Spectro 1-A Potential Spectrophotometer for Measuring Color and Myoglobin Forms in Beef.

The objective of this study was to compare the Color Muse Spectro 1 sensor to the HunterLab MiniScan XE Plus spectrophotometer for evaluating beef color. Color coordinates (lightness (L*), redness (a*), yellowness (b*), chroma (C*), and hue (h*)), myoglobin redox forms (metmyoglobin (MMb), deoxymyoglobin (DMb), and oxymyoglobin (OMb)), and metmyoglobin reducing ability (MRA) were measured on beef steaks over a 5-days storage period. The results indicated that L*, b*, C*, MMb%, OMb%, and MRA% values obtained with Spectro 1 were comparable to those of MiniScan. However, Spectro 1 values for a* were overestimated compared to MiniScan (p < 0.05), whereas those for h* and DMb% were underestimated (p < 0.05). Regardless, Spectro 1 had the capability to detect changes in color coordinates, myoglobin forms, and MRA throughout the storage period. Bland−Altman plots demonstrated that L*, b*, and C* are interchangeable between the two instruments, but it was not the case for a*, h*, myoglobin forms, and MRA. Color coordinates measured by Spectro 1 exhibited excellent stability over time, evidenced by the low total color difference (ΔE*ab) values. Collectively, these findings indicate that Spectro 1 is a potential alternative spectrophotometer for studying meat color and myoglobin redox forms.

The effects of age, sex, and hot carcass weight on cooked lamb flavor and off-flavor in four muscle cuts.

The present study used 48 lambs originating from three different locations in the Western United States (16 lambs per location; 8 ewes and 8 wethers per location). Each consisting of similar breed composition (Suffolk cross) that were selected to represent weight by age at harvest treatments: light weight carcasses at 5 mo (LW5, 31.81 ± 1.88 kg), light weight carcasses at 12 mo (LW12, 35.09 ± 4.45 kg), heavy weight carcasses at 12 mo (HW12, 57.89 ± 4.70 kg) with different carcass weight compositions. Older heavy weight lambs (HW12) had greater (P ≤ 0.01) hot carcass weight, ribeye area, backfat and body wall thickness, and yield grade compared with light weight lamb carcasses (LW5 and LW12). The longissimus thoracis longissimus thoracis (LT) from older lamb carcasses (LW12 and HW12) had a greater (P ≤ 0.01) total lipid percentage compared with younger lamb carcasses (LW5). Across harvest weight and age treatments, wether carcasses had greater (P ≤ 0.05) total lipid percentage compared with ewe carcasses. Slice shear force values were greater (P ≤ 0.01) for both the LT and semimembranosus from older lambs (LW12 and HW12) compared with LW5 lambs, with no differences between ewes and wethers. Lamb flavor intensity was greater (P ≤ 0.05) for the LT of LW12 lambs and tended (P = 0.08) to be greater for HW12 lambs, compared with the LT from LW5 lambs. The off-flavor intensity of the LT was greater (P ≤ 0.01) for older lambs (LW12 and HW12) compared with LW5 lambs. Interestingly, the lamb flavor and off-flavor intensity scores of the ground shoulder exhibited a treatment × sex interaction. Lamb flavor intensity of LW12 lamb was greater (P ≤ 0.05) from ewes compared with wethers, whereas wethers had a greater (P ≤ 0.05) lamb flavor intensity compared with ewes for HW12 lambs, and LW12 ewe lambs had a greater (P ≤ 0.05) off-flavor intensity compared with all other treatment × sex treatment combinations. Overall, lambs in the present study possessed a mild lamb flavor, typically with greater lamb flavor and off-flavor intensities for older animals; while slice shear force and LT lipid percentage increased as animal age increased at the time of harvest.

At physiological concentrations, AMP increases phosphofructokinase-1 activity compared to fructose 2, 6-bisphosphate in postmortem porcine skeletal muscle.

Phosphofructokinase-1 (PFK-1) is the most important enzyme controlling postmortem glycolysis in living skeletal muscle and is the most likely candidate for regulation of postmortem glycolysis. We investigated the regulation of PFK-1 activity by F-2, 6-BP and AMP under simulated postmortem conditions in porcine skeletal muscle. Six pigs were harvested and longissimus lumborum samples were collected immediately post-slaughter. PFK-1 activity was assayed using increasing concentrations of F-2, 6-BP or AMP, added to the buffer adjusted to different pH. Both F-2, 6-BP and AMP increased PFK-1 activity with increasing buffer pH from 5.5 to 7.0. A concentration of 50 µM F-2, 6-BP was required to increase PFK-1 activity which is very high compared to physiological concentration in the porcine skeletal muscle. However, physiological concentrations (50-150 µM) of AMP resulted in increased PFK-1 activity compared to 1-2 µM F-2, 6-BP. Thus, AMP may play a greater role in dictating the rate and extent of postmortem muscle glycolysis and pH decline as compared to F-2, 6-BP.

Potassium carbonate improves fresh pork quality characteristics.

Meat enhancement strategies like sodium tripolyphosphate (STP) are used to improve fresh meat quality attributes like color, water-holding capacity, and tenderness. However, alternatives are necessary because of reduced consumer acceptance of STP. One alternative is potassium carbonate (K2CO3). A study was conducted to evaluate K2CO3's impact on fresh, boneless, center-cut pork loins enhanced with one of five treatments: a negative control, positive control (0.3% STP), and three concentrations of K2CO3 (0.1, 0.3, and 0.5%). Loins were cut into chops, stored under simulated retail display, and analyzed for color (L*, a*, b*), pH, cook loss, and tenderness. For each quality characteristic measured, the 0.3% and 0.5% K2CO3 maintained redness (a*), decreased yellowness (b*), reduced cooking loss, and maintained tenderness compared to STP. SDS-PAGE analysis further determined that both K2CO3 and STP extracted myosin heavy chain. Combined, these data suggest that K2CO3 may function as an alternative to STP in the fresh pork industry provided microbial safety and shelf-life are appropriately controlled.

Postmortem glycolysis and glycogenolysis: insights from species comparisons.

After an animal is harvested for meat, the skeletal muscle initiates a complex series of biochemical reactions in an attempt to maintain energy homeostasis. In order to maintain energy homeostasis, enzymes within the skeletal muscle metabolize stored carbohydrate into energy under predominantly anaerobic conditions in a process known as postmortem energy metabolism. Because these biochemical reactions have the potential to affect meat quality attributes either positively or negatively, evaluating their regulation postmortem is fundamental to understanding meat quality. In this review, the regulation of postmortem glycolytic enzymes will be evaluated. Special consideration will be given to species-specific quality defects in pigs, poultry, and fish as a means to further discuss similarities and differences in postmortem glycolysis and glycogenolysis.

Phosphofructokinase and mitochondria partially explain the high ultimate pH of broiler pectoralis major muscle.

During postmortem metabolism, muscle pH gradually declines to reach an ultimate pH near 5.6 across most meat species. Yet, broiler pectoralis major (P. major) muscle generates meat with high ultimate pH (pH ∼ 5.9). For better understanding of the underlying mechanism responsible for this phenomenon, we evaluated the involvement of breast muscle chilling on the extent of postmortem metabolism. Broiler breast muscles were either subjected to chilling treatment (control) or left at room temperature (RT) for 120 min. P. major muscle from the RT treatment had lower ultimate pH, greater glycogen degradation and lactate accumulation. While these findings suggest that carcass chilling can contribute to the premature termination of postmortem metabolism, chilling did not fully explain the high ultimate pH of P. major muscle. Our results also revealed that glucose-6-phosphate (G6P) was very low at 24 h, and therefore we hypothesized that G6P was limiting. To test this hypothesis, muscle samples from P. major and porcine longissimus lumborum (LL) muscle were homogenized into a reaction buffer that mimics postmortem glycolysis with or without 0.5 mg/mL isolated mitochondria. While samples containing porcine LL muscle reached the normal level of ultimate pH, P. major muscle samples reached a value similar to that observed in vivo even in the presence of excess G6P, indicating that G6P was not limiting. Mitochondria enhanced the glycolytic flux and pH decline in systems containing muscle from both species. More importantly, however, was that in vitro system containing chicken with mitochondria reached pH value similar to that of samples containing LL muscle without mitochondria. To investigate further, phosphofructokinase (PFK) activity was compared in broiler P. major and porcine LL muscle at different pH values. PFK activity was lower in P. major muscle at pH 7, 6.5, and 6.2 than LL muscle. In conclusion, carcass chilling can partially contribute to the high ultimate pH of broiler P. major muscle, while low PFK activity and mitochondria content limit the flux through glycolysis.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

OBJECTIVE: Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. METHODS: We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. RESULTS: We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). CONCLUSIONS: Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.

Presence of oxygen and mitochondria in skeletal muscle early postmortem.

Anaerobic glycolysis dominates energy metabolism postmortem. Even so, however, recent studies suggest mitochondria can modify postmortem energy metabolism and may contribute to pH decline, possibly affecting the transformation of muscle to meat and fresh meat quality development. Because oxygen is a necessary component of mitochondrial function, oxygenation of porcine and bovine longissimus thoracis et lumborum was determined postmortem using NIR spectroscopy. The ratio of oxy- to deoxymyoglobin decreased with time postmortem in both species. Metabolic analyses of muscle samples collected over the same timeframe also revealed fluctuations in TCA intermediates. Finally, mitochondria collected from muscle of electrically stimulated carcasses differed from those of non-stimulated muscle. Collectively, these data support the thesis that muscle mitochondria function early postmortem and may play a more active part in pH decline and possibly meat quality development.

A mitochondrial protein increases glycolytic flux.

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.

Excess glycogen does not resolve high ultimate pH of oxidative muscle.

Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles.

Net lactate accumulation and low buffering capacity explain low ultimate pH in the longissimus lumborum of AMPKγ3 R200Q mutant pigs.

Postmortem lactate accumulation in skeletal muscle is linearly associated with the extent of pH decline. Yet, pigs harboring the AMPKγ3(R200Q) mutation produce meat with similar lactate levels to that of wild-type pigs but have a lower ultimate pH. We hypothesized that lower initial lactate levels and (or) lower buffering capacity in muscle of these pigs may help explain this discrepancy. Longissimus lumborum muscle samples were harvested at 0 and 1440 min postmortem from AMPKγ3(R200Q) and wild-type pigs. As expected, AMPKγ3(R200Q) muscle exhibited a lower ultimate pH but similar lactate levels to that of wild-type pigs at 1440 min postmortem. However, the total net lactate produced postmortem was greater in the AMPKγ3(R200Q) muscle due to lower initial lactate levels at 0 min postmortem. Buffering capacity measured over the pH range of 5.5-7.0 was also lower in AMPKγ3(R200Q) muscle. Greater net lactate accumulation postmortem (i.e., glycolytic flux) coupled with a lower buffering capacity explains the lower ultimate pH of meat from AMPKγ3(R200Q) pigs.

Mitochondria influence postmortem metabolism and pH in an in vitro model.

Our objective was to determine the influence of mitochondria on metabolites and pH decline using an in vitro model of postmortem muscle metabolism. Mitochondria were isolated from porcine longissimus lumborum and added (0, 0.5, or 2.0mg) to powdered muscle in reaction media containing either a combination of inhibitors for mitochondria complexes (I, IV, and V) or diluent (without inhibitors). In the absence of inhibitors, adding mitochondria (0.5 and 2.0mg) reduced ATP loss from 30 to 120 min, but did not alter glycogen or lactate during this time. In reactions with mitochondria, inhibitors decreased ATP levels by 30 min and increased glycogen degradation by 60 min. Regardless of mitochondria content, inhibitors enhanced lactate accumulation from 15 to 240 min, and decreased pH from 15 min to 1440 min. In the in vitro model, mitochondria influence the maintenance of ATP, and inhibition of mitochondria enzyme activity contributes to accelerated metabolism and pH decline.

pH inactivation of phosphofructokinase arrests postmortem glycolysis.

Fresh meat quality development is influenced by pH decline that results from muscle glycolyzing energy substrates postmortem. The exact reason why glycolysis stops in the presence of residual glycogen remains unclear. We hypothesized that a critical glycolytic enzyme loses activity near the ultimate pH of meat. Porcine longissimus muscle samples were subjected to an in vitro system that mimics postmortem anaerobic metabolism at buffered pH values (7.0, 6.5, 6.0, 5.5 or 5.0). At pH7.0, 6.5, and 6.0, glycogenolysis and glycolysis proceeded normally while pH5.5 stopped lactate formation. Additional experimentation indicated that phosphofructokinase lost activity at pH5.5 while all other glycolytic enzymes remained active. A similar inactivation of phosphofructokinase was observed when using chicken and beef muscle. Elevated temperature hastened pH decline and phosphofructokinase activity loss. Thus, pH inactivates phosphofructokinase and arrests postmortem glycolysis, which may explain the similar ultimate pH across meat of different species.

Contribution of the phosphagen system to postmortem muscle metabolism in AMP-activated protein kinase γ3 R200Q pig Longissimus muscle.

Pigs with the AMP-activated protein kinase γ3 R200Q (AMPKγ3(R200Q)) mutation generate pork with low ultimate pH (pHu). We hypothesized that reducing muscle creatine (Cr) and phosphocreatine (PCr) may accelerate postmortem ATP consumption and prevent extended pH decline in AMPKγ3(R200Q) longissimus muscle. Wild type and AMPKγ3(R200Q) pigs were assigned to control diet or diet supplemented with the creatine analog ß-guanidinopropionic acid (ß-GPA, 1%) for 2 wk. ß-GPA reduced muscle PCr (P = 0.006) and total Cr (P<0.0001). In general, AMPKγ3(R200Q)+ß-GPA exhibited more rapid metabolism than control, AMPKγ3(R200Q), and ß-GPA treatment, evidenced by more rapid loss of ATP, more rapid increase in IMP, and decreased pH during the first 90 min postmortem. Overall, pHu was similar despite elevated glycogen (AMPKγ3(R200Q)), reduced total Cr (ß-GPA) or both (AMPKγ3(R200Q)+ß-GPA). Thus, reducing muscle phosphagens did not affect pHu in AMPKγ3(R200Q) muscle, but it hastened ATP depletion and pH decline.