Postmortem Muscle Research Articles

Physiological and flesh quality consequences of pre-mortem crowding stress in Atlantic mackerel (Scomber scombrus).

In commercial wild capture pelagic fisheries it is common practice to crowd catches to high densities to allow efficient pumping onboard. Crowding during the final stages of purse seine capture for small pelagic species often results in intense and sustained behavioural escape responses. Such a response may trigger a shift in energy production from aerobic to anaerobic pathways and result in metabolic acid accumulation and exhaustion of intracellular reserves of ATP. Where there is insufficient time or opportunity to recover to physiological equilibrium before death, pre-mortem stress may be an important determinant of fillet quality, as has been shown for a variety of farmed fish species. However, there is currently a lack of knowledge related to the flesh quality implications of capture stress for wild captured species in European waters. Here we show that crowding results in a physiological stress response that has consequences for flesh quality in the wild captured species Atlantic mackerel (Scomber scombrus). Using small schools in tanks and aquaculture net pens in three separate experiments, we found crowding results in physiological changes in mackerel consistent with an acute stress response and anaerobic metabolism. Consequently, we found crowded fish had more acidic pre- and post-mortem muscle pH as well as indications of faster onset and strength of rigor mortis and increased cathepsin B & L activity. We examined fillet flesh quality after two and seven days of ice storage and found reduced green colouration, increased gaping (separation of muscle myotomes) and reduced textural firmness associated with fish which had been crowded. However, the effects on quality were dependant on experiment and/or storage time. These results indicate the potential of crowding capture stress to influence the flesh quality of an economically important species and may have important implications for the wild capture pelagic fishing industry.

Biomolecular Interactions Governing Fresh Meat Color in Post-mortem Skeletal Muscle: A Review

Appearance is an important sensory property that significantly influences consumers' perceptions of fresh meat quality. Failure to meet consumer expectations can lead to rejection of meat products, concomitant loss in value, and potential production of organic waste. Immediately after animal harvest, skeletal muscle metabolism changes from aerobic to anaerobic. However, anoxic post-mortem muscle is biochemically active, and biomolecular interaction between myoglobin, mitochondria, metabolites, and lipid oxidation determines meat color. This review examines how metabolites and mitochondrial activity can influence myoglobin oxygenation and metmyoglobin reducing activity. Further, the review highlights recent research that has examined myoglobin redox dynamics, sarcoplasmic metabolite changes, and/or post-mortem biochemistry.

Evaluation of Myosin Heavy Chain Isoforms in Biopsied Longissimus Thoracis Muscle for Estimation of Meat Quality Traits in Live Pigs.

Simple SummaryPork quality has become an important parameter in the industry. Traditional pork quality was assessed postmortem. It is considered that the determination of meat quality in live pigs is beneficial in order to obtain better pork quality and to reduce cost in production. In the present study, myosin heavy chain (MHC) isoforms in both of the pre- and postmortem longissimus thoracis muscle were evaluated as novel parameters for meat quality estimation in pork by correlation and clustering analysis. MHC isoforms in live pigs could be applied in a practical and useful method to predict meat quality in pork.Estimating meat quality prior to slaughter will be beneficial for the rapid identification of specific traits or poor quality pork compared to a conventional assessment at postmortem. In this study, we identified and quantified myosin heavy chain (MHC) isoforms from a biopsied longissimus thoracis muscle of pigs, and determined their correlation with postmortem muscle fiber characteristics and meat quality. MHC slow and fast isoforms proportions from biopsied samples correlated with postmortem percentage of type I and type IIB muscle fibers, respectively (p < 0.05). The percentage of the biopsied MHC slow isoform showed a positive correlation with pH at 45 min postmortem, and negative correlations with filter-paper fluid uptake and drip loss in pork (p < 0.05). Furthermore, clustering the pigs into three groups based on the biopsied MHC isoform proportions was not only significantly associated with muscle fiber number and proportions of muscle fiber area, but also correlated with pH at 45 min postmortem and the National Pork Producers Council color score (p < 0.05). Collectively, our findings indicate that the biopsied MHC isoforms serve as parameter for estimating meat quality, with the association between the higher proportion of MHC slow isoforms and pH at 45 min postmortem in particular being indicative of better pork quality.

Effect of oxidative stress on AIF-mediated apoptosis and bovine muscle tenderness during postmortem aging.

This study aimed to explore the effect exerted by oxidative stress on apoptosis-inducing factors (AIF)-mediated apoptosis and bovine muscle tenderness during postmortem aging. We investigated the reactive oxygen species (ROS) content, mitochondrial membrane permeability, AIF expression level, nucleus apoptosis, shear force, myofibril fragmentation index, pH, and energy level. According to the results, a rise in ROS content was accompanied by the rise in mitochondrial membrane permeability from 6 to 72 hr. In the meantime, the AIF expression in mitochondria was downregulated significantly within 72 hr. However, samples treated with N-acetylcysteine had significantly lower ROS content (6 to 72 hr) and mitochondrial membrane permeability (12 to 72 hr) than the control group. Moreover, during postmortem aging, the variations in AIF levels in mitochondria were closely associated with meat tenderization and nucleus apoptosis. These findings demonstrated that oxidative stress induced by ROS significantly promoted AIF release from mitochondria by enhancing the mitochondrial membrane permeability, and the released AIF mediated nucleus apoptosis that further enhanced bovine muscle tenderness. Besides, results suggest that in the early stage, the environmental factors (ATP content and pH) significantly decreased (0 to 72 hr), whereas ROS-induced oxidative stress had no significant effect on environmental factors. These observations further suggested that during postmortem aging, the decrease of pH and ATP consumption are required by AIF release. We conclude that ROS-induced oxidative stress and internal environment are vital for meat tenderization through the regulation of AIF-mediated apoptosis pathway. PRACTICAL APPLICATION: ROS-induced oxidative stress contributes to bovine muscle tenderization by promoting cell apoptosis. It is likely to lay a theoretical foundation for developing innovative tenderization techniques by altering the internal oxidation environment of postmortem muscles.

281 Effects of feeding endophyte-infected tall fescue seed to Angus steers on ergovaline concentration, lipid oxidation, and skeletal metmyoglobin reductase activity

Abstract This study was aimed to determine the ergovaline concentration, lipid oxidation, and skeletal metmyoglobin reductase activity in Angus steers fed with endophyte-infected tall fescue seed. Animals were blocked into light (n = 4, 205.5 ± 7.4 kg), medium (n = 5, 231.3 ± 8.2 kg), and heavy (n = 3, 272.7 ± 8.4 kg) groups and randomly assigned to either a KY31 seed treatment (6796 ppb ergovaline; 20 µg/kg BW; E+; n = 6) or a KY32 control (&amp;lt; 100 ppb; E-; n = 6) by Calan® gates during a 70-d feeding period from June to August of 2015 (period 1), followed by a 150-d resting period and another 64-d feeding period from January to March of 2016 (period 2). In both periods, there was a 2-way treatment × time interaction for ergovaline concentration in blood (P = 0.002 and 0.022, respectively), with E+ steers having 0.59 to 0.95 nM more than E- steers on d 3 (P &amp;lt; 0.001). In period 1, TBARS values were greater in blood serum of E+ steers than in that of E- steers (6.56 vs. 2.56 µM MDA; P = 0.048) and an overall increase in MDA from d 0 (2.39 µM) to d 3 (7.59 µM) was also observed (P = 0.049). At slaughter, E+ steers had a blood TBARS value of 2.27 µM, greater than 1.15 µM in E- steers (P = 0.013). In postmortem muscle, the TBARS value was 0.68 and 0.77 µg/kg for E- and E+ steers, respectively (P = 0.239). There was no effect of endophyte-infected tall fescue seed on metmyoglobin reductase in longissimus thoracis (4.82 µM/min/g in E- muscle vs. 3.93 µM/min/g in E+ muscle; P = 0.484). Therefore, feeding endophyte-infected tall fescue seed increased ergovaline concentration and oxidative stress in the blood of Angus steers.

Apoptotic and Proteolytic Attributes and Metabolomic Changes in Postmortem Muscles from Pigs Subjected to Post-Weaning Transport at Different Seasons

ObjectivesPost-weaning transport of pigs was commonly practiced in the swine industry, however, adversely impact animal growth and well-being due to concurrent stress from weaning and transport. Further, our recent study found that post-weaning transport may have long-term effects on final pork quality attributes in terms of inferior texture and water-holding capacity. Heat shock proteins (HSPs) are anti-apoptotic chaperone proteins, protecting against apoptosis under a variety of cell death stimuli including postmortem muscle conversion process. While a potential role of apoptosis in meat tenderization has been proposed, how early life stress influences apoptotic/proteolytic process and metabolism of postmortem muscles is largely unknown. Thus, the study objective was to evaluate apoptotic and proteolytic attributes and metabolomic changes in postmortem muscles of market weight pigs exposed to early life transport/weaning stress at two seasons.Materials and MethodsTwo repetitions of newly weaned pigs (N = 480) were transported for 12 h in a trailer truck during July 2016 (SUMMER) and April 2017 (SPRING) in north-central Indiana. Upon reaching market weight, 10 animals were randomly chosen from each season and slaughtered in January 2017 and September 2017, respectively. Pairs of longissimus dorsi and psoas major muscles from each carcass were separated at 1d and 7d postmortem. Proteolytic and apoptotic factors including desmin, troponin T, calpain 1, HSP27, and aβ-crystallin were quantified using Western-blot assays, and mitochondria membrane permeability (MMP) was evaluated. Metabolome profiles of 1d samples were analyzed using the GC-TOF-MS/MS platform. Multivariate analyses PCA and PLS-DA were used to determine changes of metabolites. Data were analyzed using PROC MIXED of SAS to compare the traits across season, muscle, and aging effects.ResultsPreviously, SUMMER pigs were reported showing decreased body weight, muscling, and fat deposition, as well as increased shear force and water loss during aging. In the present study, SPRING muscles exhibited increases in calpain 1 autolysis and structural protein degradation, coincided with accelerated apoptosis shown as higher MMP compared to the SUMMER counterparts (P &lt; 0.05). Moreover, PCA and PLS-DA clustering indicated distinct metabolome profiles affected by season and muscle. Seasonal effect mainly altered lipid, glucose, and nitrogen metabolism. A group of 16C to 18C fatty acids were increased in SPRING, probably due to increased lipid anabolism during warm growing/finishing season. Changes of urea, ornithine, aspartic acid, and 5’methylthioadenosine suggested increased amino acid catabolism in SUMMER, corroborating the decreased lean and fat accretion. Seasonal changes of key metabolites related to stress response, including histidine, GABA, and ascorbic acid, suggested increased stress defense in SUMMER pigs, which implied the suppression of apoptotic and proteolytic activities.ConclusionTaken together, SUMMER pigs showed suppressed onset of apoptosis with compromised growth and meat quality, possibly due to alternations in seasonal metabolic response. This may in turn affect the proteolytic potential of early postmortem muscles. Further studies elucidating the involvement of apoptotic process in proteolytic activities in postmortem muscles should be warranted.

Mitochondrial Function in Oxidative and Glycolytic Bovine Skeletal Muscle Postmortem

ObjectivesMitochondrial function in postmortem muscle is affected by decreasing oxygenation. Functional properties relating to energy production and integrity of mitochondria may influence development of meat quality characteristics. Therefore, the objective was to evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Materials and MethodsSteers (n = 6) of primarily Angus (80 to 100%) genetics were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were collected at 1, 3, and 24h postmortem. Fresh-preserved muscle samples were permeabilized using saponin, and muscle bundles (2–4 mg) were transferred to a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. First, pyruvate and malate were added to support the TCA cycle and assess leak respiration. Then, ADP was added to support electron flow through complex I. The influence of glutamate on NADH production (complex I) was tested, followed by complex II activation by succinate. Integrity of the mitochondria outer membrane was tested with cytochrome c. Next, an uncoupler (FCCP) was added to force the electron transport system (ETS) to maximum capacity. Citrate synthase (CS) activity (nmol/min/mg tissue) was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity (pmol/sec/U CS) to account for differences in mitochondria content. Coupling efficiency of oxidative phosphorylation was calculated as 1– (Leak/ADP-stimulated oxidative phosphorylation capacity). Raw and normalized OCR were analyzed in a randomized block design, with slaughter date as block and fixed effects of muscle, time, and the interaction. Time was considered a repeated measure.ResultsMuscle type affected (P = 0.0002) leak OCR, with Dia showing a higher rate than LL. After ADP was added, mitochondria from Dia exhibited higher OCR at all times tested and at all steps, with OCR being 4 times higher after FCCP addition. Mitochondrial content, evidenced by greater (P &lt; 0.0001) CS activity in Dia, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from Dia and LL were similar (P &gt; 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P &lt; 0.05) in Dia mitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR by 6.6%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased 52.4%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with Dia exhibiting greater efficiency.ConclusionDespite inherent metabolic differences between the LL and Dia, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of LL mitochondria are reduced compared to Dia. Declining mitochondrial function may be associated with calcium overload, mitochondrial fragmentation, and protease activation.

Characterization of the Cofactors Involved in Non-Enzymatic Metmyoglobin Reduction In Vitro

ObjectivesConsumers’ meat purchasing decisions are strongly influenced by color. Myoglobin is the primary meat pigment that contributes to meat color. Myoglobin consists of an iron-containing heme ring and amino acids in the form of globin chains. Both the state of the heme iron and the type of ligand affects meat color. The consumer-preferred bright cherry-red color oxymyoglobin is formed when the iron is in the ferrous state and oxygen bind to the heme. The oxidation of oxymyoglobin or deoxymyoglobin results in the formation of the brown color, ferric metmyoglobin. Predominant metmyoglobin accumulation negatively impacts consumer purchasing choices. Although muscle type and pre- and post-harvest factors can influence meat discoloration, meat has an inherent ability to reduce metmyoglobin through enzymatic pathways, mitochondria-mediated pathways, and non-enzymatic mechanisms. In the enzymatic pathway, an electron from NADH is transferred to metmyoglobin by an enzyme and an electron carrier; while in mitochondria-mediated pathway, an electron from the electron-transport chain is transferred via cytochromes. Previous research speculated the role of non-enzymatic pathway in meat color; however, limited studies have characterized the cofactors present in a meat system. The objectives of this study were to characterize cofactors in non-enzymatic metmyoglobin reduction and determine the effect of storage temperature and postmortem muscle pH in-vitro.Materials and MethodsPurified equine metmyoglobin was reduced in the presence of combinations of electron carriers and donors. Methylene blue and cytochrome c were evaluated as the electron carriers, and NADH and ascorbate were considered as the electron donors. The cofactors were held at 4 and 25°C to determine temperature effects on the reduction of metmyoglobin, and the same cofactor combinations were evaluated at pH of 5.2, 5.6, 6.0, and 6.4 to reflect postmortem muscle pH. Spectrophotometry was utilized to monitor the rates of metmyoglobin reduction. The experiments were replicated five times, and the data were analyzed using the Mixed Procedure of SAS.ResultsThe results indicated that methylene blue was a significantly more effective electron carrier than cytochrome c with both electron donors, ascorbate and NADH. EDTA had no impact on the non-enzymatic metmyoglobin reducing the ability of methylene blue (P = 0.91). Temperature and pH had cofactor specific effects on the non-enzymatic reduction of metmyoglobin. Lower temperature resulted in an increased non-enzymatic metmyoglobin reduction for methylene blue regardless of electron donor (ascorbate, P = 0.03, NADH, P = 0.04). As pH increased, the non-enzymatic metmyoglobin reducing activity reduced significantly in the presence of NADH and methylene blue.ConclusionIn conclusion, the characteristics of the cofactors at specific temperatures and pH impacted the non-enzymatic reduction of metmyoglobin. Further, current in vitro research indicated that non-enzymatic metmyoglobin reduction is possible at lower temperature and meat pH.

Ractopamine-Induced Changes in the Mitochondrial Proteome of Postmortem Beef Longissimus Lumborum

ObjectivesRactopamine is a β-adrenergic agonist approved as growth promotant in beef cattle, and it increases muscle deposition while limiting fat deposition. Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem beef skeletal muscles is influenced by fiber type. Therefore, dietary ractopamine may potentially affect mitochondrial functionality. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from beef cattle.Materials and MethodsPen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 d prior to the harvest. The animals were harvested, and carcasses were chilled for 24 h. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. Mitochondrial proteome was analyzed using two-dimensional electrophoresis, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P &lt; 0.10) between RAC and CON were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsSeven differentially abundant proteins were identified in the mitochondrial proteome. Three proteins were over-abundant (P &lt; 0.10) in RAC, whereas four spots were over-abundant in CON. The proteins over-abundant in RAC mitochondrial proteome was complement component 1 Q subcomponent-binding protein, very long-chain specific acyl-CoA dehydrogenase, and aconitate hydratase. On the other hand, ATP synthase subunit β, prohibitin, Cytochrome b-c1 complex subunit, and thioredoxin-dependent peroxide reductase were over-abundant in CON samples. The differentially abundant proteins belong to four functional groups; i.e., energy metabolism (ATP synthase subunit β, Cytochrome b-c1 complex subunit 1, and aconitate hydratase), chaperone activity (complement component 1 Q subcomponent-binding protein and prohibitin), antioxidant activity (thioredoxin-dependent peroxide reductase), and lipid metabolism (very long-chain specific acyl-CoA dehydrogenase).ConclusionDietary ractopamine impacts mitochondrial proteome in postmortem beef LL muscle and influences the abundance of proteins involved in cellular metabolism and protective mechanisms. The increased protein synthesis and leanness previously reported in ractopamine-fed cattle may be attributed to the decreased expression of enzymes involved in respiratory electron transport pathways and the increased expression of enzymes involved in lipolysis.

Functional/Physicochemical Properties and Oxidative Stability of Ground Meat from Broilers Exposed to Different Photoperiods

ObjectivesProlonged photoperiod (light) is a common practice in the broiler industry to maximize feed intake, growth and yield. Several studies, however, have found negative impacts of extended photoperiod on animal welfare-related characteristics (e.g., leg abnormalities). While the previous research has primarily focused on animal growth/welfare aspects, the effect of photoperiod on functional properties and quality attributes of broiler meat has not been evaluated. Thus, this study was aimed to determine functional properties, physicochemical attributes and oxidative stability of ground meat from broilers reared under different photoperiod conditions.Materials and MethodsRoss 308 broiler chicks (n = 432) were assigned to 4 rooms with 6 pens per treatment, which were equipped with one of the following photoperiods (T20, T18, T16, and T12; the hours of lighting per day), started from Day 15. At 42 d of age, the broilers (n = 12/treatment) were randomly taken, slaughtered and chilled for 24 h at 2°C. At 1 d postmortem, tenderloins and leg muscles were separated from the carcasses and stored at –40°C until further processing. In three batches, meat samples were ground using 1/4 in plate and formed into patties (100 g each). The ground samples were measured for pH, protein solubility, emulsion activity index, protein denaturation, salt-induced water uptake and subsequent cooking loss and final yield. The patties were displayed at 2°C under light (l × 1800) and color stability, lipid oxidation (TBARS) and thiol contents were examined. The patties were also measured for purge/cooking loss and texture profile analysis (TPA). All data were analyzed using the PROC MIXED procedure of SAS (v.9.4). Means were separated using least significant differences (P &lt; 0.05).ResultsT20 samples had the lowest sarcoplasmic protein solubility among treatments, while T18 had a lower myofibrillar protein solubility compared to other treatments (P &lt; 0.05). The emulsion activity index of T20 was higher in sarcoplasmic fraction than T12 (P &lt; 0.05). T20 group also had a lower extractable protein concentration compared to other treatments, which subsequently resulted in an increase in protein denaturation (P &lt; 0.05). T20 samples had a lower value of pH, salt-induced water uptake, and cooking loss, while T18 had a lower final yield than T16 and T12 (P &lt; 0.05). No differences in physicochemical traits of patties were found between treatments, indicated by TPA, purge and cooking loss results (P &gt; 0.05), however T20 had a greater display weight loss than T12 (P &lt; 0.05). T20 patties maintained the highest L* and hue angle values during entire display, which could be attributed to its inferior water-holding capacity (P &lt; 0.05). Both lipid (TBARS) and protein oxidation (thiol content) were increased with display (P &lt; 0.05), but no significant photoperiod effect was found (P &gt; 0.05).ConclusionThe results from the present study indicate that extended photoperiod would result in adverse impacts on functional/technological properties and oxidative stability of broiler meat. This is the first study reporting the importance of broiler housing condition (photoperiod) and its subsequent impacts on final meat quality and processing properties. The findings would provide insights into development of mitigating strategies for the poultry industry to prevent quality deteriorations of broiler meat due to the extended photoperiod.

Possible Role of Myoglobin in Regulating Calpain-1 Activity in Postmortem Beef Muscle

ObjectivesPrevious research revealed a relationship between meat color and beef tenderness and indicated that myoglobin can inhibit calpain-1 in solution. The objective of this study was to determine the extent to which myoglobin and beef color are associated with calpain activity and beef tenderness.Materials and MethodsBeef Longissimus dorsi samples from the left side of Holstein beef carcasses (n = 21) were collected immediately post exsanguination on the processing floor for 0 h analyses. Muscle temperature and pH was measured at 0, 24, and 48 h postmortem. After USDA quality and yield grade determination, steaks (n = 6) were removed from the right side of each carcass (n = 21) at 48 h for analyses at 48 and 336 h postmortem. Color (L*, a*, and b* values), surface myoglobin redox forms, metmyoglobin reducing activity (MRA), total myoglobin concentrations, slice shear force (SSF), Warner-Bratzler shear force (WBSF) were measured. Calpain-1 concentrations and autolysis were determined via Western blot at 0, 48, and 336 h.ResultsDecline in muscle pH was 6.4, 5.8, and 5.6 at 0, 24, and 48 h, respectively. Shear force values at 48 h were 73.19 N for WBSF and 384.21 N for SSF and at 336 h were 48.75 N for WBSF and 260.47 N for SSF. Myoglobin reducing activity at 336 h was positively correlated to WBSF at 48 h and negatively correlated to calpain-1 concentration at 0 h (P &lt; 0.05; Table 9). Color measurements of L* and b* at 48 h were moderately correlated with WBSF at 336 h (P &lt; 0.05; Table 9). The b* measurement at 336 h showed a moderate relationship to calpain-1 concentration at 0 h (P &lt; 0.05; Table 9).ConclusionModerate correlations between color and tenderness measurements taken at 48 h with those taken at 336 h were discovered indicating that myoglobin may impact calpain-1 in vivo.Table 9Correlations (P-values) between selected color and tenderness measurements (n = 21)

Contribution of Protein Degradation and Sarcomere Length to Aged Pork Loin Warner-Bratzler Shear Force

ObjectivesPostmortem aging of fresh pork loins improves tenderness through protein degradation. Sarcomere length (SL) of postmortem muscle can vary between animals, and this could impact access and efficacy of proteinases to degrade proteins within, but not outside of the myofibril. The relationship between SL and protein degradation is not well documented in pork. Therefore, the objective of this experiment was to compare protein degradation of troponin-T with desmin and SL in aged pork loins over 21d.Materials and MethodsPaired sides of fresh pork loins (n = 20) were collected 1 d postmortem. Criteria for inclusion in the study were a pH between 5.85 and 6.10 and a visual color score (NPPC) between 3 and 4. Eight loin chops (2.54 cm) containing only the longissimus muscle were fabricated. Two chops from each pair of loins were aged for 1, 8, 14 or 21 d and immediately evaluated. After aging, chops were cooked to 68°C and Warner-Bratzler shear force (WBSF) was measured. Whole muscle proteins were solubilized from samples at each aging period (10mM sodium phosphate, pH 7.0 and 2% wt/vol sodium dodecyl sulfate). Abundance of degraded troponin-T (30 kDa) and intact desmin (55 kDa) in the whole muscle sample was determined by immunoblotting. Abundance of troponin-T degradation product and intact desmin was normalized by a reference sample on each gel. A helium-neon laser diffraction method was used to determine SL (total of 36 SL per sample were recorded). The distance between primary diffraction bands was used to calculate SL. Correlation coefficients were determined using PROC CORR of SAS 9.4 and significance determined by P &lt; 0.05.ResultsOverall and across all days aging, SL was not strongly correlated to intact desmin (r = –0.198; P = 0.07) or troponin-T degradation (r = 0.236; P = 0.04). Troponin-T degradation was not detected at d1 in any samples but overall and across all days was highly correlated with WBSF (r = –0.671; P &lt; 0.01)). Intact desmin was correlated with WBSF (r = 0.661; P &lt; 0.01). Across all samples, SL was correlated with WBSF (r = –0.445; P &lt; 0.01). Intact desmin and troponin-T degradation were correlated (r = –0.818; P &lt; 0.01).Correlations within day of aging revealed that protein degradation was not significantly correlated with WBSF at d 1. In contrast, troponin-T degradation was correlated (P &lt; 0.01) with WBSF at 8, 14, 21 d postmortem (r = –0.733, –0.641, and –0.772, respectively). Similarly, intact desmin was correlated (P &lt; 0.01) with WBSF at 8, 14, and 21 d postmortem (r = 0.447, 0.553, and 0.824, respectively). SL was correlated (P &lt; 0.01) with WBSF at each d postmortem (r = –0.445, –0.562, –0.714, and –0.512, respectively).ConclusionThe correlation results suggest that SL is consistently correlated with WBSF across aging periods and is more strongly correlated with WBSF early postmortem than protein degradation. After aging, troponin-T degradation and intact desmin demonstrate greater correlations with WBSF than SL. Finally, SL correlation to troponin-T and desmin were generally similar and not strong, suggesting that SL does not affect the efficacy of proteinases to degrade proteins within the myofibril differently than extra-myofibrillar proteins.

Retail Display Lighting and Packaging Type May Influence Beef Flavor and Oxidative Stability

ObjectivesThis study aimed to evaluate the impact of retail display lighting and packaging type on beef flavor and lipid oxidation in five muscles.Materials and MethodsSubprimals (n = 40 strip loins, 60 shoulder clods, 60 tenderloins, 24 inside rounds, 60 top butts) were randomly collected from separate carcasses. At 7d postmortem muscles (Longissimus lumborum, LL; Triceps brachii, TB; Psoas major, PM; Semimembranosus, SM; Gluteus medius, GM) were fabricated and sliced to 2.54cm steaks. Per muscle, 120 steaks were randomly assigned to packaging treatments: vacuum rollstock (ROLL); high-oxygen (80% O2/20% CO2; HIOX); overwrapped in a motherbag with carbon monoxide (0.4%CO/30%CO2/69.6%N2; CO); and traditional overwrap (OW), which was vacuum packaged until immediately prior to display. Packages were stored in the dark at 2°C an additional 13 d prior to retail display, then were displayed under fluorescent lights (FL) or light-emitting diodes (LED) with a third treatment in dark storage (DARK). All were held in their respective light treatments at 2°C for 72h, then assigned for trained panels or chemical analysis, vacuum packaged and frozen at –20°C. For sensory analysis steaks were thawed to 4°C and cooked to 71°C. Panelists (n = 8) were trained to evaluate twelve flavors, overall juiciness and tenderness, which were scored on a 100-point scale (0 = not present; 100 = extremely present). Lipid oxidation of raw steaks was quantified as 2-thiobarbituric acid reactive substances (TBARS; mg malondialdehyde (MDA)/kg beef).ResultsNo three-way interaction (P ≥ 0.10) or lighting effect (P ≥ 0.09) was observed for trained panels or TBARS. Cardboard flavor had a muscle×lighting interaction (P = 0.02). In GM, FL had greater (p &lt; 0.05) cardboard than other lighting; in other muscle types lighting was similar. Muscle×packaging influenced three attributes (P ≤ 0.02). Steaks in ROLL were sweeter (p &lt; 0.05) than other packaging in GM, PM and TB; ROLL was juicier (p &lt; 0.05) than other packaging in GM, PM, and SM. Across all packaging types tenderness was greatest for PM, while SM was least tender (p &lt; 0.05) in CO, HIOX and OW packaging. Packaging influenced nine flavors (P ≤ 0.01); ROLL was greatest in beef ID, bloody/serumy, fat-like, umami, and salty, while HIOX scored greatest for oxidized, bitter, and sour. Brown/roasted was greatest (p &lt; 0.05) in HIOX and CO. Muscle impacted liver-like flavor (P = 0.01), which was lower (p &lt; 0.05) in SM than all other muscle types; LL, TB, PM and GM were similar (p &gt; 0.05) for liver-like. Packaging influenced TBARS (p &lt; 0.01); HIOX had the greatest concentration of MDA, followed by CO, OW and ROLL with the lowest (p ≤ 0.05). Muscle influenced TBARS (P &lt; 0.01), where TB was greatest (p &lt; 0.05), followed by SM, PM, and GM, which were similar (p &gt; 0.05); LL had the lowest MDA concentration. Oxidized (P &lt; 0.01, r = 0.34), cardboard (P &lt; 0.01, r = 0.30), bitterness (P &lt; 0.01, r = 0.23), and sourness (P &lt; 0.01; r = 0.22) were positively correlated with TBARS, while beef ID (P &lt; 0.01, r = –0.23), umami (P &lt; 0.01, r = –0.23), and tenderness (P &lt; 0.01; r = –0.21) were negatively correlated.ConclusionRetail display lighting did not directly influence sensory characteristics or lipid oxidation; lighting only impacted cardboard flavor in an interaction with muscle type. These results suggest after 72h retail display, flavor differences between steaks of similar muscle and packaging displayed under LED or fluorescent lights may not be distinguishable.

Comparison of Oxidizing Solutions in the Evaluation of Metmyoglobin Reducing Activity in Dark-Cutting Beef

ObjectivesMetmyoglobin reducing activity (MRA) is an inherent muscle biochemical property that can influence color stability. Hence, MRA is used in color research to better understand meat color changes. A greater postmortem muscle pH can affect inherent biochemical properties, including the conventional methodologies to determine MRA. The MRA methodology described in the American Meat Science Association Color Guide utilize changes in nitric oxide induced-metmyoglobin level pre- and post-incubation. However, a greater muscle pH can limit initial metmyoglobin formation. Hence, the methodology discussed in the AMSA color guide may not provide accurate results. Therefore, the objectives of this research were to compare different conditions to induce initial metmyoglobin formation.Materials and MethodsIn the first objective, normal-pH and dark-cutting steaks were dipped in 0.3%s (level recommended in the AMSA color guide) and 1% nitrite solution to induce metmyoglobin formation. Metmyoglobin formed steaks were blotted dry, vacuum packaged, and incubated at 30°C to induce metmyoglobin reduction. In the second experiment, 1% ferricyanide solution was used as an oxidizing agent. The methodology used in objective 1 was used to measure MRA. The experiments were replicated three times and the data were analyzed using the Mixed Procedure of SAS.ResultsThere was no effect (P &gt; 0.05) of nitrite concentration on MRA of dark-cutting beef. However, when 1% ferricyanide was used as an oxidizing agent, initial metmyoglobin was more (P &lt; 0.05) in dark-cutting beef compared with 0.3% sodium nitrite solution.ConclusionThe results suggest that the use of a strong oxidizing agent can impart more metmyoglobin formation in high-pH/dark-cutting beef.

Investigation of the relationships between different enzymes and postmortem duck muscle tenderization

Postmortem tenderization is induced by the cooperation of various related enzymes. In this study, CaCl2 and MDL-28170 (Cbz-Val-Phe-H, a calpain inhibitor) were used to regulate the postmortem tenderization process of duck breast muscle. Then the relationship between muscle tenderness change and different enzymes was investigated. At day 0, day 1, and day 4 of the postmortem ageing, the shear force, myofibril fragmentation index (MFI), enzymatic activities of calpains, cathepsin-B, caspase-3, Na+/K+-ATPase, and Ca2+-ATPase were respectively determined. The results showed that duck muscle tenderness could be significantly raised by CaCl2 and reduced by MDL-28170, respectively (P < 0.05). The CaCl2 treatment did not promote the calpains activity as predicted (P > 0.05), but significantly up-regulated the activity of cathepsin-B (P < 0.05). The MDL-28170 significantly inhibited the activity of the calpain system at day 1 (P < 0.05) and raised the activities of the selected 3 apoptosis-related enzymes, including caspase-3, Na+/K+-ATPase, and Ca2+-ATPase, at both day 1 and day 4 (P < 0.05). These data indicated that in duck, the calpain system could be well activated after death, and cathepsin-B might also play an important role in postmortem muscle tenderization. The caspase-3, Na+/K+-ATPase, and Ca2+-ATPase probably have no significant effects on duck muscle tenderness. It might provide useful information for duck production and further research on postmortem muscle tenderization.

Study on the effect of CaMKKβ-mediated AMPK activation on the glycolysis and the quality of different altitude postmortem bovines longissimus muscle.

This study investigated the activity of adenosine monophosphate-activated protein kinase (AMPK), glycolysis, and meat quality index in three altitude bovines during postmortem aging process. Local cattle (altitude:1,500m), Gannan yak (3,000m), and Yushu yak (4,500m) postmortem Longissimus Dorsi (LD) muscle were used. Results indicated that CaCl2 significantly increased the AMPK activity by increasing the calcium-regulated protein kinase kinase (CaMKKβ) activity. Besides, AMPK activation enhanced the activity of lactate dehydrogenase (LDH) and Ca2+ -ATPase and accelerated the rate of muscle maturation during postmortem aging. Moreover, the expression of HIF-1, PRKAA2, and GLUT4 genes in high-altitude Yushu yak was higher than that of low-altitude bovines. CaCl2 activates AMPK by activating CaMKKβ cascade and accelerates postmortem glycolysis affecting the intramuscular environment, color, and muscle protein degradation to accelerate postmortem muscle maturation, suggesting that AMPK has essential effects on postmortem muscle glycolysis and quality, and can regulate muscle quality by regulating postmortem muscle AMPK activity. PRACTICAL APPLICATIONS: Insufficient postmortem glycolysis usually leads to DFD (dark, firm, and dry) meat. Beef have relatively high incidences of DFD meat, which has an unattractive dark color and causes significant loss to the meat industry. Therefore, AMPK, which can regulate postmortem glycolysis to affect meat quality, is a valid research target.

In-depth mapping of the proteome of Tibetan pig tenderloin (longissimus dorsi) using offline high-pH reversed-phase fractionation and LC-MS/MS.

In recent years, Tibetan pig breeding and meat processing have developed rapidly. However, the basic physiological and biochemical characteristics of Tibetan pork have not been systematically explored. The present study conducted a high-throughput analysis of the tenderloin (longissimus dorsi) proteome of the Tibetan pigs and performed a functional annotation and bioinformatics analysis of the identified proteins. Based on offline two-dimensional liquid chromatography fractionation and MS/MS identification, a total of 1,723 proteins were identified in the tenderloin of Tibetan pigs. Gene ontology analysis and pathway enrichment analysis revealed that the proteins involved in respiration (oxidative phosphorylation, glycolysis/gluconeogenesis, citric acid cycle, and pyruvate metabolism) and protein synthesis and metabolism (proteasome, amino acid biosynthesis, endoplasmic reticulum protein processing, and ribosomes) were significantly enriched, indicating that the energy production and protein metabolism are the most important physiological processes in Tibetan pig tenderloin. Practical applications The in-depth mapping of the tenderloin (longissimus dorsi) proteome of the Tibetan pigs gives a panoramic perspective at the protein molecular level and provides important information on the mechanisms of postmortem muscle physiology and meat quality formation. Furthermore, the development of Tibetan pork storage and processing technologies would also benefit from the characterization of the biochemical properties of Tibetan pork.

Acetylome profiling reveals extensive involvement of lysine acetylation in the conversion of muscle to meat

Protein lysine acetylation is an post-translational modification that regulates gene expression, metabolism, cell signaling, and diseases, but its implication in the postmortem (PM) meat quality development is basically unclear. In the present study, a quantitative proteomic analysis was conducted to profile acetylome in porcine muscle within 24 h PM. In total 595 acetylation sites assigned to 163 proteins were identified in porcine muscle, of which 460 sites distributing to 110 proteins significantly changed in acetylation levels in the conversion of muscle to meat. The dynamic acetylation/deacetylaion of muscle proteins was closely associated with critical chemical-biophysical changes in PM muscle. Bioinformatic analysis revealed that protein lysine acetylation likely regulated postmortem meat quality development by regulating glycolysis and muscle pH, cell stress reponse and apoptosis, muscle contraction and rigor mortis, calcium signaling and proteolysis, IMP synthesis and meat flavor development, and even the stability of pigment proteins and meat color. This study provided the first overview of protein lysine acetylation in PM muscle and revealed its significance in the conversion of muscle to meat. Future exploration of the exact role of protein lysine acetylation at specific sites will further our understanding regarding the underlying mechanisms and be helpful for meat quality control. SignificanceThis is the first analysis of acetylome in farm animal and postmortem muscle. Our data showed that the dynamic acetylation/deacetylation of muscle proteins was closely related to the postmortem changes of muscle that affect the final quality of raw meat. Proteins related to glucose metabolism and muscle contraction were the two largest clusters of acetylproteins identified in postmortem porcine muscle. Networks of acetylproteins involved in apoptosis, calcium signaling and IMP synthesis were identified in postmortem porcine muscle at the same time. Our results revealed that protein lysine acetylation regulated the conversion of muscle to meat. It likely regulated meat quality development by regulating postmortem glycolysis, mitochondrion initiated cell apoptosis, calcium signaling, rigor mortis, meat flavor compound sysnthesis and meat tenderization. Our study broadened our understanding of the biochemistry regulating the postmortem conversion of muscle to meat and final meat quality development, which may be helpful for future meat quality control.

Proteomic analysis reveals that lysine acetylation mediates the effect of antemortem stress on postmortem meat quality development

To explore the involvement of protein lysine acetylation in the conversion of muscle to meat, a quantitative analysis of the acetylome in postmortem porcine muscle with or without antemortem stress was conducted. In total, 771 acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified. Acetylproteins were enriched in muscle contraction, carbohydrate metabolism, cell apoptosis and calcium signaling. Bioinformatic analysis suggests that preslaughter handling may be associated with glycolysis in postmortem muscle and the overall meat quality, via acetylation of multiple enzymes of glycogenolysis/glycolysis, regulate rigor mortis via acetylation of contractile, ATP production and calcium signaling-related proteins, and regulate stress response, cell apoptosis and meat tenderization via regulating the functions of heat shock proteins and permeability transition pore complex. This study provides the first overview of the acetylome in postmortem muscle as affected by preslaughter handling and broadens knowledge of the biochemistry regulating meat quality development.

Mitochondrial functionality and beef colour: A review of recent research

The bright-red colour of meat that consumers prefer depends on the depth of oxygen diffusion into the tissue and myoglobin oxygenation. Interestingly, both processes are influenced by mitochondrial activity in postmortem muscle. The transition of muscle metabolism from aerobic to anaerobic pathways affects various cellular processes including mitochondrial functionality. Numerous studies report that even with compromised structure, mitochondria continue to influence postmortem beef colour via oxygen consumption and metmyoglobin reducing activity. Hence, an in-depth understanding of the pre- and post-harvest factors affecting mitochondrial functionality can significantly enhance existing knowledge of meat colour and colour stability. Several comprehensive reviews have discussed the biochemical factors affecting meat colour, but there are only a few that have sections on the impact of mitochondria on beef colour. Furthermore, advancement of high-throughput techniques such as metabolomics and proteomics has enabled researchers to elucidate metabolite and protein changes related to mitochondria. Therefore, the objective of this review is to provide an overview on the role of mitochondria in beef colour, with a focus on recent advances in mitochondrial research, oxygen consumption, and metmyoglobin reducing ability. Keywords: Myoglobin, metmyoglobin reducing activity, meat colour, metabolomics, oxygen consumption, proteomics