Sulaiman K. Matarneh

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.

Exploring the unknowns involved in the transformation of muscle to meat

Meat quality development, or the transformation of muscle to meat, involves a myriad of biochemical pathways that are largely well-studied in living muscle tissue. However, these pathways are less predictable when homeostatic ranges are violated. In addition, there is far less known about how various management or environmental stimuli impact these pathways, either by substrate load or altered cellular environment. Likewise, it is largely accepted that oxygen plays little to no role in the conversion of muscle to meat, as anaerobic metabolism predominates in the muscle tissue. Even so, the oxygen tension within the tissues does not fall precipitously at exsanguination. Therefore, transition to an anaerobic environment may impact energy metabolism postmortem. Antemortem handling, on the other hand, clearly impacts meat quality development, yet the exact mechanisms remain a mystery. In this paper, we will attempt to review those factors known to affect postmortem energy metabolism in muscle and explore those areas where additional work may be fruitful.

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 AMPKγ3R200Q 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.

Altered AMP deaminase activity may extend postmortem glycolysis

Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3(R200Q) mutated pigs (RN⁻) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN⁻ muscle than wild-type. RN⁻ muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN⁻ pork.

Chapter 4 – Perimortal Muscle Metabolism and its Effects on Meat Quality

Muscle tissue experiences profound changes during its conversion to meat. Indeed, these changes are necessary and requisite for meat to assume those unique properties are keenly coveted by consumers of this highly nutritious and palatable food source. However, when this process is perturbed, the resulting quality is dramatically impacted. Therefore, understanding those reactions forming the basis of postmortem metabolism is incumbent to those interested in meat animal production. Initially, the biochemistry responsible for postmortem metabolism is simply an extension of those processes responsible for supplying energy to functioning skeletal muscle, responsible for locomotion. However, at some yet-to-be defined point, muscle tissues broach the physiological threshold where they begin to die and at this juncture, biochemical reactions dysregulate according to physiological normalcy. Even with the seemingly dearth of information available on the topic, our intent is to outline the biochemical processes responsible for postmortem metabolism and the conversion of muscle to meat as currently known.

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.

Mitochondrial F1-ATPase extends glycolysis and pH decline in an in vitro model

The experiment was conducted to identify the mitochondrial protein responsible for enhancing glycolytic flux. We hypothesized that mitochondrial F1-ATPase promotes ATP hydrolysis and thereby the flux through glycolysis. Porcine longissimus muscle mitochondria were incorporated into an in vitro system designed to recapitulate postmortem glycolysis with or without Na-azide to specifically inhibit the β-subunit of mitochondrial F1-ATPase that catalyzes ATP hydrolysis. Addition of mitochondria enhanced ATP hydrolysis, glycogen degradation, lactate accumulation, and pH decline in the in vitro system. However, the majority of mitochondria-mediated enhancement in glycolytic flux was abolished in the presence of Na-azide. To investigate further, myofibrillar and mitochondrial proteins were added to the in vitro system after 240min from the initiation of the reaction. Greater pH decline and lactate accumulation were observed in system containing mitochondrial protein compared to their myofibrillar counterpart. In conclusion, mitochondrial F1-ATPase is capable of increasing glycolytic flux through promoting greater ATP hydrolysis at lower pH.

Muscle characteristics only partially explain color variations in fresh hams

Fresh hams display significant lean color variation that persists through further processing and contributes to a less desirable cured product. In an attempt to understand the underlying cause of this color disparity, we evaluated the differences in muscle characteristics and energy metabolites across semimembranosus (SM) muscles differing in color variation. The L* (lightness) and a* (redness) values were highest and lowest (P<0.001), respectfully in the most caudal aspects of the muscle while the ultimate pH was the lowest (P<0.001). Correspondingly, this region possessed highest (P<0.01) glycolytic potential (GP) and lactate dehydrogenase (LDH) levels but did not differ in the amount of myoglobin or myosin heavy chain type I isoform. These data show that differences in muscle may contribute to ham color variation but suggest other factors may mitigate or exacerbate these variances.