel‐free proteomics method was used to explore the effects of differentially expressed proteins on the tenderness of yak skeletal muscle (tenderloin) during post‐mortem storage (0, 3, and 7 days) at 3 ± 1°C.ResultsThe tenderness of yak skeletal muscle improved significantly during storage. A total of 91 differentially expressed proteins of yak skeletal muscle during post‐mortem storage were identified by the following comparisons: day 3 versus 0, day 7 versus 0, and day 7 versus 3. NDUFS6, CYCS, COX6A2, LDB3, HSPB7, TPM4, TAGLN, COL1A1, LUM, MYH11, ACTC1, and MYOZ1 proteins showed a significant difference during yak skeletal muscle post‐mortem storage. Furthermore, bioinformatics analyses revealed that the identified proteins were related to carbon metabolism, citrate cycle, glycolysis, oxidative phosphorylation, and RNA degradation.ConclusionThe results of the present study could provide proteomic insights into changes in yak skeletal muscle tenderness during storage and may be a valuable resource for future investigations.

Two-dimensional gel analysis to investigate the effect of hydroxyl radical oxidation on freshness indicator protein of Coregonus peled during 4 °C storage

The present study investigates the freshness indicator protein of Coregonus peled and the effects of oxidation on the quality of fish during 4 °C storage. Samples were stored at 4 °C after oxidation treatment and analyzed for changes in proteins, using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). A total of 382 differential proteins were identified, of which 20 proteins were quantified and studied including 12 proteins related to oxidation. The analysis of differential proteins was related to quality postmortem. Moreover, creatine kinase, malate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase were identified as potential biomarkers for protein oxidative damage. Actin can be used to represent the softening of fish in postmortem storage. Pearson correlation analysis indicated that the changes in protein structure and biomarkers were influenced by oxidation. Bioinformatics suggested that differentially expressed proteins participated in the metabolic pathway via oxidation during 4 °C storage. The results of this study can contribute to a further understanding of the changes in fish quality during storage and the use of differential proteins as indicators of fish freshness.

Myostatin gene inactivation increases post-mortem calpain-dependent muscle proteolysis in mice

Myostatin deficiency leads to extensive skeletal muscle hypertrophy, but its consequence on post-mortem muscle proteolysis is unknown. Here, we compared muscle myofibrillar protein degradation, and autophagy, ubiquitin-proteasome and Ca2+-dependent proteolysis relative to the energetic and redox status in wild-type (WT) and myostatin knock-out mice (KO) during early post-mortem storage. KO muscles showed higher degradation of myofibrillar proteins in the first 24 h after death, associated with preserved antioxidant status, compared with WT muscles. Analysis of key autophagy and ubiquitin-proteasome system markers indicated that these two pathways were not upregulated in post-mortem muscle (both genotypes), but basal autophagic flux and ATP content were lower in KO muscles. Proteasome and caspase activities were not different between WT and KO mice. Conversely, calpain activity was higher in KO muscles, concomitantly with higher troponin T and desmin degradation. Altogether, these results suggest that calpains but not the autophagy, proteasome and caspase systems, explain the difference in post-mortem muscle protein proteolysis between both genotypes.

The role of endogenous serine proteinase on disintegration of collagen fibers from grass carp (Ctenopharyngodon idellus)

Softening, causing by the degradation of collagen, leads to serious quality deterioration of fish fillets during post-mortem storage. In order to study the role of endogenous enzymes in softening of fish muscle, collagen fibers from grass carp (Ctenopharyngodon idellus) were degraded by serine proteinase. The structural and biochemical changes of collagen fibers with serine proteinase digestion were investigated in terms of scanning electron microscopy, fourier transform infrared spectroscopy, electrophoresis and chemical analysis. In the present study, an endogenous proteinase (approximately 250 kDa) with gelatinolytic activity was isolated from grass carp muscle by ammonium sulfate precipitation and column chromatographies, and identified as serine proteinase. Collagen fibers were partly disaggregated into collagen fibrils after serine proteinase treatment for 48 h, coinciding with a release of hydroxyproline (5.56 ± 0.02%) and glycosaminoglycans (9.04 ± 0.03%). However, almost all gelatin and 40% of acid-soluble collagen were degraded within 1 h and 6 h, respectively. Meanwhile, serine proteinase-treated collagen fibers maintained the triple-helical structure of collagen molecule, while their thermal stability was gradually decreased with digestion time. All results indicated that disintegration of collage fibers by serine proteinase might be due to both the degradation of proteoglycans and collagen molecules during textural deterioration of chilled grass carp fillets.

Proteomic Changes in Sarcoplasmic and Myofibrillar Proteins Associated with Color Stability of Ovine Muscle during Post-Mortem Storage.

The objective of this study was to investigate the proteomic characteristics for the sarcoplasmic and myofibrillar proteomes of M. longissimus lumborum (LL) and M. psoasmajor (PM) from Small-tailed Han Sheep. During post-mortem storage periods (1, 3, and 5 days), proteome analysis was applied to elucidate sarcoplasmic and myofibrillar protein changes in skeletal muscles with different color stability. Proteomic results revealed that the identified differentially abundant proteins were glycolytic enzymes, energy metabolism enzymes, chaperone proteins, and structural proteins. Through Pearson’s correlation analysis, a few of those identified proteins (Pyruvate kinase, Adenylate kinase isoenzyme 1, Creatine kinase M-type, and Carbonic anhydrase 3) were closely correlated to representative meat color parameters. Besides, bioinformatics analysis of differentially abundant proteins revealed that the proteins mainly participated in glycolysis and energy metabolism pathways. Some of these proteins may have the potential probability to be predictors of meat discoloration during post-mortem storage. Within the insight of proteomics, these results accumulated some basic theoretical understanding of the molecular mechanisms of meat discoloration.

Label-free proteomic analysis reveals freshness-related proteins in sea bass (Lateolabrax japonicus) fillets stored on ice

The advantages of epigallocatechin gallate incorporated in agar oligosaccharide treatment during post-mortem storage on ice for maintaining the quality and proteome stability of sea bass muscle was investigated using hardness, total volatile basic nitrogen, total viable counts, protein biochemical properties and label-free proteomic analysis. The results indicated that hardness value and sulfhydryl content of EGCG–AO-treated fish muscle were much higher than those of the samples immersed in sterile water. The total volatile basic nitrogen, total viable counts, and carbonyl content increased to 165.65%, 18.93%, and 42.37%, respectively after 8 days of storage. Proteomic analysis of muscle samples from control fillets on days 0 (CK0) and 8 (CK8), and EGCG–AO-treated fillets on day 8 (T8) revealed 708 different abundant proteins (DAPs). DAPs in CK8 vs. CK0 fillets were mainly structural proteins, metabolic enzymes, and proteins involved in protein turnover, and were closely related with freshness properties. Muscles from fillets in T8 showed delayed degradation of structural proteins, lower abundance of calpain and carbohydrate metabolic enzymes, and maintained higher levels of transcription and translation to maintain quality. Thus, EGCG–AO treatment is a possible method for maintaining freshness of sea bass fillets. Several DAPs are potential proteomic markers for monitoring fillet freshness.

PSX-B-16 Recovery of live cells from refrigerated sheep skin after different days of cold storage

Abstract Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2–3 mm2) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO2 incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with &amp;gt;80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.

Sarcoplasmic and myofibril-bound calpains during storage of pork longissimus muscle: New insights on protein degradation

The subcellular distribution of calpain-1 and -2 and the proteolytical activity of myofibril-bound calpains in pork were investigated during 12 days cold storage. The content of sarcoplasmic calpain-1 decreased during storage while myofibril-bound calpain-1 content first increased (P < 0.05) to 17% of that of 12 h-sarcoplasmic calpain-1 on day 6 followed by a gradual decrease with subsequent storage, suggesting that calpain-1 gradually translocated from sarcoplasm to myofibrils during the initial 6 days of postmortem storage. Intact desmin decreased (P < 0.05) after incubation of myofibrils with 0.05 mM Ca2+, and this was more pronounced with 5 mM Ca2+ (P < 0.05). Ca2+ titration curves of day 6 myofibrils showed two distinct proteolytic activities becoming activated in the range 0.03 to 0.06 mM and 0.4 to 0.8 mM Ca2+, respectively. The results suggest that both calpain-1 and calpain-2 binds to myofibrils during storage and subsequently degrade structural proteins including desmin.

The effect of caspase-3 in mitochondrial apoptosis activation on degradation of structure proteins of Esox lucius during postmortem storage

This study aimed to investigate the effect of caspase-3 inhibitor in mitochondrial apoptosis activation on structure protein degradation during postmortem storage. Mitochondrial dysfunction, apoptotic factors, structure protein degradation and the myofibrillar rupture index between the control and caspase-3 inhibitor groups were determined. The results show caspase-3 inhibitor repressed the mitochondrial membrane permeability and mitochondrial swelling, as well as increased mitochondrial membrane potential, causing a decrease in the release of cytochrome c from mitochondria to cytoplasm and caspase-9/3 activities (P < 0.05). Subsequently, small myofibrillar proteins (desmin and troponin-T) were susceptible to degradation, initiating texture deterioration. By contrast, giant structure proteins (titin and nebulin) were degraded during later postmortem storage, predominantly contributing to fish softening. The results further suggest that caspase-3 is involved in degradation of structure proteins during postmortem through mitochondrial apoptosis pathways.

Endogenous Proteolytic Systems and Meat Tenderness: Influence of Post-Mortem Storage and Processing.

Meat proteolytic systems play a crucial role in meat tenderisation. Understanding the effects of processing technologies and post-mortem storage conditions on these systems is important due to their crucial role in determining the quality characteristics of meat and meat products. It has recently been proposed that tenderisation occurs due to the synergistic action of numerous endogenous proteolytic systems. There is strong evidence suggesting the importance of μ-calpain during the initial post-mortem aging phase, while m-calpain may have a role during long-term aging. The caspase proteolytic system is also a candidate for cell degradation in the initial stages of conversion of muscle to meat. The role of cathepsins, which are found in the lysosomes, in post-mortem aging is controversial. Lysosomes need to be ruptured, through aging, or other forms of processing to release cathepsins into the cytosol for participation in proteolysis. A combination of optimum storage conditions along with suitable processing may accelerate protease activity within meat, which can potentially lead to improved meat tenderness. Processing technologies such as high pressure, ultrasound, and shockwave processing have been reported to disrupt muscle structure, which can facilitate proteolysis and potentially enhance the aging process. This paper reviews the recent literature on the impacts of processing technologies along with post-mortem storage conditions on the activities of endogenous proteases in meat. The information provided in the review may be helpful in selecting optimum post-mortem meat storage and processing conditions to achieve improved muscle tenderness within shorter aging and cooking times.

ITRAQ-mediated analysis of the relationship between proteomic changes and yak longissimus lumborum tenderness over the course of postmortem storage

To identify differentially expressed proteins associated with energy metabolism and tenderness during the postmortem aging of yak longissimus lumborum muscle samples, we collected tissue samples from yaks raised at different altitudes. At 12 h post-slaughter, we identified 290 differentially expressed proteins (DEPs) in these samples, whereas 436 such DEPs were detected after 72 h. Identified DEPs were clustered into four main functional categories: cell structural proteins, glycogen metabolic proteins, energy reserve metabolic proteins, and cellular polysaccharide metabolic proteins. Further bioinformatics analysis revealed that these proteins were associated with carbon metabolism, glycolysis, and the biosynthesis of amino acids. Our functional insights regarding these identified proteins contribute to a more detailed molecular understanding of the processes of energy metabolism in yak muscle tissue, and represent a valuable resource for future investigations.

Post-mortem storage conditions and cooking methods affect long-chain omega-3 fatty acid content in Atlantic mackerel (Scomber scombrus)

Long-chain omega-3 fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) are health beneficial lipids found in high concentration in pelagic fishes, including Atlantic mackerel. While EPA and DHA are sensitive to oxidation during fish storage and processing, post-mortem degradation in the first hours following fish death is poorly documented. Here, we stored fish at two temperatures (2–4 °C and 18–20 °C) and monitored EPA + DHA content in dorsal fillet 6, 12 and 24 h after fish death and after cooking (grill or steam). Storage duration was the only influencing factor, and EPA + DHA loss was faster at 18–20 °C. Six hours after fish death, EPA + DHA content decreased by 1.3 ± 1.3 mg.g−1 dw (9.6 ± 9.5% of the initial content) but it was highly variable among individuals. Handling between fishing and storage should be as short and as cool as possible to preserve EPA + DHA and food safety. Regarding cooking, EPA + DHA and mono-unsaturated fatty acids increased in grilled fillets.

Spoilage microbes’ effect on freshness and IMP degradation in sturgeon fillets during chilled storage

Bacterial spoilage is an important reason causing freshness deterioration of fish during postmortem storage. The aim of this study is mainly to investigate the role of bacteria in freshness and the degradation of inosine 5′-monophosphate (IMP) as well as nucleotide derived values in chilled sturgeon (hybrid, Acipenser schrenckiid × Huso dauricus) fillets using a microbial reduction pretreatment. During storage at 4 or -1 °C, lower total bacterial count and total volatile base nitrogen in the treated samples was observed when compared to the control samples. But very close K, Ki value and IMP was observed in both groups, although higher IMP was recorded at day 6 and 9 at 4 °C. In later storage period, accelerated decrease of inosine (Ino) and increment of hypoxanthine (Hx) occurred in two control groups, resulting in a rapid increase of G and H values, significant higher than the treated groups. Six predominant spoilage bacteria (Acinetobacter johnsonii, Acinetobacter lwoffii, Shewanella baltica, Pseudomonas Fragi, Pseudomonas koreensis and Pseudomonas antarctica) were isolated from the sturgeon fillets during storage. Based on the results of respective co-cultivation of IMP with the six species, only Acinetobacter johnsonii had ability to degrade IMP when reached a high density of 2.84 × 109 CFU/mL. Results suggested that microbial reduction pretreatment could better the freshness of chilled sturgeon fillet. Comparing to endogenous IMPase, spoilage bacteria probably played a small role in IMP degradation, but affected considerably the decomposition of Ino to Hx.

Injection of iodoacetic acid into pre-rigor bovine muscle simulates dark cutting conditions

The purpose of this study was to develop an in situ model for dark cutting beef. Iodoacetic acid (IAA) was injected at different concentrations (0, 0.625, 1.25, 2.5, 3.75, 5, or 10 μmol/g of muscle) into pre-rigor bovine longissimus thoracis et lumborum (LTL) muscle samples, and pH and color were evaluated over a 48 h period. Injection of IAA blunted muscle pH decline and lowered lightness (L*), redness (a*), and yellowness (b*) values (P ≤ 0.05) in a concentration dependent fashion. In a follow-up study, LTL muscle samples were injected with 5 μmol IAA/g of muscle to test whether IAA maintains its effect over a 336 h post-mortem storage period. In addition to inhibiting pH decline and decreasing color values, IAA increased LTL muscle water holding capacity (WHC) and firmness (P ≤ 0.05) throughout the 336 h post-mortem storage period. Collectively, these data suggest that pre-rigor injection of IAA generates beef with dark cutting-like characteristics.

Application of proteomics to understand the molecular mechanisms determining meat quality of beef muscles during postmortem aging

Proteomics profiling disclosed the molecular mechanism underlying beef poor meat quality. This study aimed to identify protein markers indicating the quality of beef during postmortem storage at 4°C. Beef longissimus dorsi samples were stored at 4°C. The meat water holding capacity (WHC), pH value and moisture content were determined at different time points during the storage period. The iTRAQ MS/MS approach was used to determine the proteomics profiling at 0, 3.5 and 7 d during storage at 4°C. Bioinformatics analysis was performed to investigate the potential correlated proteins associated with meat quality. Storage at 4°C gradually decreased the pH value, WHC, and hence the moisture content. The iTRAQ proteomic analysis revealed that a cluster of glycolytic enzymes including malate dehydrogenase, cytoplasmic, L-lactate dehydrogenase, phosphoglycerate mutase and pyruvate kinase, and another cluster of proteins involved in oxygen transport and binding (myoglobin) and hemoglobin complex (including Globin A1 and hemoglobin subunit alpha) were decreased during the postmortem storage. These results suggest that the decreased glycolysis, oxygen, and heme-binding activities might be associated with the beef muscle low quality and the decline of tenderness during postmortem storage at 4°C.

The Effect of Dietary Lycopene Supplementation on Drip Loss during Storage of Lamb Meat by iTRAQ Analysis.

This study was designed to investigate the impact of dietary lycopene (antioxidant extracted from tomato) supplementation on postmortem antioxidant capacity, drip loss and protein expression profiles of lamb meat during storage. Thirty male Hu lambs were randomly divided into three treatment groups and housed in individual pens and received 0, 200 or 400 mg·kg−1 lycopene in their diet, respectively. All lambs were slaughtered after 3 months of fattening, and the longissimus thoracis (LT) muscle was collected for analyses. The results indicated that drip loss of LT muscle increased with storage days (P < 0.05). After storage for 7 days, significantly lower drip loss of meat was found in fed the lycopene-supplemented diet (P < 0.05). Dietary lycopene supplementation increased the activity of antioxidant enzymes (total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT)) (P < 0.05) and decreased the thiobarbituric acid reactive substance (TBARS) and carbonyl contents (P < 0.05). During the storage period (days 0, 5 and 7), a number of differentially abundant proteins (DAPs), including oxidases, metabolic enzymes, calcium channels and structural proteins, were identified based on iTRAQ data, with roles predominantly in carbon metabolism, oxidative phosphorylation, cardiac muscle contraction and proteasome pathways, and which contribute to decreased drip loss of lamb meat during storage. It can be concluded that dietary lycopene supplementation increased antioxidant capacity after slaughter, and the decreased drip loss during postmortem storage might occur by changing the expression of proteins related to enzyme activity and cellular structure in lamb muscle.