Postmortem energy metabolism and meat quality development: Advances in basic pathways, endogenous regulatory factors, and exogenous management strategies

Graphical abstract

Highlights

• •Basic pathways of postmortem energy metabolism (PEM), especially via mitochondria, were presented.
• •CaMKK/AMPK and HIF-1α signaling contributed notably to activation of postmortem glycolysis.
• •Regulation of PEM by protein posttranslational modifications was comprehensively discussed.
• •Exogenous management strategies and scientific outlook of PEM were also provided.

Abstract

Background

A complex series of biochemical processes is involved during the transformation of muscle into meat, among which postmortem energy metabolism that contributes to adenosine triphosphate regeneration remains paramount. Thus, a timely and thorough overview of postmortem energy metabolism is essential for developing high-quality fresh meat.

Aim of review

The present work reviewed three basic pathways of postmortem energy metabolism and their impacts on meat quality development with special attention to mitochondria aerobic metabolism. In addition, endogenous factors and their mechanisms as well as exogenous strategies for regulating postmortem energy metabolism were also comprehensively summarized.

Key scientific concepts of review

Postmortem mitochondria, especially from livestock, remain metabolically active and exhibit oxygen consumption, which further mediates changes in muscle pH. Adenosine monophosphate-activated protein kinase and hypoxia inducible factor-1α, as energy-controlling hubs, contribute notably to the activation of postmortem glycolysis. Several protein posttranslational modifications (e.g., S-nitrosylation and lactylation) also show crucial regulatory roles for postmortem energy metabolism by altering the structure and function of metabolic enzymes. Exogenous techniques like electrical stimulation and advanced chilling can improve meat quality by optimizing energy metabolism process, while emerging technologies such as ultrasound hold promise. This work integrated existing knowledge, identified research gaps, and proposed a scientific outlook on species-specific postmortem energy metabolism.

Introduction

Fresh meat quality properties, including water holding capacity (WHC), color, and texture, significantly impact consumer purchasing behavior and the potential for meat to be further processed. Thus, the major objective of the meat industry across the globe has always been to achieve high-quality fresh meat [[1], [2], [3], [4], [5]]. However, the extreme variations in meat quality from pale, soft, and exudative (PSE) to dark, firm, and dry (DFD) meats across animal species and muscle types are consistently presented, causing substantial economic detriment to the fresh meat market. For instance, it was previously shown that PSE pork incidence in China was between 10–30 % and it is 15–20 % cheaper than that of normal pork, resulting in billions of RMB in annual losses to the pork industry [6].

Postmortem muscle involves a complex series of biochemical processes (e.g., energy metabolism, calcium release, oxidative stress, proteolysis, and apoptosis), and shedding better light on these processes is a prerequisite for reducing the occurrence of quality-deficient fresh meats. Extensive research has previously elucidated the basic biochemical pathways underlying muscle-to-meat conversion and some of the mechanisms driving the development of quality-deficient meats, as excellently reviewed in the previous literature [[7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]]. Especially, it is well-recognized that excessive discrepancy in the rate or amount of pH drop at the early postmortem directly determines product quality characteristics. Much emphasis has previously been paid to the link between postmortem energy metabolism, primarily concerning glycolysis and phosphagen system, and pH alteration [7,9]. Nonetheless, this previous viewpoint is rather restricted. Recently, a body of excellent reports has shown that mitochondria were capable of coupled respiration and function partially up to several hours postmortem, contributing to postmortem metabolism and thus changes in pH as well as meat quality [18,19]. Hence, an updated review of the basic pathways of postmortem energy metabolism, especially the contribution of mitochondria, and their implications for meat quality development is warranted.

Postmortem energy metabolism is a multi-enzyme-catalyzed biochemical process, with its levels being governed largely by the functional activities of metabolic enzymes. Abnormal alterations in this enzymatic network and the resulting metabolic status have been well-documented to be responsible for the development of quality-deficient meats, as clearly demonstrated in PSE pork, DFD beef, PSE-like poultry, and wooden breast (WB) in chicken [[20], [21], [22], [23], [24]]. Along these lines, recently, researchers have further paid more attention to the endogenous regulators responsible for the above abnormalities. To date, such as calcium/calmodulin-dependent protein kinase kinase/adenosine monophosphate-activated protein kinase (CaMKK/AMPK) as well as hypoxia inducible factor-1α (HIF-1α) signaling, protein posttranslational modifications (PTMs), heat shock proteins (HSPs), and protein DJ-1 have been discovered to mediate potentially postmortem energy metabolism through precise regulation of enzyme activation states [23,[25], [26], [27], [28], [29], [30], [31]]. These provide pivotal targets for related prevention strategies to control the development of defective meats. However, a comprehensive summary of the regulatory role of these endogenous factors on postmortem energy metabolism is still rare.

The core of this work was to deliver a recent overview of the basic pathways of postmortem energy metabolism and their endogenous regulators. Meanwhile, the impact of postmortem energy metabolism on meat quality development and exogenous strategies regarding the management of postmortem energy metabolism to produce high-quality fresh meat were critically reviewed. Finally, some recommendations for future research were also presented. Hopefully, this work can help to better understand and improve the formation of fresh meat quality by providing cutting-edge knowledge about postmortem energy metabolism.

Basic pathways of postmortem energy metabolism

After livestock and poultry are slaughtered, the ischemic and hypoxic environment triggers skeletal muscles to initiate a series of biochemical processes to achieve pre-slaughter homeostasis. During this process, enzymes metabolize stored carbohydrates into adenosine triphosphate (ATP), known as postmortem energy metabolism. Meanwhile, it is noteworthy that the ATP regeneration is crucial for maintaining energy homeostasis since ATP stores in postmortem myocytes are limited. To date, three pathways, including phosphagen system, glycolytic metabolism, and mitochondria oxidative phosphorylation (OXPHOS), are suggested to involve ATP regeneration in postmortem muscle (Fig. 1).

Fig. 1.

Postmortem energy metabolism in livestock and poultry and its effect on meat quality development. Three basic pathways, including phosphagen system, glycolytic metabolism, and mitochondria oxidative phosphorylation (OXPHOS), are suggested to involve postmortem adenosine triphosphate (ATP) regeneration, pH changes, and the resulting meat quality development. WHC, Water holding capacity; PSE, Pale, soft, and exudative; DFD, Dark, firm, and dry; WB, Wooden breast. WHC, Water holding capacity; PSE, Pale, soft, and exudative; DFD, Dark, firm, and dry; WB, Wooden breast.

Phosphagen system

The phosphagen system is composed of ATP and phosphocreatine (CP), a high-energy storage molecule in myocytes. In the presence of creatine kinase (CK), CP reversibly transfers its high-energy phosphate group to adenosine diphosphate (ADP) to produce ATP, effectively supplying energy at the early postmortem [32]. However, the storage of CP is limited, and the adenine nucleotide pool consisting of adenosine monophosphate (AMP), ADP, and ATP is readily depleted, so the phosphagen system for energy supply is only short-lived in postmortem muscle [7]. Using a kinetic model derived from beef data, Wang et al. [33] indicated that CP could be depleted within 30 min postmortem, with over 92 % of the ATP being produced during the period.

Anaerobic glycolysis metabolism

Once 70 % of the CP pool is consumed, glycolysis is recognized as the major metabolic pathway supplying ATP in postmortem muscle. Under this pathway, muscle glycogen is cleaved and metabolized to lactate with the involvement of a series of catalytic enzymes, which can be concisely summarized in the following three steps: the degradation of glycogen to a glucose unit, the generation of pyruvate, and the accumulation of lactate [34]. Among many catalytic enzymes, hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) are considered as the three rate-limiting enzymes of glycolysis. Particularly, the PFK-mediated catalytic step has been widely proven to possess a more dominant regulatory role for postmortem glycolysis as compared with other enzymes [[35], [36], [37], [38], [39]]. Phosphagen system and glycolysis are well-understood by researchers as postmortem energy supply pathways and are only succinctly described here, with details available in the previous excellent review [7,9,40,41].

Mitochondrial aerobic metabolism

Under aerobic conditions, pyruvate can be transformed into acetyl-CoA via pyruvate dehydrogenase (PDH). Acetyl-CoA then enters the mitochondrial tricarboxylic acid (TCA) cycle and yields high-energy compounds such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FADH₂). These are further oxidized in the electron transport chain (ETC) to release energy and drive ATP synthesis, known as mitochondria OXPHOS (Fig. 2). In early times, mitochondria were commonly neglected in postmortem energy metabolism due to the exsanguination abolished oxygen delivery to myocytes. However, any residual oxygen in postmortem myocytes could potentially be exploited for mitochondria OXPHOS, producing much more ATP than anaerobic glycolysis and extending postmortem metabolism. Research in recent years has demonstrated that muscle oxygen concentration in some species (e.g., pigs and cattle) could stabilize at around 18 % at 2 h postmortem after an initially rapid decline [19]. More importantly, mitochondria remained structurally intact and functionally available for several hours postmortem [[42], [43], [44]]. In an in vitro glycolysis model, the addition of isolated mitochondria differentially influenced the rate and extent of ATP hydrolysis and hydrogen proton (H⁺) accumulation, while the inhibition of mitochondrial functional activity contributed to accelerated metabolism and pH decline [[45], [46], [47]]. The electrical stimulation of beef longissimus muscle could accelerate glycolysis metabolism, primarily on account of reduced muscle oxygenation levels and impaired mitochondrial function [19]. Besides, the reduced glycogen content did not fully explain the development mechanism of dark-cutting beef. A series of recent reports indicate that the formation of typical and atypical dark-cutting beef with impaired glycolysis was always associated with upregulated mitochondrial matrix proteins and enhanced mitochondrial functional activity [[48], [49], [50], [51], [52], [53]]. These emerging data present preliminary evidence for the involvement of mitochondria in postmortem energy metabolism and thus muscle acidification.

Fig. 2.

The oxidative phosphorylation (OXPHOS) process for generating adenosine triphosphate (ATP) molecules in the postmortem mitochondria system. OXPHOS is the metabolic pathway in which mitochondria utilize energy released from the oxidation of reducing equivalents to synthesize ATP from adenosine diphosphate and inorganic phosphate. This process occurs in the inner mitochondrial membrane and is facilitated by five multi-subunit protein complexes.

Along these lines, Ramos et al. [54] further clarified that mitochondria from Angus and Brahman steer muscles showed partial competence for OXPHOS during 24 h postmortem, with especially greater OXPHOS capacity in Brahman steer muscle contributing to stable ATP levels and delayed glycolysis at the early postmortem [55,56]. Likewise, in the study of Kiyimba et al. [57], glycogen replenishment with the inhibition of mitochondria complexes I, IV, and V produced a stronger stimulus to glycolysis metabolism, reverting the metabolome of longissimus lumborum muscle (glycolytic fibers) from dark-cutting beef toward a normal postmortem state. In other words, postmortem mitochondria OXPHOS was not only available but also presented at higher levels in dark-cutting beef. As ATP regeneration from the OXPHOS pathway was inhibited, glycolysis in the presence of excess glycogen could be accelerated to supply energy with concomitant muscle acidification. It is, however, worth noting that glycogen replenishment and subsequent reshaping of normal postmortem metabolism were not achieved in the oxidative muscle across livestock species [[58], [59], [60]]. This may be related to higher antioxidant capacity and intracellular calcium homeostasis in oxidative muscle fibers, which contribute to better maintenance of mitochondrial function [61,62]. As a result, energy supply may be ensured through the OXPHOS pathway. As Ramos et al. [63] discovered, although mitochondria from bovine diaphragm (oxidative) and longissimus lumborum (glycolytic) muscles maintained their ability to produce ATP within 24 h postmortem, those from glycolytic fibers presented earlier mitochondrial disruption and lower efficiency, leading to a higher degree of muscle acidification. Besides, the metabolites in the TCA cycle changed dynamically with extended postmortem time [64], while adding mitochondria downregulated the level of pyruvate and lactate as well as variably affected the abundance of TCA cycle metabolites under postmortem simulating conditions [46]. Following this, employing a similar in vitro model and inhibiting mitochondria to consume pyruvate via blocking PDH activity, Taylor et al. [65] further discovered that the enrichment of TCA cycle intermediates (e.g., α-ketoglutarate and malate isotopomers) was notably reduced, and the glycolysis process was then accelerated. All in all, the evidence of postmortem residual oxygen and variable metabolites in glycolytic pathway and TCA cycle highlighted a potential contribution of mitochondria OXPHOS to supply energy and delay glycolytic flux at the early postmortem.

Although the above viewpoint is favored by most researchers, controversy still exists in several investigations. It was previously reported that the cumulative contribution of postmortem mitochondria aerobic respiration to ATP production was limited to less than 1 % of total ATP production, as determined by a kinetic model based on data from beef longissimus lumborum muscle [33]. However, the validation of this model had certain limitations, including a small sample size, limited muscle variety, and incomplete metabolic profiling. Besides, studies using an in vitro muscle glycolysis system revealed that within the first 30 min postmortem, the addition of more intact mitochondria helped stabilize ATP levels, suppressed glycolysis, and reduced the accumulation of H⁺ and lactate [45]. However, as incubation time increased, the added mitochondria exhibited a markedly opposite regulation pattern as described above [45]. Further analysis revealed that a water-soluble mitochondrial protein, identified as mitochondrial F₁-ATPase, was primarily responsible for the accelerated glycolytic flux [66]. Indeed, postmortem metabolism conditions characterized by elevated calcium and oxidative stress levels are favorable for the mitochondrial swelling and subsequent rupture of the mitochondrial membrane, thus causing the release of mitochondrial proteins that can potentially modulate glycolysis [42]. These findings highlight that variations in structural integrity of mitochondria may differentially determine their biochemical regulatory function within postmortem simulation systems and even in situ muscle tissue. In parallel, this also points to a critical limitation of in vitro co-incubation models for studying postmortem mitochondrial metabolism in terms of the structural and functional integrity of isolated mitochondria.

In addition to the issues including selective enrichment, low yield (typically 20 %–40 %), and the loss of physiological interactions with other organelles (e.g., sarcoplasmic reticulum and cytoskeleton), a critical limitation associated with mitochondrial isolation involves the disruption of the mitochondrial reticular architecture [67,68]. This has been clearly established by the earlier evidence that, due to the mechanical homogenization and centrifugation as well as the necessary isolation buffer, the standard isolation methods changed protein stoichiometry of mitochondrial ETC and caused notable disruption to mitochondrial structure and function [69]. Therefore, the different integrity of the exogenously added mitochondria may account for the variability observed in some of the in vitro experiments. Indeed, many of them did not assess the integrity of the isolated mitochondria. Moreover, the in vitro glycolysis model is held at a constant temperature of 25 °C and thus fails to mimic the dynamic cooling process of postmortem muscle. In general, such potential concerns outlined above could partly compromise the applicability of in vitro findings on mitochondria modulation to postmortem metabolic dynamics at the carcass or intact muscle scale.

Another approach to investigating postmortem mitochondrial aerobic metabolism involves permeabilized myofibers. In contrast to conventional organelle isolation, the preparation of permeabilized myofibers enables in situ assessment of the mitochondria within a muscle tissue. This method is independent of mitochondrial size, reticular structure, functional status, and location, thereby avoiding selective isolation of specific mitochondria while preserving over 95 % of mitochondria intact within the normal cytoarchitectural environment [70]. Therefore, permeabilized myofibers combined with high-resolution respirometry represent a more precise in vivo approach for assessing mitochondrial aerobic metabolism in postmortem muscle. However, although the above has been employed in several studies, most have focused on comparative analyses across species and muscle types [54,56,63,71]. The specific contribution of mitochondrial aerobic metabolism to postmortem ATP supply remains insufficiently elucidated. For instance, the study by Ramos et al. [56] evidenced that the mitochondria in permeabilized myofibers from Brahman exhibited greater OXPHOS capacity and coupling at 1 h postmortem relative to Angus. This finding may further explain why the Brahman longissimus muscle displayed higher ATP levels and a slower pH decline at the early postmortem [72,73]. Moreover, although recent findings indicated sustained aerobic metabolism in chicken meat, with oxidative muscles showing greater OXPHOS capacity compared to glycolytic muscles [74], evidence for mitochondrial participation in poultry postmortem aerobic metabolism remains unfortunately less available. This lack may stem from the inherently low mitochondria density characteristic of highly glycolytic poultry muscles as compared with other livestock species.

In summary, despite significant advances in understanding the regulation of postmortem mitochondria on energy metabolism have been achieved, additional in vivo investigations are warranted given the inherent limitations of in vitro efforts.

Species- and myofiber-specific postmortem energy metabolism

The pattern of postmortem energy metabolism, including its rate and extent, varies considerably among different species with respect to their distinct muscle characteristics. Diverse functional characteristics of skeletal muscle are largely determined by muscle fiber types characterized by distinct molecular, structural, and contractile properties [[75], [76], [77]]. Early on, mammalian skeletal muscle is simply classified into slow-twitch dark red muscle and fast-twitch light white muscle. Most subsequent studies employ myosin heavy chain (MyHC) isoforms as molecular markers for muscle fiber typing. For instance, using electrophoretic separation technology, our lab separated four MyHC isoforms, including MyHC I, MyHC IIA, MyHC IIB, and MyHC IIX, in three porcine skeletal muscles [78]. Similar results were also presented in other reports [[79], [80], [81], [82]]. Based on the major expression of MyHC isoforms, mammalian skeletal muscle is characterized into four types including type I, IIA, IIB, and IIX fibers. It has been widely recognized that slow-oxidative fibers correspond to type I, fast-oxidative-glycolytic fibers to type IIA, and fast-glycolytic fibers to type IIB and IIX [62]. The type I fibers, due to their greater levels of myoglobin and blood capillaries, mainly produce ATP through oxidative metabolism, while type II fibers, particularly type IIB, depend heavily on anaerobic glycolytic metabolism for ATP generation [62,82]. Another important point is that type I fibers also display a higher density of mitochondria and richer oxidative enzymes, whereas type IIB fibers encompass more glycolytic enzymes as well as more extensive sarcoplasmic reticulum responsible for the intensified calcium release [74,75,83]. These intrinsic factors further support the unique energy metabolism characteristics of various muscle fiber types. As previously mentioned by Stienen et al. [84], the ATP hydrolysis rate in type IIB fibers is about four times higher than that of type I fibers, while type IIA and type IIX fibers fall at intermediate levels. More importantly, the rate and extent of ATP hydrolysis and thereby H⁺ accumulation are critical contributors to the decline of postmortem pH. For instance, the rapidly decreased pH in type IIB fibers from poultry breast and pork longissimus muscle is largely accounted for by their greater anaerobic glycolytic capacity and the consequently higher rate of ATP hydrolysis [78,85,86]. In contrast, the slower decline of postmortem pH in type I fibers such as beef longissimus muscle is closely correlated with the lesser glycolytic flux and stronger mitochondrial functional activity [79,87].

Overall, from a macro perspective, a moderate suppression of mitochondrial aerobic metabolism and acceleration of glycolytic flux is conducive to improving meat sensory characteristics and texture properties in ruminant species with a higher proportion of type I fibers, such as cattle and ovine. On the other hand, in species dominated by type IIB muscle fibers (e.g., pork and poultry), the enhancement of mitochondrial aerobic metabolism and the slowing of glycolytic rate can avoid undesirable meat quality characteristics, i.e., typical PSE characteristics. Further understanding of the species- and myofiber-specific responses to postmortem energy metabolism is essential for improving and maintaining the meat quality of animals with different characteristics.

Effects of postmortem energy metabolism on meat quality attributes

pH

As aerobic metabolism and phosphagen system cease to supply energy, glycolysis is initiated and coupled with ATP hydrolysis and H⁺ accumulation, which subsequently results in a decrease in muscle pH from about 7 to a normally ultimate pH around 5.5–5.7 at 24 h postmortem in most species. Furthermore, the net H⁺ production of the phosphagen system is zero, so it does not directly mediate pH alteration. The effect of the phosphagen system and mitochondria OXPHOS on postmortem pH is widely perceived to be through the regulation of glycolysis. In general, when the muscle contains more CP or shows higher OXPHOS levels, the rate and extent of glycolysis as well as thereby pH decrease could be delayed, elevating the ultimate pH of postmortem muscle [54,88]. In the opposite case, the glycolysis process is exacerbated, thus reducing the postmortem ultimate pH. For instance, a recent study with pork muscle has proposed that increased reactive nitrogen species (RNS) could potentially inhibit CK-mediated phosphagen system and disrupt mitochondria OXPHOS, thus intensifying postmortem glycolysis and pH reduction at the early postmortem [89]. However, this section only provides a broad overview of how the three basic pathways involved in energy metabolism synergistically mediate postmortem pH. To elaborate, changes in postmortem pH are also controlled by multiple known endogenous factors, including glycogen, PFK, AMP, AMP deaminase, glycolytic flux, buffering capacity, glycolytic capacity, and mitochondrial F1 ATPase [45,60,66,[90], [91], [92]]. In this regard, David Gerrard and his collaborators have made significant contributions over the past decade, innovatively establishing a working model for controlling postmortem pH based on the aforementioned factors (Fig. 3). Although the establishment of this model relies in part on in vitro simulation experiments, its contribution to advancing this field is undeniable.

Fig. 3.

Working model of the factors controlling the extent of postmortem metabolism. The ultimate pH of postmortem muscle is dictated by the amount of glycogen present, as long as levels are ≤53 μmol/g. If glycogen exceeds this threshold level, the ultimate pH is determined by the amount of glycolytic metabolites passing phosphofructokinase (PFK) before inactivation. Greater glycolytic enzyme and mitochondria F1 ATPase activities as well as lower AMP-deaminase activity may increase glycolytic flux and decrease ultimate pH. Figure adapted with permission by Matarneh et al. [66]; copyright 2018 Elsevier.

In addition, the rate and extent of pH variation, especially at the early postmortem, has a substantial influence on meat quality attributes, including WHC, tenderness, and color. Any deviations from the normal levels of glycolysis and pH decline can cause impaired meat quality. Typically, PSE meat caused by excessive glycolysis and rapid pH decline is commonly identified in pigs and poultry [23,28], and high prevalence of WB in chicken [21] as well as DFD meat in beef [22] is always associated with insufficient glycolysis and delayed pH reduction.

WHC

WHC, defined as the ability of fresh meat to retain its inherent moisture, is an important quality characteristic of fresh meat. It has been assessed that that about half of all fresh retail cuts of pork or poultry suffered unacceptably high purge loss, and the average weight loss during purge could average as much as 5 % or even nearly 10 % in PSE-like meat [[93], [94], [95]]. Water, a dipole molecule, is attracted to charged muscle proteins at a higher pH. Once the postmortem pH decreases to reach the isoelectric point of major proteins (pH < 5.4), in particular myosin, the net charge of the proteins becomes zero, accompanied with the impairment of repulsion within the myofibrils. These alterations make the protein molecules less capable of attracting water and simultaneously reduce the filament space, forcing the water within myofibrils to flow outward [96]. Also, at higher carcass temperatures, the hastened drop in pH induces the formation of massively denatured rigor cross-bridges, which could further weaken WHC via altering the contraction levels of intracellular volume [97,98]. However, it is noteworthy to mention that a higher pH is not always better, and muscle with a high ultimate pH (>6.0) could exhibit abnormally increased WHC, readily causing darker color and spoilage-prone characteristics of meat.

Tenderness

Tenderness is commonly considered the most crucial textural attribute determining consumer perception and eating quality of meat. Postmortem changes in glycolysis and pH decline are the critical factors affecting meat tenderness [10]. This can be clearly demonstrated, for example, by the abnormal textural characteristics related to PSE pork and typical DFD beef. PSE meat presented a tougher texture for cooked products as a result of over-denaturation of muscle proteins and deteriorated WHC [93]. In comparison, DFD meat exhibited a more tender texture due to minimal protein denaturation and higher water retention [22]. Additionally, numerous experimental data have suggested that the degradation of critical cytoskeleton proteins (e.g., filamin C, plectin, desmin) eliminated myofiber shrinkage and increased myofiber swelling capacity, contributing to the improvement of meat tenderness and WHC [[99], [100], [101], [102], [103], [104], [105], [106]]. This process could, however, be impaired by a rapidly decreased pH, as plainly evidenced in stress-induced quality-defective meats [96]. Indeed, the three proteolytic enzyme systems, including calpain, caspase, and cathepsin, are responsible for the impaired structural integrity of myofibrils as well as the improved meat tenderness and WHC, which can be controlled by postmortem pH. Especially, the calpain system is commonly regarded as the most important contributor and actively responds to pH variations. Its activity, especially calpain-1, under low pH conditions (pH 6) could be notably decreased as compared with neutral conditions (pH 7.5). Meanwhile, a rapid pH drop could also result in μ-calpain inactivation. However, the opposite regulation was noted for caspase, with a lower postmortem pH favoring an optimal environment for caspase activation [107]. Likewise, lysosomal cathepsin was mainly active under acidic conditions with an optimal pH of 5.0 to 6.0, but its contribution to meat tenderization was speculated to be at the later stages of aging owing to the delayed lysosomal cleavage [108].

Interestingly, energy metabolism enzymes and mitochondria can also contribute to proteolysis and meat tenderization independently of their involvement in energy metabolism and thus pH alterations. It has been demonstrated that several energy metabolic enzymes (e.g., pyruvate kinase M type (PKM), phosphoglycerate kinase (PGK), and hexokinase (HK)) can act as protein kinases to induce protein phosphorylation, presenting a novel pathway for regulating proteolysis and meat tenderization. As an example, Ren et al. [109] utilized PKM2 to incubate lamb myofibrillar protein and discovered that PKM2 treatment notably inhibited the degradation of desmin and actin by increasing their phosphorylation levels, potentially impairing meat tenderization. Besides, calcium can be absorbed and released by mitochondria, which affects sarcoplasmic calcium concentration and thereby regulates calpain activation process [110,111]. Recent studies have concluded that calpain activity, proteolysis, and the resulting meat tenderization could be increased or decreased, respectively, in response to either inhibiting mitochondrial calcium uptake or exogenously supplementing intact mitochondria [[112], [113], [114], [115]]. Besides, as postmortem time extends, mitochondrial structure gradually disintegrates, with spontaneous initiation of a series of biochemical processes within it. Among these, oxidative stress and apoptosis are the most typical, and their contributions to postmortem meat tenderization have also been emphasized as reviewed by Scheffler [18], Lee et al. [76], Ouali et al. [116], and Huang et al. [117].

Color

Fresh meat color is a crucial sensory attribute, and consumers commonly believe that a vibrant cherry-red meat color represents freshness and wholesomeness. Myoglobin content and the interconversion of its redox forms, including deoxymyoglobin, oxymyoglobin, and metmyoglobin, underlie the development of meat color. Furthermore, mitochondria aerobic respiration and metmyoglobin reduction (MR) ability are critical deep-seated factors influencing the development of oxymyoglobin and the resulting meat cherry-red color [51,118,119]. Higher mitochondria respiration could limit myoglobin oxygenation through competitively consuming oxygen, leading to an excessive deoxymyoglobin accumulation. This is extensively evident in typical and atypical DFD beef, where the higher ultimate pH favored mitochondria respiration, impaired the oxygen available for myoglobin oxygenation, and consequently led to the development of darker meat color [49,53,[120], [121], [122], [123], [124], [125]]. Conversely, the inhibition of mitochondrial respiration utilizing rotenone, an organic compound capable of blocking ETC complex I, markedly reduced oxygen consumption and deoxymyoglobin formation, thereby furthering the transformation of dark-cutting beef from a dark into a red color [126,127]. In addition, NADH, as the product of glycolysis and TCA cycle, can be coupled with MR. Regeneration of NADH can enhance MR ability, and there has been great interest in using glycolysis and TCA cycle intermediates (e.g., lactate, succinate, pyruvate, and malate) as substrates for NADH regeneration to improve meat color [[128], [129], [130], [131]].

On the other hand, as the weakening of meat WHC in response to rapid pH decline, a large amount of myoglobin can be lost with purge loss, while additional moisture on the meat surface can further increase light reflection, resulting in a pale meat color. In comparison, the darker meat color at a higher ultimate pH is partly attributed to void myofiber shrinkage and increased WHC, which restrained myoglobin loss and elevated light absorption [132]. Meanwhile, the lipid oxidation could be exacerbated at lower pH levels, which could further lead to an undesirable brown meat color via loosening myoglobin structure and inducing excessive oxidation of myoglobin [133,134].

Protein biomarkers discovery from energy metabolic pathways

Currently, research in meat biochemistry has shifted its focus to exploring molecular biomarkers in live animals or sampled muscle tissues. These biomarkers can be identified in the genome, transcriptome, proteome or metabolome and further reveal unique biological characteristics or changes within postmortem muscle, enabling their association with typical heterogenous meat or exogenous intervention treatment [[135], [136], [137], [138]]. In particular, protein biomarkers derived from proteomics have emerged as a novel approach for precisely evaluating and predicting meat quality, thereby ensuring the production of high-quality meat [139,140]. Recently, numerous studies utilizing proteomics have discovered energy metabolism enzymes as potential biomarkers for characterizing fresh meat quality in multiple species. In beef [[141], [142], [143]] and pork [95,144,145], the protein biomarkers, including creatine kinase M−type (CKM), enolase 3 (ENO3), aldolase A (ALDOA), phosphoglucomutase 1 (PGM1), PKM1, malate dehydrogenase (MDH1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH), were critically screened. Phosphoglycerate kinase-1 (PGK1), PKM2, PGM1, ALDOA, GAPDH, and ENO3 were highlighted in lamb and goat muscles [146,147], PKM, PK, CKM, LDHA, and ENO in chicken muscle [148], CKM, GAPDH and ATP synthase in goose muscle [149], GAPDH, PFKM, ALDOA, and PKM in foal and donkey muscles [150,151]. Among these, as shown in Fig. 4, protein biomarkers such as GAPDH, CKM, ENO, and PKM were not only identified in postmortem muscle of several species but also concurrently characterized tenderness, WHC, and color of meat. Therefore, they could be regarded as critical protein biomarkers related to postmortem energy metabolism for controlling meat quality attributes.

Fig. 4.

Potential protein biomarkers associated with postmortem energy metabolism and meat quality traits. Note: the above data is derived from a synthesis of multiple reviews and recent research articles comparing meat proteomics of varying quality characteristics [52,89,95,105,[143], [144], [145],147,180,[299], [300], [301], [302], [303], [304], [305], [306], [307], [308], [309], [310], [311], [312], [313], [314], [315], [316], [317], [318], [319], [320], [321]]. Glyceraldehyde-3-phosphate dehydrogenase, GAPDH; Creatine kinase M−type, CKM; Enolase, ENO; Triosephosphate isomerase, TPI; Pyruvate kinase isozymes M, PKM; Fructose-bisphosphate aldolase, ALDOA; Phosphoglucomutase-1, PGM1; UTP-glucose-1-phosphate uridylyltransferase, UGP2; Phosphoglycerate mutase 2, PGAM2; AMP deaminase 1, AMP D 1; Lactate dehydrogenase, LDH; Fructose-1,6-bisphosphatase, FBP; ATP-dependent 6-phosphofructokinase, PFK; Isocitrate dehydrogenase, IDH; Malate dehydrogenase, MDH; ATP synthase subunit alpha, ATP5F1A; Dehydrogenase/reductase SDR family member 7B, DHRS7B; Glycogenin 1 NADH-cytochrome, CYG1; Water holding capacity, WHC; Pale, soft, and exudative, PSE; Dark, firm, and dry, DFD; Wooden breast, WB.

In addition, the above protein biomarkers can also be involved in various postmortem biochemical pathways and disclose underlying molecular mechanisms during the transformation of muscle into meat. However, it is worth noting that postmortem muscle is a pretty complex system of biochemical reactions, especially the metabolic enzymes within it. Instead of being solely dependent on their abundance, their postmortem biological functions are collectively determined by their abundance as well as their structure, stability, and activity as regulated by multiple endogenous factors. In parallel, such endogenous factors as AMPK, HlF-1α, reactive oxygen species (ROS), RNS, lactate, and others are notably activated or accumulated in response to postmortem ischemia-hypoxia, a process that is coupled with the initiation of energy metabolism process (Fig. 5).

Fig. 5.

Postmortem changes in biochemical, physical, and energy metabolism during the conversion of muscle to meat. During the early postmortem time, phosphocreatine (PCr) and mitochondria oxidative phosphorylation (OXPHOS) help potentially maintain adenosine triphosphate (ATP) levels. As aerobic metabolism and phosphagen system cease to supply energy, glycolysis is initiated and coupled with ATP hydrolysis and H⁺ accumulation, which subsequently results in a decrease in muscle pH. As ATP is gradually depleted, irreversible actomyosin cross bridges form and thus increase muscle tension (rigor mortis onset). Tension is maximal when ATP is exhausted (completion). Afterwords, muscle tension decreases, and meat quality improves (resolution and aging) due to proteolysis mediated by endogenous enzyme system. Importantly, mitochondria are implicated in these changes through their potential roles in mediating energy metabolism, calcium regulation, apoptosis, and oxidative stress. In parallel, at the early postmortem, many endogenous regulatory factors were activated and accumulated in response to hypoxic-ischemic environment. Such as adenosine monophosphate-activated protein kinase (AMPK) as well as hypoxia inducible factor-1α (HIF-1α) signaling, protein posttranslational modifications (PTMs), heat shock proteins (HSPs), and protein DJ-1 could potentially control postmortem energy metabolism through regulation of enzyme activation states.

Endogenous factors and their mechanisms regulating postmortem energy metabolism

AMPK signaling

AMPK, as an energy-sensing kinase, is a heterotrimer complex composed of one catalytic α-subunit and two regulatory β- and γ-subunits, exerting a central role in the maintenance of cellular energy homeostasis. Among the all AMPK-mediated pathways that preserve cellular energy homeostasis in response postmortem ischemia and hypoxia, its regulation of glycolysis is considered as the most critical in meat biology [8]. Utilizing exercised wild-type and AMPK knockout mice, the team of Shen et al. [152] first proposed that AMPK signaling, especially AMPKα2, was involved in controlling glycolysis of postmortem muscle [153]. At that time, these findings provided crucial clues to a half-century-long debate centered on what mechanisms may control the prolonged glycolysis in postmortem muscle, especially in the highly glycolytic muscles of pigs and poultry. A number of subsequent studies, particularly those addressing pre-slaughter stress in pigs and poultry, have reinforced the aforementioned data and further supported that the activation of AMPK signaling was a pivotal contributor to the aggravated glycolysis and thus rapid pH drop in postmortem muscle [8]. Meanwhile, it is generally agreed that the AMPK-mediated increase in glycolysis flux was predominantly achieved by improving glycolytic enzyme activities such as glycogen phosphorylase (GP) and PFK.

AMPK activation is initiated through two upstream AMPK kinases, named liver kinase B1 (LKB1) and CaMKK. Of these, AMPK is activated by LKB1 responding to an improved cellular AMP/ATP ratio. In broilers, Duan et al. [154] demonstrated that the dietary supplementation of creatine nitrate could improve myocyte energy status and reduce AMP/ATP ratio, thereby limiting the activation of LKB1-mediated AMPK signaling, slowing down postmortem glycolysis, and ultimately improving meat quality. However, CaMKK phosphorylates and thereby activates AMPK not in an AMP-dependent manner, but rather in response to elevated levels of intracellular calcium. In C2C12 myotubes, the increased intracellular calcium promoted CaMKK binding to AMPK, increased AMPK phosphorylation levels, and thereby improved AMPK activity, yet the phosphorylation level of AMPK could be markedly reduced when chelating calcium or silencing CaMKK signaling [155]. Postmortem hypoxia–ischemia could induce the production of ROS and RNS in myocytes, with a notable exacerbation in animals exposed to severe antemortem stress [89,96]. The overproduced ROS and RNS could participate further in the regulation of the sarco-endoplasmic reticulum channels, accounting for the dysregulation of postmortem intracellular calcium homeostasis and consequently cascading activation of the CaMKK/AMPK pathway. Recent investigations on postmortem muscles of pigs, yak and broilers have extensively shown that the upregulated calcium/CaMKK/AMPK pathway, due to pre-slaughter transport stress and postmortem oxidative stress, was notably responsible for accelerated glycolysis [23,[156], [157], [158]]. Meanwhile, CaMKK, a family member of the calcium/calmodulin-dependent protein kinase, is composed of the α and β subtypes. The activation of AMPK driven by specific isoforms of CaMKK differed in various types of tissues or cells. Unlike poultry, where both isoforms could be upregulated in response to pre-slaughter stress to activate AMPK signaling, this process in livestock primarily involved the β isoforms of CaMKK [157,158].

Besides, AMPK signaling can activate nicotinamide phosphoribosyltransferase (NAMPT) and enhance the NAD⁺/NADH ratio, activating silent information regulation 1 (SIRT1), known as another energy receptor [159]. This pathway has, thereby, been named the AMPK/SIRT1 pathway. Importantly, the activated SIRT1 can also deacetylate and in turn activate LKB1, which improves the AMPK activity via phosphorylating AMPK at the Thr172 site [160]. In other words, SIRT1 and AMPK can mutually activate each other via developing a positive feedback loop, consequently reinforcing catabolism and maintaining cellular energy homeostasis. In the study of Yang et al. [161], NAD⁺ and NADH were added to yak muscle as activators and inhibitors of SIRT1, respectively. It was observed that NAD⁺ treatment improved AMPK activity and thereby postmortem glycolysis flux, ultimately intensifying the meat tenderization, whereas the opposite result was discovered with NADH treatment. In summary, AMPK serves as an energy control hub, and its activation as well as subsequent positive regulation of glycolysis across species are critical for postmortem pH drop and meat quality development. Specifically, due to differences in postmortem energy metabolism patterns among various species, expectations regarding the modulation of glycolysis by AMPK also vary. For poultry and pigs, it is preferred to delay glycolysis by inhibiting AMPK activity, consequently controlling the occurrence of PSE syndrome in meat. However, an increased AMPK activity in yak and cattle can promote the ultimate pH escape from the intermediate range of 6.1 to 5.8 that is unfavorable for meat tenderization.

HIF-1α signaling

HIF-1, a heterodimeric factor composed of HIF-1α and HIF-1β, is a pivotal modulator of the cellular energy homeostasis in response to hypoxia or ischemia. As mentioned by Chen et al. [162], HIF-1α can bind to hypoxia response element (HRE) within the nucleus, thereby improving the gene expression and enzymic activity of most glycolytic enzymes such as HK, PFK, and PK. Initially, Gao et al. [163] demonstrated a positive relevance between HlF-1α expression and altitude in yak muscle and identified HlF-1α as a major protein biomarker characterizing meat quality in yak at different altitudes. Likewise, in the subsequent examinations of protein biomarkers associated with meat WHC and tenderness, HlF-1α was also typically identified in mutton [164] and beef [165]. These findings indicate a critical involvement of HlF-1α signaling in the development of meat quality.

Recent research on sheep and yak muscles incubated with YC-1 (a HIF-1α inhibitor) have further demonstrated that HIF-1α inhibition could alter the mode of energy replenishment in postmortem myocytes via impairing glycolysis process and reinforcing mitochondria aerobic metabolism [[166], [167], [168]]. Similarly, the dietary supplementation of taurine reduced HIF‐1α expression of pig skeletal muscle, and importantly, this reduction coincided with lower glycolytic enzyme activities [169]. In the same study, further experiments using porcine myotubes demonstrated that DMOG treatment (a HIF-1α activator) enhanced HIF-1α expression and accelerated glycolytic flux. However, co-treatment with taurine effectively counteracted these DMOG-induced effects, confirming that taurine delayed postmortem glycolysis mainly through suppressing HIF‐1α signaling [169]. Evidence has suggested that postmortem ROS and RNS could stimulate HIF-1α expression, which in turn initiated glycolytic metabolism in response to postmortem ischemic and hypoxic environment [25]. Thus, such findings from Chen et al. [169] might be related to the high antioxidant capacity of taurine that induced scavenging of ROS and RNS in postmortem muscle.

Interestingly, ROS is an upstream regulator of the classic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Under hypoxic conditions, ROS-derived oxidative stress could stimulate PI3K/AKT and MAPK/ERK pathways to stabilize HIF-1α signaling [170]. This process is associated with the binding of HIF-1α to HRE, suppression of HIF-1α degradation, and promotion of HIF-1α nuclear translocation. According to Chen et al. [162], increased glycolytic potential and improved meat tenderization in bovine muscle treated with hydrogen peroxide (H₂O₂) could account for oxidative stress-mediated activation of the PI3K/AKT pathway and therefore HIF-1α accumulation. Likewise, studies on pigs also showed that postmortem glycolytic flux could be reduced by downregulation of the PI3K/AKT/HIF-1α pathway with pre-slaughter dietary supplementation of pea starch [171]. In a separate study, Zhu et al. [172] incubated yak muscle employing H₂O₂ and H₂O₂ plus U0126 (a MAPK/ERK pathway inhibitor), respectively. The authors observed that H₂O₂ treatment upregulated the MAPK/ERK pathway, accompanied with upregulated HIF-1α signaling, accelerated glycolysis, and decreased postmortem pH. However, these phenomena were notably attenuated in the treatment containing U0126.

Besides, HIF-1α was also demonstrated to indirectly regulate glycolysis by managing calcium transporters such as ryanodine receptor (RyR) and sarcoplasmic reticulum calcium-ATPase (SERCA). Hyperoxia-induced reduction in HIF-1α accumulation could decrease RyR levels and strengthen SERCA activity, causing a cascade of reduced sarcoplasmic calcium levels and subsequent inhibition of CaMKK/AMPK/glycolysis pathway in yak muscle [158,173]. Altogether, under postmortem hypoxic conditions, HIF-1α signaling could be activated in oxidative stress-dependent pathways, including PI3K/AKT and MAPK/ERK, thereby accelerating glycolytic flux. Despite the existence of well-defined research, these mainly concern yaks and sheep grazing under extremely harsh conditions and low-oxygen environments. It remains largely undefined whether HIF-1α signaling plays an equally significant role in postmortem metabolism of animals raised on large-scale farms such as pigs and poultry, and whether it can serve as an AMPK-like energy hub to control the development of PSE meat.

AMPK and HIF-1α signaling crosstalk

Considering the above discussion together, it is apparent that there are many similarities in postmortem initiation mechanisms and action targets of HIF-1α and AMPK signaling, implying the possibility of complex crosstalk between them. Granted, a recent study on yak muscle confirmed the existence of such crosstalk [174]. Based on inhibitor and activator injection experiments similar to those described above, the researchers revealed that AMPK could phosphorylate seven sites on HIF-1α and promote HIF-1α nuclear translocation. In parallel, this effect was found to inversely amplify AMPK activity. Thus, a mutually reinforcing regulatory loop was formed between the two signaling, which collectively exacerbated glycolysis and induced rapid pH decline of postmortem yak muscle [174]. However, no studies have yet revealed the potential mechanism of AMPK reversible activation by HIF-1α. From one of the perspectives presented by the author, it is proposed that HIF-1α activation could promote glycolysis and inhibit mitochondrial OXPHOS, leading to reduced ATP production. This, in turn, elevated the AMP/ATP ratio, thereby indirectly improving AMPK activation. Together, all these strongly support the function of AMPK and HIF-1α signaling in controlling postmortem energy metabolism (Fig. 6) and shed much insight into the origin of extended glycolysis in postmortem muscle. Also, these may provide critical targets for related prevention strategies to control the development of quality-defective meats.

Fig. 6.

Adenosine monophosphate-activated protein kinase (AMPK) and hypoxia inducible factor-1α (HIF-1α) signaling contribute significantly to the activation of glycolysis in postmortem muscle. AMPK activation and the resulting glycolysis improvement are initiated through two upstream AMPK kinases, named liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase (CaMKK). Of these, AMPK is activated by LKB1 responding to an improved cellular adenosine triphosphate/adenosine monophosphate (AMP/ATP) ratio. However, CaMKK phosphorylates and thereby activates AMPK not in an AMP-dependent manner, but rather in response to elevated levels of intracellular calcium. HIF-1α signaling could be activated in oxidative stress-dependent pathways, including phosphoinositide 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase (PI3K/AKT and MAPK/ERK), thereby accelerating glycolytic flux.

PTMs

PTMs refer to covalent processing events that alter spatial conformation, molecular interaction, and subcellular localization of protein mainly through adding chemically modified groups to the amino acid residue, conferring more complex biological functions to the protein. As an emerging research field in meat science, several PTMs, encompassing phosphorylation, S-nitrosylation, acetylation, lactylation, and carbonylation, have been documented to regulate the development of meat quality by participating in multiple biochemical pathways in postmortem muscle, as illustrated in Fig. 7. Among these PTMs, protein phosphorylation is the most extensively studied, with the most consistent and functionally validated role in meat quality development. The results from a comprehensive review by Li et al. [175] have shown that protein phosphorylation negatively mediated the development of meat tenderness, color, and WHC during postmortem aging. However, compared to phosphorylation, other PTMs have received less attention in early-stage research, and their regulation of postmortem biochemical processes and meat quality across species has not yet been fully established. Despite the above, it is worth noting that postmortem energy metabolism pathways have been identified as highly responsive to all the aforementioned PTMs, as numerous glycolytic enzymes and mitochondrial matrix proteins are extremely susceptible to these modifications. Moreover, certain PTMs and their precursor substrates (e.g., RNS, ROS, and lactate) exhibited notably higher levels in stress-induced defective meats, especially extreme PSE meat, as evidenced by our series of studies [20,22,28,89,96,176]. Therefore, given these considerations, it is believed that the resolution of S-nitrosylation, carbonylation, and lactylation holds greater practical significance for understanding and improving meat quality, and these processes warrant priority research in the future. In the following part, we would present a comprehensive summary of the role of PTMs in mediating postmortem energy metabolism, with a particular emphasis on protein S-nitrosylation. Moreover, the order in which PTMs are discussed below is based on our assessment of their importance in this field.

Fig. 7.

Protein post-translational modifications (PTMs) and their crosstalk on the regulation of postmortem biochemical processes. PTMs refer to covalent processing events that alter spatial conformation, molecular interaction, and subcellular localization of protein mainly through adding chemically modified groups to the amino acid residue, conferring more complex biological functions to the protein. Several PTMs, encompassing S-nitrosylation, lactylation, carbonylation, phosphorylation, and acetylation, have been documented to regulate the development of meat quality by participating in multiple postmortem biochemical pathways (e.g., energy metabolism, proteolysis, calcium homeostasis, apoptosis, actomyosin dissociation, and myoglobin stability). Meanwhile, the combinatorial action of multiple PTMs on the same or distinct proteins to achieve higher-order regulation is called PTMs crosstalk.

Protein S-nitrosylation

Nitric oxide (NO) is generated in skeletal myocytes through the catalytic action of NO synthase (NOS) and has been shown to be a critical modulator of muscle metabolism and contraction. At the earliest, Warner et al. [177] carried out a series of research on exploring the function of NO in regulating meat tenderization. They demonstrated that antemortem NO release from skeletal myocytes could affect meat tenderness primarily by modulating glycolysis and proteolysis. The above interesting findings shed initial light on the role of NO in regulating meat quality formation and were subsequently progressed by our team at the level of NO-induced S-nitrosylation [34,111,[178], [179], [180], [181], [182]]. S-nitrosylation refers to the covalent binding of NO to the cysteine group to produce S-nitrosothiol, which is the classical signaling pathway whereby NO performs its postmortem biological functions to regulate energy metabolism, proteolysis, apoptosis, and calcium release.

Particularly, relevant studies on regulating postmortem energy metabolism by NO and its-induced protein S-nitrosylation have been conducted in pigs, cattle, yaks, sheep, and broilers, as exhibited in Table 1. Liu et al. [183] first identified 339 proteins modified by S-nitrosylation in postmortem pork and discovered that they were primarily implicated in the energy metabolism pathway, especially glycolysis. This is supported by Liu et al. [184] and Zhu et al. [22] when studying differential S-nitrosylated proteins in pork and beef with different ultimate pH, respectively. Besides, they also identified the upregulated S-nitrosylation levels of glycolysis enzymes, including ALDO, TPI, PGK, and LDH, in postmortem muscle with high ultimate pH, implying a negative correlation between S-nitrosylation and glycolytic flux. Indeed, the postmortem biological significance of several glycolytic enzymes modified by S-nitrosylation has been shown to be remarkably altered. For example, utilizing an in vitro incubation experiment with S-nitrosoglutathione (GSNO, an NO donor) and a comparative analysis between PSE and normal meats, our lab reported that higher S-nitrosylation extents of GP, GAPDH, PFK, and PK were partly responsible for their lower catalytic activity and thus impaired glycolysis rate in postmortem pork [20,185].

Table 1.

Summary of relevant studies involving the regulation of postmortem energy metabolism by NO and its-induced S-nitrosylation based on timeline.

Species	Sample	Management of NO and protein S-nitrosylation levels		Critical statements	References
		NO enhancer	NOS inhibitor
Ovine	LTL and SM muscles	Pre-slaughter exercise	Pre-slaughter injection with 30 mg/kg L-NAME	Pre-slaughter suppression of NOS accelerated glycolysis within 24 h postmortem	[292]
Broiler	Pectoralis muscle	Postmortem incubation with 0.1 mM GSNO	Postmortem incubation with 0.06 M L-NAME	The pH at 24 postmortem was not affected by postmortem alteration of NO levels	[293]
Lamb	LTL and SM muscles	Pre-slaughter injection with 500 mg/kg L-Arginine	Pre-slaughter injection with 30 mg/kg L-NAME	LTL muscle lactate levels were not affected by NO levels, but initial lactate levels were lower in SM muscle treated with L-NAME	[294]
Pig	SM, PM, and LT muscles	Muscle fiber types		Glycolytic fiber proportion was positively related to nNOS content	[78]
Pig and cattle	LT muscle	Postmortem aging or incubation with GSNO		S-nitrosylation in postmortem muscle was identified as being primarily involved in energy metabolism, especially glycolysis	[180,183,295]
Pig	LT muscle	PSE and normal pork		Increased RyR1 and SERCA1 S-nitrosylation levels induced the imbalance of calcium in the cytoplasm, promoting pH decline and PSE pork formation	[176]
Pig	LT muscle	Incubation with 200 μM GSNO	Incubation with 0.1 M L-NAME	Protein S-nitrosylation inhibited glycogen metabolism via impairing GP, GAPDH, and PK activities	[185]
Pig	LT muscle	PSE and normal pork		Lower S-nitrosylation of GP, PFK, and PK corresponded with higher enzyme activities in PSE pork	[20]
Pig and cattle	LT muscle	PSE pork or DFD beef		Protein S-nitrosylation levels were higher in quality-defective meats, and more different proteins were energy metabolism enzymes	[22,184]
Cattle	SM muscle	Incubation with 200 μM GSNO	Incubation with 0.1 M L-NAME	S-nitrosylation could retard the decrease of MMP maintaining mitochondria function	[296]
Pig	LT muscle	NO gas treatment	—	Muscle pH decreased as NO gas concentration increased from 20 to 80 μL/L at 1 d of storage	[297]
Pig	LT muscle homogenate	Incubation with 0.4 and 1 mM NOR-3	Incubation with 0.1 M L-NAME	Protein S-nitrosylation elevated postmortem glycolysis flux	[26,186]
Pig	LT muscle	Pre-slaughter transport stress	—	Increased S-nitrosylation levels caused by transport stress might trigger premature activation of glycolysis via impairing OXPHOS and phosphagen system	[89,94]
Yak	LTL muscle	Postmortem injection with 200 μM GSNO	—	NO-mediated activation of HIF-1α S-nitrosylation accelerated glycolysis and pH drop	[25]
Yak	LT muscle	Incubation with 200 μM GSNO	Incubation with 0.1 M L-NAME	S-nitrosylation exacerbated mitochondria dysfunction of yak muscle via impairing antioxidant systems and improving sarcoplasmic calcium levels	[228]
Bull or pigs	GQ or LT muscle	Pre-slaughter transport stress	Injection with 20 mM L-NAME	NO and its induced S-nitrosylation activated CaMKK/AMPK pathway and thus accelerated glycolysis	[158,298]

Notes: LT, longissimus thoracis; LTL, longissimus thoracis et lumborum; SM, semimembranosus; GQ, gluteal quadriceps; GSNO, S-nitrosoglutathione; L-NAME, Nω-nitro-L-arginine methyl ester hydrochloride; MMP, mitochondria membrane potential. OXPHOS, oxidative phosphorylation. nNOS, neuronal nitric oxide synthase. PSE, Pale, soft, and exudative; DFD, Dark, firm, and dry; RyR, Ryanodine receptor; SERCA, sarcoplasmic reticulum calcium-ATPase; NO, Nitric oxide; CaMKK, Calcium/calmodulin-dependent protein kinase kinase; AMPK, Adenosine monophosphate-activated protein kinase; HIF-1α, Hypoxia inducible factor-1α; GP, Glycogen phosphorylase; PK, Pyruvate kinase; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Furthermore, it was recently revealed that antemortem stress elevated protein S-nitrosylation levels in postmortem muscle by increasing NO production, regulating pork quality, especially pH and WHC [94]. Along these lines, the authors further discovered that the S-nitrosylation extents of CK and several mitochondria matrix proteins were notably upregulated in response to antemortem stress [89]. Although the role of S-nitrosylation on these proteins has not been studied in the meat field, it is clear in biomedical and exercise physiology that their activity could be markedly suppressed by NO and its mediated S-nitrosylation. Therefore, the aggravated glycolysis and thus pH decline caused by pre-slaughter stress might be associated with the inhibition of ATP regeneration pathways by S-nitrosylation modification, including phosphagen system and mitochondria respiration metabolism.

Beyond the direct regulatory role, reports have suggested that S-nitrosylation could indirectly affect postmortem energy metabolism by modifying HIF-1α signaling and calcium transporters such as RyR1 and SERCA1. The activation of RyR1 and the inhibition of SERCA1 triggered by their increased S-nitrosylation levels could result in the elevated sarcoplasmic calcium, which in turn accelerated glycolysis of postmortem pork via upregulating CaMKK/AMPK pathway [158,176]. Using postmortem yak muscle injected with GSNO, Sun et al. [25] concluded that NO could activate HIF-1α signaling via S-nitrosylation pathway and thus promote glycolytic flux and pH drop by improving glycolytic enzymes activity.

However, it is important to mention that some inconsistent results do appear among the various investigations applying NO donors to manage postmortem protein S-nitrosylation levels. For instance, after adding the same NO donor (GSNO) to postmortem muscle, the delayed glycolysis was observed in pork muscle [185], but an opposite phenomenon was noted for yak muscle [25]. Besides, building on an in vitro glycolysis model with NOR-3 (a NO donor) treatment, Lu et al. [26] demonstrated that NOR-3 improved the activity of GP and LDH via intensifying their S-nitrosylation as well as therefore increased energy metabolism of early postmortem pork [186], in contrast to the investigation mentioned above using GSNO as the NO donor [185]. Altogether, the association between protein S-nitrosylation and postmortem energy metabolism has been preliminarily established (Fig. 8). Nevertheless, the management of NO and S-nitrosylation levels by various NO donors in different animal species and muscle types does exert inconsistent effects on the modulation of postmortem energy metabolism and thus meat quality development owing to diverse aging characteristics.

Fig. 8.

Schematic illustration of the role of S-nitrosylation and lactylation in the regulation of postmortem energy metabolism. S-nitrosylation regulates postmortem energy metabolism not only by directly acting on its three basic pathways, but also indirectly through mediating calcium homeostasis and muscle contraction. The E1A binding protein p300 (p300) serves as primary lactyltransferase for the onset of postmortem lactate-derived lactylation. Also, energy metabolism and muscle contraction were the two most critical biochemical pathways mediated by lactylation modification during muscle to meat conversion.

Protein lactylation

Lactate-derived lactylation, a newly found PTMs in 2019, occurs on the lysine residue of protein [187] and exists in three isomers, including L-lactylation, D-lactylation, and N-ε-(carboxyethyl)-lactylation. Among them, L-lactylation catalyzed by lactyltransferase is the main isomer that critically responds to glycolysis [188], and so the ensuing discussion here is concerned with L-lactylation. Lactylation was first identified on histones, and follow-up investigations have incrementally evidenced its prevalent presence on non-histone proteins, particularly metabolic enzymes involved in TCA cycle and glycolysis [[189], [190], [191]]. For instance, in the biomedical field, lactate has been indicated to increase PK and ALDOA activities via upregulating the lactylation levels of PKM2 and ALDOA, respectively [192,193]. Lactylation of mitochondrial proteins could impair their activity, thus limiting the mitochondria OXPHOS process and reducing ATP generation [194,195].

Inspired by the above emerging findings, the lactylation of postmortem muscle may be worth considering, as lactate levels in some quality-deficient meats are notably different from normal meat [8,89]. Recently, Wang et al. [196] observed a significant reduction in postmortem muscle pH in broilers following pre-slaughter intraperitoneal injection of sodium lactate (a lactylation donor) and attributed this to the upregulation of the expression and activity of glycolytic enzymes such as PFK, PKM and LDH. In another study exploring the relevance of protein lactylation to postmortem glycolysis, they further illustrated that lactylation levels were positively related to the activity of LDH and PFK, indicating that lactylation might develop a positive feedback pool on glycolysis and pH decline via modifying glycolytic enzymes [197,198]. In the same line, our lab for the first time identified protein lactylation in livestock meat science, disclosing that the degree of protein lactylation corresponding to lactate levels in PSE pork was higher relative to that of normal pork [28]. In addition, postmortem energy metabolism and rigor mortis contraction were highly responsive pathways for lactylation modification [199,200], and E1A binding protein p300 was the dominant lactyltransferase of protein lactylation in postmortem muscle (Fig. 8) [199]. Next, it is of interest to identify lactylated proteins and sites, especially those involving energy metabolism, in muscle samples along with the postmortem aging time and varying lactylation extents among muscles and species. The discovery of postmortem protein lactylation can contribute to a novel lactate-based postmortem biochemistry theory, and therefore, it could be a fascinating topic for future research.

Protein carbonylation

Protein carbonylation is an irreversible and non-enzymatic PTMs involving the formation of carbonyl compounds (e.g., aldehydes or ketones), which is a major biomarker of ROS-driven oxidative damage in postmortem muscle [11,15,201]. A strong relationship among increased protein carbonylation, abnormal skeletal muscle performance, and impaired meat quality has been demonstrated in many animals suffering from pre-slaughter stress [93,96,202,203]. Meanwhile, several glycolytic enzymes, encompassing ENO, TPI, ALDO, PGM, LDH, and GAPDH, were identified to be carbonylated in postmortem skeletal muscle [30,204]. Carbonylation levels of these enzymes were upregulated in postmortem muscle from broilers exposed to pre-slaughter heat stress, accounting for the elevated glycolytic enzyme activity and thus exacerbated postmortem glycolysis [205]. In a study on yak muscles incubated by H₂O₂, Zhu et al. [157] stated that increased oxidative stress levels could enhance glycolytic enzyme activity and thus glycolysis rate in postmortem muscle. As for the reasons, beyond the activation of the CaMKK/AMPK pathway claimed by the authors, the direct regulatory role of oxidative stress-driven carbonylation on glycolytic enzymes might be another critical point. The above findings highlight the importance of protein carbonylation with respect to the augmentation of glycolytic flux in postmortem muscle.

Moreover, the regulatory effect of carbonylation on intracellular calcium homeostasis is an indirect pathway for its impact on postmortem glycolysis. Carbonylation of multiple calcium-handling proteins (e.g., SERCA1, RyR1, and ankyrin) could impair their ability to manage intracellular calcium and further cause sarcoplasmic calcium overload, exacerbating postmortem glycolysis by disrupting mitochondria integrity as well as activating CaMKK/AMPK signaling [202,206]. Although protein carbonylation is not unfamiliar in the meat field, previous studies have concentrated on its effects on the functional properties and nutritional value of proteins [201,207,208]. Investigations on its postmortem biochemical regulation, especially on energy metabolism, still need to be further established.

Protein phosphorylation

Phosphorylation involves the transfer of phosphate groups mainly derived from ATP to protein amino acid residues (e.g., serine and tyrosine) in the presence of protein kinase, which is the most reported PTMs in the meat field up to now. It has been previously shown that muscle protein phosphorylation could be used as a dynamic metabolic feature to reflect the activity status of energy metabolism enzymes during muscle-to-meat conversion [209]. Using ovine muscle treated by phosphatase and kinase inhibitors, Chen et al. [210] further analyzed in detail the regulatory role of phosphorylation on glycolysis in terms of enzyme activity. The results showed that glycolytic enzymes such as GP, PK, and PFK activities were notably strengthened in the phosphatase inhibitor group, suggesting that phosphorylation possessed positive regulation on postmortem glycolysis. Previous work on the regulation of glycolysis and other biochemical processes by phosphorylation has been well-summarized by Li et al. [175], and only a representative few are presented here.

Recently, research in this area has focused on delving into the role of phosphorylation of specific sites of energy metabolism enzymes on their structure and function. As Bai et al. [211] argued, the improved catalytic activity of GP induced by phosphorylation was mainly attributed to its increased ordered secondary structure after phosphorylation, as evidenced by increased α-helix and β-sheet contents. In addition, applying computerized molecular simulation and targeted mutagenesis, it was also demonstrated that postmortem biological function of PKM was determined more by the acetylation of its lysine 137 site than by the phosphorylation of its serine 99 site [212]. As for the catalytic activity of PFK, however, it depended mainly on the phosphorylation of its threonine 704 site instead of the acetylation of its lysine 678 site [213].

Besides, mitochondrial proteins are also important targets for phosphorylation. In recent research performed by Li et al. [214], a total of 191 mitochondrial proteins in beef muscle were identified as being phosphorylated, with 47 proteins being differentially enriched in meat with varied color stability. By correlation and bioinformatics analyses, the authors further stated that the mitochondrial protein phosphorylation could positively affect beef color by modulating the OXPHOS pathway. However, the detailed regulation of phosphorylation on mitochondria OXPHOS and thereby energy metabolism in postmortem muscle is still unclear and remains to be further explored.

Protein acetylation

Protein acetylation is the addition of an acetyl group to the ε-NH₂ of a protein lysine residue catalyzed by acetyltransferases, while this process can be reversed by the action of deacetylases, i.e. protein deacetylation. Investigations conducted by Li et al. [215] have shown that antemortem stress could influence glycolysis of postmortem muscle by regulating acetylation/deacetylation of glycolytic enzymes. In their study, inhibition of mouse protein acetylation by pre-slaughter injection of acetyltransferase inhibitors eliminated the swimming stress-induced augmentation of postmortem muscle glycolysis. Later, the authors further identified 595 acetylation sites mapping to 163 proteins in pork muscle and discovered that energy metabolism enzymes were the largest group of acetylated proteins [216]. This was validated by three additional acetylated proteomic analyses of postmortem muscle from pigs exposed to pre-slaughter stress, including acute transport [217] and violent driving [218], and from beef with varying color stability [219].

Furthermore, the effect of protein acetylation on the meat quality of lamb at different postmortem stages was investigated by Zhang et al. [220]. They demonstrated that regulation of meat quality by protein acetylation was primarily manifested at the early postmortem (1–12 h), and particularly, the acetylation of sarcoplasmic proteins could negatively affect pH and WHC by regulating glycolysis. This is supported by Yan et al. [29], who evidenced that inhibiting acetylation by pre-slaughter injection of curcumin alleviated glycolysis at the early postmortem, improving pH in specific muscles (e.g., lumbar longissimus and trapezius muscles), and ascribed these mainly to the inhibition of CK acetylation. Meanwhile, the authors emphasized that muscles with more oxidative muscle fibers, but fewer glycolytic muscle fibers, were more likely to achieve the above process. On the basis of molecular biology methods such as site-specific mutation, further research has indicated that the lysine 141 site in PKM and the lysine 681 site in PFKM were essential targets for acetylation, which largely controlled their catalytic activity and postmortem glycolysis [219,221].

Altogether, the current study, mainly on pre-slaughter stressed animals, evidenced the potential implication of protein acetylation on postmortem energy metabolism. Despite these findings, however, there has been a gap in research on protein acetylation in other quality-defective meats related to abnormal energy metabolism such as DFD beef and WB chicken. In addition, detailed information on the effect of protein acetylation on meat quality attributes is sorely lacking.

PTMs crosstalk

The regulation of individual PTMs on postmortem energy metabolism has been discussed in detail above. However, PTMs do not occur in isolation, and several PTMs can concurrently modify a single protein at multiple sites. The combinatorial action of multiple PTMs on the same or distinct proteins to achieve higher-order regulation is called PTMs crosstalk [222]. As stated by Li et al. [175], there were at least two types of PTMs acting in the same biochemical pathway to regulate meat quality development. Granted, phosphorylation, acetylation, and S-nitrosylation of muscle proteins involved in glycolysis, TCA cycle, and calcium-handling pathway were recently identified to cooperatively regulate meat quality from pre-rigor to post-rigor [223]. In particular, the crosstalk between phosphorylation and acetylation has been more frequently mentioned [218]. Based on an in vitro model of acetylation, Ren et al. [224] stated that acetylation treatment could increase the phosphorylation level of lamb muscle proteins, which subsequently exerted a significant improvement on glycolysis. A study using lamb muscles with different glycolysis rates showed that the phosphorylation and acetylation of HK were positively coupled with its activity, whereas the phosphorylation and acetylation of PFK negatively regulated its activity [225].

Besides, AMPK is of importance in controlling postmortem energy metabolism and meat quality development. The activation of AMPK in a phosphorylated manner could subsequently increase acetylation levels of total proteins and thereby intensify glycolysis flux of postmortem muscle [226]. A similar statement was presented by Liu et al. [227], who examined the effect of guanidinoacetic acid administration of pre-slaughter on postmortem muscle glycolysis in mice. Additionally, the positive crosstalk of S-nitrosylation and carbonylation could exacerbate mitochondria dysfunction of yak muscle via impairing antioxidant systems and improving sarcoplasmic calcium levels, which potentially interferes with postmortem energy metabolism [228]. It is also interesting that both lactylation and acetylation occur on lysine residues with a 36.7 % overlap of their modification sites, implying a greater likelihood of crosstalk between them in postmortem muscle [229]. However, the modulation of PTMs crosstalk on postmortem energy metabolism and meat quality formation is still poorly understood, and more studies, especially on PTMs occurring within one protein, should be performed in the future.

Other endogenous factors

HSPs and protein DJ-1 are other potential endogenous factors regulating postmortem energy metabolism, except for those stated above. HSPs are a group of widespread and highly conserved proteins that are quickly synthesized in response to various stressors. It can be classified into five major families, including small HSPs, HSP60, HSP70, HSP90, and HSP110. Among them, HSP27, HSP70, and HSP90 have been identified in postmortem muscles of several animal species and are considered protein biomarkers for predicting meat quality [141,230]. Additionally, the abundance of HSP20, HSP70, and HSP90 in PSE-like meat was lower as compared with normal meats and was positively correlated with pH at the early postmortem [231]. Induction of HSP70 overexpression by pre-slaughter injection of glutamine inactivated AMPK signaling and thus impaired glycolytic flux, ultimately mitigating the pH decline of postmortem muscle from broilers subjected to pre-slaughter stress [27]. On the other hand, HSP70 could interact with SERCA, which further contributed to a protective effect on the functional activity of SERCA and therefore maintained intracellular calcium homeostasis, potentially delaying postmortem glycolysis [8].

Protein DJ-1, a proteolytic peptidase belonging to the C56 family, exists as a homodimer composed of two 20 kDa subunits. As presented by Liu et al. [93], protein DJ-1 could be detected in various muscle types from pigs, cattle, and sheep and was widely recognized as a biomarker for monitoring meat quality, especially for PSE meat. Based on data from several proteomics studies, Gagaoua et al. [232] constructed a relevance network between protein biomarkers and further showed that protein DJ-1 was strongly correlated to several energy metabolism enzymes such as ENO3, LDHB, and malate dehydrogenase (MDH). Similar findings were recently stated by Liu et al. [31], who identified a direct interaction of protein DJ-1 with energy metabolism enzymes (e.g., ALDOC, PGAM2, GADPH and MDH2). A higher extent of protein DJ-1 potentially contributed to an incremental pH of postmortem pork via modulating the glycolysis process and oxidative stress. Meanwhile, the authors discovered that the protein DJ-1 interacted notably with ATP synthase F1 subunit, which might favorably maintain ATP synthase activity and thereby mitochondria function, improving ATP regeneration efficiency [233].

In summary, HSPs and protein DJ-1, as molecular chaperone proteins, hold the potential to modulate postmortem energy metabolism. However, the current knowledge about them is mainly focused on identification using proteomics, and more direct evidence needs to be further explored regarding their postmortem protective functions on certain energy metabolism-regulating proteins.

Exogenous strategies managing postmortem energy metabolism

Management of postmortem energy metabolism is primarily categorized into two strategies: pre-slaughter (e.g., dietary feeding and stress management) and postmortem (e.g., electrical stimulation, very fast chilling, superchilling, and ultrasound technology) (Fig. 9). Among them, dietary feeding and stress management of pre-slaughter are well-known and widely investigated in the meat field. For instance, recent work has suggested that feed supplementation with guanidinoacetic acid [227,234], creatine [88,235], taurine [169,236], and chlorogenic acid [237] could slow postmortem muscle glycolysis by maintaining energy homeostasis in pigs and poultry, thereby improving ultimate pH and meat quality. Dietary supplementation with Codonopsis pilosula agro-waste improved yak meat quality via increasing oxidative stability and balancing energy metabolism efficiency during postmortem aging [238]. Optimization of pre-slaughter management conditions such as ambient temperature, transport, and resting, repelling, and stunning to manage postmortem energy metabolism and improve meat quality was also largely established [8,96,218,[239], [240], [241]]. Consequently, some of the pre-slaughter management strategies would not be specifically described in this section.

Fig. 9.

Exogenous strategies managing postmortem energy metabolism.

Electrical stimulation

Postmortem electrical stimulation (ES), including constant voltage ES and constant current ES, is defined as the process of passing an electric current through the carcasses of freshly slaughtered animals via the application of contact electrodes [242,243]. However, the constant current ES system does not seem to be yet commercially applied. By comparison, in many countries, constant voltage ES of over 110 V is more commonly applied in the livestock industry, typically for cattle and lamb. Especially, a new generation medium voltage (300 V peak) ES has been commercially applied in Australia [244,245]. This postmortem handling technology can accelerate postmortem muscle glycolysis and promote pH fall below 6.0 within 3 to 4 h postmortem, ultimately preventing cold shortening and reducing time to rigor mortis process [142,246]. For example, a recent study claimed that muscle pH rapidly dropped by about 0.6 units in lamb carcass treated with ES (15 Hz, 700 mA, 45 s), whereas this process lasted more than 4 h in unstimulated carcass [247]. Similarly, another investigation on beef demonstrated that muscle pH in carcass subjected to medium voltage ES (15 Hz, 300 V, 30–64 s) was nearly 1 unit lower at 2 h postmortem than that in unstimulated ones [248]. Gadiyaram et al. [249] also observed that castrated goats treated with a high voltage ES (580 V, 120 s, 11 × 5 s intervals) exhibited a lower muscle glycogen content and postmortem pH. In addition, ES can also exert a positive effect on meat color, especially redness, which can be primarily attributed to increased light scattering and impaired mitochondrial aerobic metabolism [250]. The improvement of meat quality, especially meat tenderness and color, by ES is not new in the meat field, and additional research in this area has been well-reviewed by recent publications [142,251].

However, apart from the positive effects of ES mentioned above, there remain some potential concerns regarding the impact of ES on meat quality. First, it is noteworthy that sufficient muscle glycogen must be available before the slaughter of animals to maximize the efficacy of the ES [249]. In ruminants subjected to severe stress prior to slaughter, ES application may not have a significant effect on postmortem pH and other meat quality attributes due to glycogen depletion. Besides, the effect of ES on meat quality is highly relevant to its power intensity as well as the carcass size and anatomical location [252]. Some studies have indeed found that inappropriate ES parameters did not yield any significant beneficial outcomes. For instance, a limited efficacy of high voltage ES (e.g., 470 V, 60 Hz, 1.5 A for 1 min) in heavier carcasses to improve beef tenderization and color was reported by Juárez et al. [253] and Djimsa et al. [254]. Similarly, relatively low ES intensity (100 V, 60 Hz for 30 s) also failed to promote postmortem proteolysis and tenderness improvement in beef [255]. On the other hand, it is worth emphasizing that the primary commercial risks to meat quality associated with improper application of ES system are excessive stimulation, which can notably increase the incidence of extreme PSE-like symptoms [256,257]. Under conditions of rapid drop of postmortem pH and high temperature established immediately after ES, protein denaturation could be intensified, leading to a reduced WHC. This has been mentioned in several reports [258,259]. Overall, although ES has been widely commercialized, it requires continuous development to provide greater flexibility and more predictable outcomes for creating appropriate parameters across animal species and carcass sizes.

Advanced chilling

Very fast chilling (VFC) involves the rapid reduction of the center temperature of the carcass or meat of pre-rigor to near freezing point within 5 h postmortem. As compared with conventional chilling, VFC possesses the potential advantages of minimizing chilling time, decreasing carcass weight loss, and extending meat shelf-life [260]. Specially, superchilling (SC) is an update of VFC technology, a process that rapidly reduces the temperature of meat to 1–2 °C below its initial freezing point [261], which has been reported to bring a more positive effect on meat tenderness [262]. However, whether VFC or SC contributes to the significant inhibition of energy metabolism, preventing a rapid decrease in muscle pH at the early postmortem period. A study using quantitative proteomic analysis indicates that VFC (14.43 °C/h) could reduce the glycolysis rate of postmortem pork via downregulating the abundance of glycolytic enzymes such as ALDOA and PKM [263]. Likewise, another study on beef muscle demonstrated that the activities of metabolism enzymes related to glycolysis and TCA cycle, including PK, LDH, MDH, succinate dehydrogenase, could be significantly impaired after SC treatment (center temperature up to −3 °C), thereby inhibiting postmortem energy metabolism [264]. In terms of the mechanism of applying VFC (−35 °C blast chilled for 2 h 31 min to core muscle temperature of −1 °C) in improving sheep meat quality, Bai et al. [265] concluded that VFC slowed down glycolysis mainly by changing phosphorylation and acetylation levels of glycolytic enzymes, which further promoted proteolysis and improved meat tenderness [[266], [267], [268]].

On the other hand, the hydrolysis of ADP and AMP in postmortem muscle could be notably augmented after SC or VFC treatment, which subsequently led to the accumulation of phosphates, NH₃, and inosine monophosphate (IMP) [269,270]. As a result, the phosphates and NH₃ transiently improved the muscle pH via consuming H⁺ at the early postmortem, as observed by Jacob et al. [271]. Also, the increased IMP could bind to the myosin head allowing dissociation of myosin from actin [270]. Besides, recent work conducted by Xiao et al. [272] has demonstrated that VFC treatment (13.33 °C/h) could trigger sarcoplasmic calcium overload of postmortem pork through the modulation of calcium-handling proteins (e.g., RyR1 and dihydropyridine receptor), inducing sarcomere super-contraction and resulting in myofibril fragmentation. Consequently, such intensified actomyosin dissociation and myofibril fragmentation at the early postmortem may delay ATP depletion and thus weaken stimulation of the glycolysis pathway, improving postmortem muscle pH and meat quality.

Despite considerable efforts regarding VFC and SC in improving meat quality [260,262], research findings remain inconsistent and their specific effect mechanisms are unclear, which has, to some extent, limited the industrial application of these technologies. Concretely speaking, in addition to the positive effects discussed in the preceding two sections, the application of VFC and SC can potentially lead to cold shortening of postmortem muscle that is a classic problem in meat science. It is defined as a phenomenon that exposure of pre-rigor muscle to temperatures near the freezing point triggers excessive muscle contraction [273,274]. As shown by Van Moeseke et al. [275], after the VFC treatment, significant cold shortening occurred, with the sarcomere length of beef muscle reduced by over 30 % compared to the treatment of conventional cold storage. Similar observations were also presented in other literature [276,277]. These variations in different reports may be related to the cooling object (e.g., carcass, half-carcass, or anatomical cuts), cooling method, cooling rate, and cooling endpoint temperature. Among these, the cooling endpoint temperature appears particularly significant, and most of the reported studies on enhancing tenderness used cooling endpoint temperatures below 0°C. However, its underlying mechanisms have not yet been fully elucidated.

Ultrasound

Ultrasound, an effective and promising non-thermal technology, involves the use of sound waves beyond the audible frequency range of 20 kHz. In recent years, the exploitation of the mechanical and cavitation effects generated by ultrasound to speed up processing (e.g., tenderization and curing) or to improve product quality characteristics (e.g., flavor, emulsification, and digestibility) has been increasingly explored in the field of meat [[278], [279], [280], [281], [282], [283]]. Also of particular interest is that the appropriate ultrasound could improve enzyme activity by modifying the structure of enzyme and substrate or by altering the interaction between enzyme and substrate or by changing the conditions where the enzyme operates [[283], [284], [285]]. Accordingly, ultrasonic technology can potentially improve postmortem energy metabolism, a multi-enzyme driven process, at the early postmortem and thus meat quality.

After applying ultrasound (40 kHz, 200 W for 40 min, and 4 °C) to beef muscle within 1 h postmortem, Gao and co-workers discovered that relative to non-ultrasound group, lactate levels in ultrasound group were increased by nearly 0.5 units during postmortem aging [286], along with a marked decrease in muscle pH [287]. Similar results were also discovered by Kent et al. [288] when evaluating the effect of ultrasonic treatment (30 min, 25/45 kHz, below 10 °C) on the muscle pH of bovine muscle at the early postmortem. However, it is worth noting that no significant changes were observed when the authors further utilized an in vitro model system to assess glycolytic enzyme activity and pH drop rate of postmortem muscle following exposure to ultrasound [288]. This indicates that the effect presented in intact muscle was not due to a permanent alteration in glycolytic enzyme activity but might be related to the micro-environment in the muscle where the enzyme operated. Additionally, the ultrasonication of postmortem muscle could induce mitochondria dysfunction by enhancing sarcoplasmic calcium and impairing mitochondria oxygen consumption rate [113]. As a result, ultrasound-mediated weakening of the mitochondria energy supply pathway might stimulate glycolysis and thereby decrease postmortem muscle pH, which could be another reason for the outcomes by Kent et al. [288] and Gao et al. [287] in intact muscle.

Inconsistently, in a subsequent study, Kent et al. [289] introduced bovine muscle homogenates subjected to ultrasound treatment into a similar in vitro buffer system to monitor the change in glycolytic enzyme activity in response to ultrasound. They showed that ultrasound treatment (20 kHz, 19 W/cm² for 30 min, and 32 °C) notably inhibited lactate accumulation as well as pH decline and attributed the result in part to the loss of catalytic activity of HK. Some of the reported inconsistencies are perhaps closely associated with the frequency, intensity, duration, and operating temperature of ultrasound. It is indeed true that the degree of oxidative denaturation of muscle proteins and lipid peroxidation can be notably exacerbated under inappropriate ultrasound conditions [278,284,290], with potentially negative effects on metabolic enzyme activity. For instance, following ultrasonic pre-treatment (20 kHz, 500 W for 40 min, and 4 °C), Fang et al. [291] observed a remarkable increase in lipid oxidation levels in beef during the aging period of 12 d. Furthermore, there are several limitation factors impeding the application of ultrasonic technology in the meat industry. First, large-scale meat production may involve carcasses with intact skin and fat or frequently be accompanied by dense stacks of meat, which can potentially hinder the propagation of ultrasonic energy. Second, it remains necessary to develop sophisticated temperature control systems to avoid meat quality deterioration caused by thermal effects induced by ultrasound during scaled-up production. Thirdly, current research on ultrasound in meat science primarily focuses on meat after rigor mortis, with limited studies on its application before rigor mortis. Whether ultrasound can regulate energy metabolism during the early postmortem period and thereby improve fresh meat quality requires extensive research to establish.

Conclusion and future prospects

Postmortem energy metabolism, composed of glycolysis, phosphagen system, and mitochondria OXPHOS, is a central biochemical process that determines meat quality development. Among the three basic pathways, glycolytic metabolism directly contributes to the decline in postmortem pH and meat quality changes, mediated by phosphagen system and mitochondria OXPHOS process. CaMKK/AMPK and HIF-1α signaling show a significant improvement of glycolytic enzyme activity and thereby postmortem glycolysis. Importantly, PTMs and their crosstalk have been highlighted in recent years in the field of meat. Their remarkable contribution to postmortem energy metabolism was also comprehensively discussed in this review. Additionally, the exogenous management techniques such as ES, VFC, and SC contribute to a high efficiency in modifying energy metabolism and meat quality, while ultrasonic technology also presents potential feasibility.

Based on the comprehensive review presented, we propose the following strategic research priorities to advance the field of postmortem energy metabolism and meat quality control. These directives aim to translate mechanistic understanding into practical applications.

• (1)Elucidate the multifaceted role of mitochondria and develop targeted strategies

Future research must move beyond descriptive studies to define the precise contribution of mitochondrial aerobic metabolism to postmortem energy supply across species, breed, age, and muscle type by employing techniques including near infrared reflectance spectroscopy, permeabilized fibers, and real-time cellular energy metabolism detection (e.g., Oxygraph-2 k and Seahorse XF analyzers). Next, understanding the regulatory role of mitochondria on calcium homeostasis, protein degradation, apoptosis, and oxidative stress in postmortem myocytes as well as the crosstalk of these biochemical pathways with postmortem energy metabolism warrant future emphasis. Finally, this knowledge should be leveraged to develop novel mitochondria-targeted strategies for improving meat quality. These may involve either physically disrupting mitochondrial function at the early postmortem, or conversely, boosting it through pre-slaughter dietary supplements containing mitochondrial enhancers such as rutin, resveratrol, and curcumin, depending on the differences in muscle samples.

• (2)Decipher the regulatory code of postmortem PTMs with a focus on novel modifications

Given their significant upregulation in quality-deficient meats, validating the postmortem biochemical regulatory significance of S-nitrosylation, carbonylation, and particularly lactylation modifications should be an immediate priority. Meanwhile, a dual-technological approach is required: First, employing a subcellular fractionation strategy to isolate proteins located in subcellular organelles (e.g., mitochondria) or regions can potentially improve detection coverage and discover more PTMs sites included in low-abundance metabolic enzymes. Second, utilizing techniques such as computational simulation and targeted mutagenesis to in-depth elucidate the regulation of PTMs on energy metabolism enzymes from the level of specific-sites are expected to provide a reference for the precision breeding of livestock and poultry.

• (3)Investigate the non-metabolic functions of energy metabolism enzymes

Several energy metabolism enzymes (e.g., PKM, HK, ALDOA) have dual functions of metabolic catalytic activity and non-metabolic kinase activity. Future studies can further investigate the function of these enzymes as protein kinases involved in the modulation of postmortem proteolysis and the meat tenderization process. This may deliver a possible pathway for achieving meat tenderization via regulating the glycolytic enzyme.

• (4)Optimize exogenous technologies for precision management

The potential of techniques like ultrasound must be transitioned from promise to practice. Research should focus on optimizing parameters (e.g., frequency, intensity, duration) that effectively modulate early postmortem energy metabolism. This effort should specifically target challenging meat types with low metabolic rates and poor tenderness such as atypical DFD beef and WB chicken, with the goal of developing non-invasive technologies to enhance tenderness and overall quality.

Overall, with a focus on these strategic priorities, we expect to accelerate the development of innovative and evidence-based solutions to mitigate meat quality defects and meet evolving consumer demands.

CRediT authorship contribution statement

Chao Ma: Conceptualization, Writing – original draft, Writing – review & editing, Visualization, Funding acquisition. Wangang Zhang: Conceptualization, Project administration, Supervision, Writing – review & editing, Funding acquisition. Jian Zhang: Writing – review & editing, Funding acquisition. Lei Zhou: Writing – review & editing. Lujuan Xing: Writing – review & editing. Rui Liu: Writing – review & editing.

Funding

This work was funded by the National Key Research and Development (R&D) Program of China (2024YFD2100401), the Earmarked fund for China Agriculture Research System (CARS-35), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX25_1018), and the National Natural Science Foundation of China (32502247).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Chao Ma is currently a PhD student at the State Key Laboratory of Meat Quality Control and Cultured Meat Development at Nanjing Agricultural University in China, under the supervision of Prof. Zhang Wangang. His research focuses on postmortem muscle biochemistry and meat quality control.

Wangang Zhang is a professor at the College of Food Science and Technology of Nanjing Agricultural University in China. In 2009 he got his PhD from Iowa State University of United States with the major of Meat Science. His research mainly focuses on meat and muscle biology. He serves currently as the Associate Editor of Meat Science, International Editor of Food Science of Animal Resources, and Editorial Board Member of Trends in Food Science & Technology and Current Opinion in Food Science. He has published 242 peer reviewed SCI journal articles with over 17,000 citations and an H-index of 67. He also has published 15 book chapters as editor or coauthor.

Jian Zhang is currently a postdoctoral fellow at Nanjing Tech University in China focusing on innovative meat processing and flavor enhancement. He, as the first author, has published a total of 14 peer reviewed SCI journal articles, with an H index of 17.

Lei Zhou obtained his doctorate degree from Nanjing Agricultural University in 2024 and joined the School of Food Science and Technology of Hunan Agricultural University in the same year. He mainly studied the development of high-quality meat products and high-tech food processing technology. He was invited to serve as a guest editor of Gels and Frontiers in Nutrition. He has published 57 articles, including 52 peer reviewed SCI journal articles, 16 SCI articles as the first author or corresponding author, and H index 19.

Lujuan Xing is an associate professor at the College of Food Science and Technology, Nanjing Agricultural University in China. Her research focuses on the high-value utilization of animal source by-products. She has published more than 20 articles as the first author, including 15 peer reviewed SCI journal articles.

Rui Liu is an associate professor at the College of Food Science and Engineering, Yangzhou University in China, where his research interests focus on meat biochemical metabolism and muscle protein posttranslational modification. He currently serves as the Editorial Board Member of Meat Science. He has published 70 articles, including 22 peer reviewed SCI journal articles as the first author or corresponding author.