Role of postmortem bioenergetics in beef colour chemistry

Abstract Meat colour is one of the primary factors affecting the acceptability of retail meat cuts. Therefore, displaying the natural bright-red colour of meat is a major goal of the meat industry. Consumers frequently reject to retail meat cuts with discolouration even at low levels of surface browning and/or darkening, which in turn results in significant food waste and economic loss. Thus, understanding the factors that influence meat colour deviations such as discolouration or darkening is crucial in developing effective strategies to minimise the display colour-related quality issues. Numerous factors influence series of biochemical reactions of post-mortem muscles and the chemical state of myoglobin, which ultimately affects meat colour and colour stability. Recent studies have demonstrated the incidence of dark-cutting beef post-mortem can be attributed to the combination of lower glycolytic potential, less glycolytic metabolites, enzymes and dysregulated energy metabolism. This review will focus on the role of post-mortem bioenergetics and mitochondria dynamics that affect beef colour chemistry.


Introduction
Colour of food impacts anticipated and perceived sensory characteristics (Wei et al. 2012).This is particularly relevant in acceptance and purchasing decisions of muscle foods.Any deviation from expected sensory characteristics leads to less consumer acceptance.Meat colour is an important sensory attribute in all facets of beef merchandise (Ramanathan et al. 2022).Consumers rank-colour as an important sensory quality that influences purchasing decisions.A recent study noted that the US beef industry annually loses approximately $3.9 billion (USD) due to discolouration and discounts (Ramanathan et al. 2022).The study also noted that discolouration led to discarding 194.70 million kg of meat.Another report noted that globally $14.2 billion was lost due to discolouration (Maia Research Analysis 2020).Discarding nutritious beef also leads to waste of natural resources and energy used to produce meat.Hence, understanding the factors that influence meat colour deviations such as discolouration or failure to have bright red colour is vital to maximise the benefits of producing beef.Interestingly, development of bright red colour is related to series of biochemical reactions that originate pre-and post-mortem.The use of highthroughput techniques has greatly enhanced our knowledge related to muscle to meat conversion.This review will focus on the role of post-mortem bioenergetics in beef colour chemistry, particularly focusing on dark-cutting muscle phenomenon.

Basic meat colour chemistry
The presence of haem in a structure within a molecule allows light to be absorbed and reflected.This is particularly relevant in meat due to the presence of haem containing proteins such as myoglobin, haemoglobin and cytochromes.Physiologically, both myoglobin and haemoglobin are related to oxygen storage and transport, while cytochromes are present in mitochondria and aid in electron flow.The major role of myoglobin is to store oxygen in tissue and transfer to mitochondria to produce adenosine triphosphate (ATP) by oxidative phosphorylation.However, after animal harvest oxygen flow ceases, and these biomolecules contribute to appearance of meat.In animals that are exsanguinated well, haemoglobin content is much less.Myoglobin is the primary sarcoplasmic protein responsible for meat colour.Although contribution of haemoglobin and cytochromes are relatively lower, these two proteins also influence meat colour to a lesser extent but can increase oxidative processes such as lipid oxidation.The spectral features of both haemoglobin and myoglobin are similar, hence spectral approach to characterise these two proteins are challenging.
Meat colour is determined by the redox state of myoglobin and meat ultra-structure.Myoglobin has a haem prosthetic group and globin (protein) part.Myoglobin is composed of 153 amino acids, which are folded into eight helices segments around haem.The protein part is colour-less, but haem portions, which are hydrophobic, impart colour.Haem can coordinate with six different bonds, of which four are formed with pyrrole ring, fifth with histidine, and sixth with ligand.The valence state of iron and the ligand present at the sixth site ultimately determines meat colour.Four primary forms of myoglobin exist including: deoxymyoglobin, oxymyoglobin, carboxymyoglobinnd ferrous iron.A dark purplish-red colour is the result of myoglobin being in the deoxymyoglobin state and is typically found within the centre of fresh meat as well as anaerobic type packaging systems.If oxygenation of deoxymyoglobin occurs, oxymyoglobin is formed as the sixth coordination site is bound with oxygen (O 2 ) and ferrous iron.Oxymyoglobin imparts a bright cherry-red colour in fresh beef and is the colour that most consumers associate with fresh product (Carpenter et al. 2001).Additionally, oxymyoglobin can be converted back to deoxymyoglobin through mitochondrial oxygen consumption by a lowering of oxygen partial pressure (Tang et al. 2005;2005).Through oxidation and the change of ferrous iron to ferric iron, metmyoglobin if formed imparting a tan to brown colour in fresh beef products (Ledward 1970).Metmyoglobin typically forms at low (1-3%) oxygen partial pressure (Ledward 1970;King et al. 2023).If meat comes into the presence of carbon monoxide (CO) gas, carboxymyoglobin can be formed as CO will attach to the vacant sixth coordinate.Carboxymyoglobin results in a stable bright cherry-red colour in beef and is used in modified-atmosphere packaging systems.However, carboxymyoglobin can also be oxidised to metmyoglobin when low oxygen partial pressure exists (King et al. 2023).Ultimately, the conversion of the four primary forms of myoglobin is affected by numerous parameters of which several will be further covered in the sections below.
Oxygen consumption is one of the many factors that play a pivotal role in colour stability and the conversion of deoxymyoglobin to metmyoglobin and oxymyoglobin to deoxymyoglobin.In meat, most oxygen consumption is attributed to mitochondria respiration and oxygen consuming enzymes.Holistically oxygen consumption is attributed to the use of oxygen by myoglobin, oxygen consumption enzymes, microbes, lipids and mitochondria (Faustman and Cassens 1990;Baron and Andersen 2002;Tang et al. 2005;2005).Oxygen consumption has been shown to be an indicator of colour stability in fresh meat systems (Sammel et al. 2002;2002) and can be measured in a variety of ways.Typically, high oxygen consumption results in decreased colour stability and greater deoxymyoglobin formation (Ramanathan et al. 2020).In practice, oxygen consumption can be influenced by a variety of factors including pH (Ramanathan and Mancini 2018;Kiyimba et al. 2022) , temperature (Tang et al. 2005;Tang et al. 2005), muscle type (Abraham et al. 2017), packaging (Ramanathan et al. 2012) and ageing (English, Mafi, et al. 2016;English et al. 2016).
Metmyoglobin reducing activity is another critical component to colour stability and the conversion of metmyoglobin back to deoxymyoglobin.The ability of intact muscle to reduce and limit the formation of surface discolouration (metmyoglobin) is critical when we consider fresh meat systems.Three pathways are ultimately responsible for the reduction of metmyoglobin and include: enzymatic reduction (Arihara et al. 1995), non-enzymatic reduction (Brown and Snyder 1969) and electron-transport metmyoglobin reducing activity (Tang et al. 2005;Tang et al. 2005).Oxygenation plays a role in both enzymatic (Mikkelsen and Skibsted 1992) and nonenzymatic (Denzer et al. 2020;Denzer et al. 2020) reduction.However, electron transport metmyoglobin-reducing activity is mediated primarily by mitochondria (Tang et al. 2005;Tang et al. 2005).Numerous ways to evaluate metmyoglobin-reducing activity have been assessed and vary widely among labs and investigators (Bekhit and Faustman 2005).Due to the vast variations in methodology, interpreting metmyoglobin reducing activity must be done with caution and aided by retail display characteristics.

Conversion of muscle to meat
In antemortem muscle, the circulation system transports oxygen and glucose to the muscle.Glucose is stored in the muscle as glycogen at approximately 1-2% of the total muscle mass (Matarneh, England, Scheffler, and Gerrard, 2017).The stored glycogen is mobilised and utilised to generate several energy substrate metabolites such as pyruvate that are incorporated into the tricarboxylic acid cycle (TCA) in the mitochondria and further oxidised producing ATP (Ramanathan et al. 2019).The oxygen bound by myoglobin is released to the mitochondria and serves as the final electron acceptor in the mitochondrial electron transport chain.Hence, prior to slaughter, energy present in the muscle is dependent upon circulation and presence of oxygen and energy-providing metabolites such as glycogen or glucose.From exsanguination, there is a loss of circulation resulting in loss of homeostasis in the body.Hence, due to the lack of oxygen, the metabolism converts from aerobic to anaerobic as the muscle rapidly utilises ATP to attempt to return to homeostasis (Scheffler et al. 2015;Matarneh, England, Scheffler, Yen, et al. 2017;Matarneh et al. 2018).The resultant anaerobic conditions post-mortem promotes pyruvate conversion into lactate via lactate dehydrogenase (Kim et al. 2006).The hydrogen ion formed due to the hydrolysis of ATP during glycolysis (Matarneh, England, Scheffler, and Gerrard, 2017) as well as in the mitochondria (Matarneh et al. 2018) plays a role in post-mortem pH decline.More specifically, pH decline occurs within 1-24 h post-mortem depending on species and results in a decline from physiological muscle pH of approximately 7.2 to a pH around 5.6 (Matarneh, England, Scheffler, and Gerrard, 2017).Therefore, the change in pH has a significant impact on the meat quality attributes.Hence, metabolic changes antemortem and early post-mortem combined with post-mortem pH decline results in the conversion of muscle to meat and influence meat quality (Kim et al. 2018).

Role of fibre type on colour
Muscle fibre type directly impacts the predominant metabolism (Hunt and Hedrick 1977a).Such that, greater type I fibres have more oxidative metabolism and type IIb fibres are glycolytic (Picard and Gagaoua 2020).Research has evaluated the effects of muscle types on post-mortem metabolome and stability.Mitochondria content is greater in the oxidative psoas major muscle compared with the glycolytic m. longissimus which influences the stability of muscles (Ke et al. 2017).Mitochondrial degradation was reported to be faster in the psoas major muscle than the longissimus muscle leading to greater oxidative stress in the psoas major muscle (Ke et al. 2017).Furthermore, oxidative muscles such as m.masseter have been reported to have lower glycolytic flux than glycolytic cutaneous trunci muscles due to an early termination of glycolysis and glycogenolysis (Chauhan et al. 2019).In support, post-mortem oxidative muscles have greater glycogen (Lefaucheur 2010;Chauhan et al. 2019;Picard and Gagaoua 2020) and lower glucose-6-phosphate content (Chauhan et al. 2019).Oxidative muscles (psoas major and masseter) have higher postmortem pH in comparison to glycolytic muscles (longissimus and cutaneous trunci; Abraham et al. 2017;Chauhan et al. 2019).Thus, the use of glycogen post-mortem must extinguish through a separate mechanism beyond pH decline.Several studies on the proteome of post-mortem muscles have supported the variations in post-mortem glycolytic metabolismbased muscle type.Triosephosphate isomerase was reported to be higher in more glycolytic muscles (longissimus and semitendinosus) compared with oxidative muscles (psoas major and masseter; Oe et al. 2011;Joseph et al. 2012).Furthermore, the glycolytic longissimus muscle has more of the glycolytic enolase enzyme than the oxidative psoas major muscle (Joseph et al. 2012;Nair et al. 2018;Zhai et al. 2020).The variation in muscle type influences the post-mortem metabolism thus the glycogen content.
Beyond muscle type, dark-cutting muscle has shown deviation in post-mortem metabolism to normal-pH muscle metabolism.Dark-cutting beef is a condition which occurs due to antemortem stress resulting in a higher-than-normal ultimate pH (Hunt andHedrick 1977b, 1977c;Ramanathan et al. 2020;Wu et al. 2020;Kiyimba et al. 2021Kiyimba et al. , 2022)).Such stress results in changes to metabolome and proteome impacting the appearance and resulting in a dark appearance.Dark-cutting beef has been reported to have greater mitochondrial content (McKeith et al. 2016;Ramanathan et al. 2020;Kiyimba et al. 2021Kiyimba et al. , 2022) ) along with greater oxygen consumption compared with normal-pH beef (Ramanathan et al. 2020;Wu et al. 2020;Kiyimba et al. 2021Kiyimba et al. , 2022)).Hence, there is less oxygen available for binding to myoglobin and more deoxymyoglobin formation (English et al. 2016;English et al. 2016).These differences in mitochondria are supported by the greater number of oxidative muscle fibres found in dark-cutting beef than normal-pH beef (Zerouala and Stickland 1991).Furthermore, dark-cutting beef has been reported to have an upregulation in mitochondrial proteins related to biogenesis (Kiyimba et al. 2021).In support, seven glycogen degradation enzymes are down-regulated in dark-cutting beef compared with normal-pH beef (Ramanathan et al. 2020).Less phosphorylation of glycolytic enzymes such as glycogen phosphorylase and ATP-dependent 6-phosphofructokinase could inhibit glycolysis in dark-cutting beef (Ijaz et al. 2022;Ijaz et al. 2022).These support the lower glycolytic potential (Wulf et al. 2002;Holdstock et al. 2014;Apaoblaza et al. 2015;Cônsolo et al. 2021) and glycogen content (Apaoblaza et al. 2015;Ijaz et al. 2022;Ijaz et al. 2022) of dark-cutting beef.Glycolytic metabolites such as glucose-6-phopshate (Apaoblaza et al. 2015;Ramanathan et al. 2020;Cônsolo et al. 2021;Ijaz et al. 2022;Ijaz et al. 2022) were lower in dark-cutting beef compared to normal-pH beef along with ribose-5-phosphate and adenosine monophosphate associated with energy metabolism and ATP degradation (Ramanathan et al. 2020;Ijaz et al. 2022;Ijaz et al. 2022).Several studies supported a lower energy metabolism in dark-cutting beef (Cônsolo et al. 2021).Hence, the combination of lower glycolytic potential, less glycolytic metabolites and enzymes, and dysregulated energy metabolism could lead to formation of dark-cutting beef post-mortem (Hunt and Hedrick 1977c).

Practical implications of post-mortem bioenergetics in meat colour
Previous sections discussed the biochemical basis of post-mortem bioenergetics on metabolite formation and their subsequent impacts on ultimate muscle pH and meat colour.However, any deviation from normal range of pH along with various intrinsic and extrinsic factors can influence meat quality attributes, particularly meat colour.Some of the scenarios are discussed below.

Ground beef discolouration in vacuum package
Vacuum or anaerobic packaging is a common type of packaging for chilled or frozen extended storage.Anaerobic conditions within package limit microbial and oxidative changes in meat and extend shelf-life longer than oxygen-containing or permeable packages.However, any deviation from normal meat characteristics, such as low pH, lower oxygen consumption and metmyoglobin-reducing activity, incomplete removal of oxygen, poor cold chain management, microbial load, post-mortem age of the meat or residual antimicrobial can influence acceptable colour in vacuum packaged meat.Most industries follow strict sanitary and good manufacturing practices; hence the role of microbial load and antimicrobial residue in meat colour might be minimal.
Various biochemical processes are involved to have acceptable purple or dark-red colour in vacuum-packaged ground beef (King et al. 2023).The time delay between grinding, lower processing room temperature and packaging allows meat to bloom.As indicated in the previous sections, meat is biochemically active to a certain extent, and still exerts oxygen consuming activity.Oxymyoglobin is the primary source of oxygen for cellular activity when bloomed meat is vacuum packaged.Hence, various processes start utilising available oxygen, primarily from oxymyoglobin.The conversion of oxymyoglobin to deoxymyoglobin is not a single step, but it goes through metmyoglobin (George and Stratmann 1952;Ledward 1970;Brantley et al. 1993).More specifically, oxygen consumption leads to lower oxygen partial pressure within package, promoting metmyoglobin.The formation of metmyoglobin is a function of myoglobin's inherent property of being oxidised at 5-7 mm Hg (Ledward 1970).When oxygen is consumed there will be mixture of deoxygenated and oxygenated myoglobin.In addition, superoxides are formed during deoxygenation, which can react with some of the deoxymyoglobin to form metmyoglobin (Brantley et al. 1993;Richards 2013).However, metmyoglobin formation and conversion to deoxymyoglobin is very quick process and depends on metmyoglobinreducing activity (King et al. 2023).Hence, lower temperature (decrease oxygen consumption and metmyoglobin-reducing activity), imperfect vacuum (inability to remove all oxygen and creates myoglobin oxidising partial pressure range) and increased post-mortem ageing period (lower substrates for oxygen consumption and metmyoglobin reducing activity) can promote discolouration on the edges and surfaces of vacuum packaged meat.The easiest way to test whether discolouration of surface is due to imperfect vacuum or altered colour chemistry is to make a perpendicular cut of ground beef patty or chub and examine the interior colour for any visual observation of metmyoglobin (brown) ring formation.The formation of a subsurface metmyoglobin layer has been reported previously using digital image photography (Limsupavanich et al. 2004(Limsupavanich et al. , 2008)), nearinfrared oximeter (Mohan et al. 2010;Mohan et al. 2010) and near-infrared diffuse reflectance spectroscopy (Piao et al. 2021).This subsurface layer is critical to the further development of surface discolouration.Troubleshooting discolouration in vacuum packaged meat, however, is often challenging as several factors can influence metmyoglobin formation.

Variation of colour within muscle
The metabolism and biochemical characteristics of individual muscles in distinct anatomical and physiological locations within a beef carcase differ, resulting in varying colour of different muscles.The colour and colour stability of each muscle is affected by various endogenous factors such as myoglobin content, myoglobin redox stability (e.g.MRA) and mitochondrial functionalities.The concentration of myoglobin within a carcase can vary based on the type of muscle fibre (type I vs. type IIb) with different energy metabolism (oxidative vs. glycolytic) as discussed above.Also, muscles used for locomotion, which require more oxidative metabolism and extensive muscle movement, tend to have a higher myoglobin concentration and thus appear darker than support muscles (Meng et al. 1993).Although muscles possess the metabolic machinery to reduce and consume oxygen, their ability to reduce oxidised myoglobin states through inherent ability (MRA) is limited.Also, while the consumption of oxygen and MRA mediated by mitochondria affects colour stability, myoglobin's redox stability of each muscle and subsequent impacts on meat colour are a muscle-specific factor.
Muscles that exhibit superior colour stability, such as the longissimus lumborum, demonstrate higher levels of MRA and nicotinamide adenine dinucleotide (NADH), which is the primary reducing substrate for MRA.They also exhibit lower mitochondrial respiratory enzyme activity and lipid oxidation compared to muscles with less colour stability, such as the psoas major.Additionally, emerging evidence suggests that enzymes and substrates involved in the glycolytic and TCA pathways are linked to muscle-specific meat colour differences and discolouration (Ma et al. 2017).

Impacts of post-mortem ageing on meat colour
Post-mortem ageing is a crucial process that enhances the eating quality attributes of fresh meat.The primary goal of post-mortem ageing is to improve tenderness, making it a widely used practice in the meat industry.Wet-ageing, the most common ageing type, involves vacuum packaging primal or subprimal muscle sections and holding them at a low temperature for several days to weeks (Kim et al. 2018).The average ageing time of beef subprimals at retail establishments in the United States is 25.9 d, with an increasing trend towards extended ageing time up to 102 d (Martinez et al. 2017).
Despite the benefits of post-mortem ageing on palatability attributes, it can also affect the surface colour development and subsequent colour stability of meat during display.Initially, aged meat shows improved surface redness compared to non-aged meat, likely due to a decrease in mitochondrial oxygen consumption.This leads to increased oxygen availability for myoglobin to form a thicker layer of oxymyoglobin, showing enhanced bloomed colour appearance (Kim et al. 2018).However, the transient improvement of fresh redness associated with post-mortem ageing can be rapidly disappeared during display under retail light conditions (Kim et al. 2011).The rate and extent of discolouration are elevated with long-term aged meat, which can be a significant economic problem as consumers prefer fresh-looking meat, as bright cherry/pink, red colour.While more research is needed to fully understand the underlying mechanisms by which extended ageing affects colour and colour stability of muscles, it can be attributed to the exhaustion of endogenous reducing substrates or antioxidants along with the accumulation of pro-oxidants, which also would be related to different energy metabolic conditions of muscles, with ageing (Ma et al. 2017).

Impact of oxygen exposure on meat colour
Oxygen plays a critical role in the development oxymyoglobin, post-mortem mitochondrial functionality and myoglobin oxidation.As mentioned in previous sections, post-mortem mitochondrial oxygen consumption influences myoglobin state (Tang et al. 2005;Tang et al. 2005).Abundance of oxygen such as conditions in high oxygen modified atmospheric packaging reported deeper oxygen penetration (Denzer et al. 2020;Lu et al. 2020;Yang et al. 2022).The greater presence of oxygen decreased oxygen consumption (English et al. 2016;English et al. 2016;Yang et al. 2022) supporting the deeper penetration due to decreased competition between myoglobin and mitochondria.Oxygen exposure during retail display of the psoas major muscles reported a decrease in oxygen consumption during retail display while there were minimal impacts on the longissimus muscle (Denzer et al. 2022).Furthermore, high oxygen (80%) conditions decreased MRA of steaks (English et al. 2016;English et al. 2016;Yang et al. 2022).Similarly, oxygen exposure during retail display decrease MRA of the oxygen-exposed surface compared to a more biochemically active non-oxygen exposed interior (Denzer et al. 2022).Hence, oxygen exposure could negatively influence colour stability.Oxygen exposure also influenced the metabolome of the longissimus muscle during retail display (Denzer et al. 2023, accepted).
Metabolites such as citric acid and gluconic acid were impacted by oxygen exposure with both in greater abundance on the oxygen-exposed surface.Hence, oxygen exposure resulted in changes in the TCA cycle and pentose phosphate pathway during retail display.These shifts in metabolome change the bioenergetics of post-mortem muscle and can impact colour stability.

Dark-cutting condition
Carcase values in several countries such as Canada, Australia and the US depend on acceptable bright red colour or pH during grading.Dark-cutting beef has worldwide occurrence and economic impact can be considerable.For example, based on the National Beef Quality Audit, in 2022 the US beef industry loses approximately $202 million.In Canada, discounts cost approximately $10 million 2016-2017 (Bergen 2023).In Australia, it costs approximately $55 million Australian dollars in 2014 (Jose et al. 2015;Steel et al. 2022).Greater than normal muscle pH will influence both muscle structure and activity of oxygen-consuming enzymes.More specifically, a greater than normal muscle pH leads to less shrinkage of muscle bundles after post-mortem (Hughes et al. 2017) ultimately resulting in less space between muscle bundles and also less free water (Hughes et al. 2017).This can decrease oxygen diffusion from the surface to interior, and also limit light reflectance (Hughes et al. 2017).Thus, beef appears darker in colour (Hughes et al. 2017).Several studies noted lower L Ã values (lower lightness) in dark-cutting beef compared with normal-pH beef (English et al. 2016;English et al. 2016;Wills et al. 2017;Kiyimba et al. 2021, Kiyimba et al. Kiyimba et al. 2023).Greater muscle pH favours mitochondrial respiration, thus limiting available oxygen to myoglobin (Ashmore et al. 1972;McKeith et al. 2016).Thus dark-cutting beef has greater deoxymyoglobin than normal-pH beef, and results in darker colour (English et al. 2016;English et al. 2016).
Improving redness of dark-cutting beef primarily focus on to lower pH, increase oxygen availability within package, form other bright red colour forms such as nitric oxide myoglobin and carboxymyoglobin, and increase spacing between muscle bundles.Various acid enhancements using citric or lactic acid has been used to lower pH and improve colour (enhancement represents injecting solution to meat using multi-needle injector) (Sawyer et al. 2008(Sawyer et al. , 2009;;Apple et al. 2011;Stackhouse et al. 2016).However, use of acid enhancement/injection solution leads to localised discolouration (Sawyer et al. 2009;Apple et al. 2011).Enhancing with rosemary improved redness of dark-cutting steaks in poly vinyl chloride packaging (English et al. 2016;English et al. 2016).Using modified atmospheric packaging was reported to improve the redness of dark-cutting steaks through increased oxygen (40-80%) (Wills et al. 2017;Mitacek et al. 2018;Lu et al. 2020) or the addition of carbon monoxide (Wills et al. 2017;Mitacek et al. 2018).Including greater oxygen levels within package (40-80%) along with antioxidant enhancement increased a Ã values of dark-cutting steaks (Wills et al. 2017).Wills et al. (2017) reported enhancing meat with rosemary and packaged in 80% oxygen increased redness as well as lightness of dark-cutting steaks.With the increased amount of oxygen present, the greater demand for oxygen by the mitochondria can be met, improving the colour of dark-cutting beef (Wills et al. 2017).However, increased oxygen resulted in the formation of greater lipid oxidation (Mitacek et al. 2018).The storage of rosemary-enhanced dark-cutting steaks in 0.4% carbon monoxide improved redness through the formation of carboxymyoglobin (Wills et al. 2017).Rosemary enhancement was combined with nitriteembedded packaging to form nitric oxide myoglobin and bright-cherry red appearance in dark-cutting beef (Ramanathan et al. 2018).The use of nitrite-embedded packaging did not improve the surface colour of darkcutting steaks (Ramanathan et al. 2018).Glucono-delta lactone enhancement coupled with rosemary and nitrite-embedded packaging resulted in improved redness and lightness during retail display (Denzer et al. 2022b) and dark storage (Denzer et al. 2022a).Nonetheless, use of the nitrite-embedded packaging resulted in unstable red appearance upon repackaging in aerobic packaging along with a red external cooked appearance of dark-cutting steaks (Denzer et al. 2022a(Denzer et al. , 2022b)).Furthermore, addition of rosemary was reported to result in the formation of off-flavours in dark-cutting beef steaks (Denzer et al. 2020;Denzer et al. 2020).A recent study reported the use of highpressure processing at 300 MPa can result in improved redness and lightness of dark-cutting steaks with no impact on juiciness or tenderness (Reesman et al. 2023).Therefore, high-pressure processing has the potential for improving the colour characteristics of dark-cutting beef without negatively impacting palatability.Lastly, extended ageing of dark-cutting beef results in improved oxygenation of muscle possibly due to the decrease in substrates available for mitochondrial function or decreased mitochondrial function (English et al. 2016;English et al. 2016).Hence, the bioenergetics of post-mortem dark-cutting beef is critical to colour development.

Feeding condition
Significant changes in colour chemistry are impacted by feeding regime, especially when comparing grassfinished vs. grain-finished, high concentrate diets.Grass-fed diets are less energy dense than grainfinished diets, in addition the increase in physical activity as animals forage (Dunne et al. 2011;Apaoblaza et al. 2020).Consequently, significant differences in live and carcase weights, ribeye area, fat thickness, kidney, pelvic and heart fat, marbling score, as well as lean colour/maturity are observed between grass-and grain-finished cattle (Mandell et al. 1998;Realini et al. 2004;Faucitano et al. 2008;Garmyn et al. 2010).Generally, grass-finished cattle are lighter weight, leaner, and have less intramuscular fat than traditional conventional fed cattle (Garmyn et al. 2010;Lucherk et al. 2022).More specifically, cattle finished on grass typically have yellow fat and soft-coarse-dark lean (Davis et al. 1981;Crouse et al. 1984;Miller et al. 1996;Lucherk et al. 2022), leading to decreased consumer appeal at retail.In general, the longissimus muscle of grass-finished carcases are less red and darker in colour than grain-finished cattle (Realini et al. 2004;Duckett et al. 2007;Duarte et al. 2022).Darker coloured lean in grass-finished cattle is thought to be primarily caused by the decrease of muscle glycogen levels attributed to the low energy diet (Duckett et al. 2007), especially as forage quality decreases or is lacking in abundance (Knee et al. 2007).Some studies have found increased pH in grassfinished carcases along with differences in colour (Tansawat et al. 2013;Apaoblaza et al. 2020), however, others have noticed no difference in pH with significant differences in colour (Lafreniere et al. 2021;Duarte et al. 2022).In addition, the slowed growth rate of pasture finished cattle may be partially attributed to the darker colour, as energy metabolism and associated pathways are altered in post-mortem muscle metabolism (Antonelo et al. 2022;G omez et al. 2022).

Chilling rate
Carcase temperature decline in addition to pH decline within the first 24 h can have the primary impacts on the muscle biochemistry and ultimate colour of beef.Spray chilling, which is the largely preferred method in most commercial US packing plants, allows for a steady rate of carcase temperature decline with minimal effects to quality and colour (Greer and Jones 1997).A slowed rate of temperature decline leads to a more rapid pH decline and ultimately leads to increased protein denaturation within the muscle causing subsequent reduced colour stability (Seyfert et al. 2004;Ferguson and Gerrard 2014;Kim et al. 2018).Specifically, a paler-coloured lean with decreased water-holding capacity is the result from slowed temperature decline and rapid pH decline (Kim et al. 2014).However, rapid chilling can also produce detrimental effects to colour including muscle darkening (Bowling et al. 1987;Aalhus et al. 2002).Utilising methods such as blast chilling leads to less cooler shrink and increased marbling visibility, but at the expense of darker-coloured lean and slowed pH decline (Aalhus et al. 2002).Furthermore, small shifts in air temperature during chilling can impact the ultimate proportions of oxymyoglobin, deoxymyoglobin and metmyoglobin within the muscle when evaluated during retail display (Holdstock et al. 2023).
When carcases are cooled at a steady temperature, the ability to take oxygen from myoglobin is retained through the electron transport chain, ultimately allowing for optimal bloomed colour (Ramanathan and Mancini 2018).Some post-harvest interventions have been implemented in order to aid in the conversion of muscle to meat and consequently improve the post-mortem colour of meat.Electrical stimulation at both high and low voltage is prevalent in nearly all commercial plants prior to carcases entering the chilling cooler and has been shown to improve instrumental colour (Savell et al. 1978;Roeber et al. 2000;Djimsa et al. 2022).Electrical stimulation aids in decreasing the effects of cold shortening and improving the brightness of fresh muscles (Savell et al. 1978;Bakker et al. 2021).
Beef carcase weights in the Unites States have steadily increased over the last several decades resulting in more variability in carcase size (Boleman et al. 1998;McKenna et al. 2002;Garcia et al. 2008;Boykin et al. 2017).The increased variability among carcases has posed challenges in terms of chilling and achieving adequate temperature decline.As carcases increase in size, there is also generally a subsequent increase in fat thickness which acts as insulation and a subsequent slower rate of temperature decline (Aalhus et al. 2001;Kim et al. 2014).In addition, the added weight of some carcases comes from larger, heavier muscle rounds resulting in delayed temperature decline of muscle deep within the round (Kim et al. 2014;Lancaster et al. 2020).Deep tissue temperatures such as that of m. semimembranosus on heavy weight carcases may take more than 24 h to drop below the optimal 4 C temperature threshold potentially causing exacerbating issues to post-mortem metabolism (Djimsa et al. 2022).Moreover, increased surface discolouration occurs during retail display in muscles from the round of heavy weigh carcases (Lancaster et al. 2020), however, more studies need to address a wider range of cuts and muscles primarily those from the chuck.As beef carcase weights continue to increase, additional chilling measures and strategies will be needed in order to maintain desirable colour and mitigate some detrimental changes to postmortem muscle metabolism.

Molecular evidence from omics approaches in understanding the biochemical and molecular markers of muscle darkening
Depending on the ultimate pH level, muscle darkening can range from a very dark colour with a high pH (typical dark-cutting beef) to a dark colour with slightly elevated muscle pH (atypical dark-cutting beef; Mahmood et al. 2018;Roy et al. 2022).Early studies of muscle darkening by Hedrick et al. (1959) relied on active manipulation of the animals' physiology specifically utilising drug treatment of adrenaline to induce stress.In this study, they showed that cattle fasted for 24 h and injected with adrenaline for 5 h before slaughter presented with symptoms of stress.Furthermore, administration of adrenaline at 2.5 or 5 mg reduced muscle glycogen producing a shady meat surface colour.Therefore, pre-slaughter stress is characterised as the major contributing factor of muscle darkening in farm animals and the current evidence for muscle darkening is thus associated with defective glycogen metabolism pre-slaughter (Immonen et al. 2000;Rosenvold et al. 2001;Ponnampalam et al. 2017;Mahmood et al. 2018;Jerez-Timaure et al. 2019;Fuente-Garcia et al. 2020;Kiyimba et al. 2021).Although a greater than normal muscle pH (in excess of 5.8) is one of the benchmark characteristics of dark meats, muscle darkening has also been reported at pH close to normal (pH ¼ 5.6; Ijaz et al. 2022;Ijaz et al. 2022;Kiyimba et al. 2023).Thus, this raises a possibility of multi-pathway cross talks in the aetiology of dark-cutting beef.
In the following section, we will discuss some of the findings from proteomics and metabolomics approaches in characterising typical and atypical darkcutting beef, with a particular focus on the role of defective glycogen metabolism on post-mortem metabolism and muscle acidification.We propose a paradigm through which substrate preference and switching could be tested as a new area of focus to further our understanding of the mechanistic basis for the occurrence of the dark-cutting phenotypes.

Use of proteomics to characterise dark cutting beef
Integrative and system biology approaches, including proteomics, metabolomics and transcriptomics are currently incorporated in meat science research to elucidate the molecular and biochemical basis of muscle darkening.Recent proteomics studies utilising labelfree quantification coupled with liquid chromatography-mass spectrometry (LC-MS) have identified severa markers associated with energy metabolism, muscle contraction, stress response and mitochondrial function differentially abundant in dark-cutting phenotypes compared with normal-pH beef (Kiyimba et al. 2021(Kiyimba et al. , 2022(Kiyimba et al. , 2023)).Glycogen metabolism enzymes more specifically those involved with glycogen degradation such as glycogen phosphorylase (PYGM), bis-phosphoglycerate mutase (PGAM1), amylo-alpha-1-6-glucosidase (AGL), phosphorylase b kinase regulatory subunit alpha, beta and gamma (PHKa, PHKb and PHKg), exhibit low abundance in dark-cutting muscles.However, the control mechanisms for the enzymes of glycogen metabolism in muscles have been described and are quantitatively similar across fibre types (Heffron 1981).
Among the glycogen degradation enzymes, glycogen phosphorylase (PYGM), a key enzyme that regulates glycogen breakdown in the muscle, has been reported in several dark-cutting proteomics studies with about 50% reduction in expression in dark-cutting muscles compared with normal-pH beef (Wu et al. 2020;Fuente-Garc ıa et al. 2021;Gagaoua et al. 2021;Kiyimba et al. 2022).The low abundance of PGYM and other glycogenolytic enzymes suggests limited capacity for the muscles to mobilise and utilise glycogen, which in turn results into greater pH levels post-mortem.Recent investigations into the PGYM regulation have identified several mutations in the PYGM gene coding region.These mutations are associated with a glycogen storage disease (type V) in humans (reviewed by Andreu et al. 2007).Based on these findings, questions to 1) whether defective glycogen metabolism in dark-cutting muscles results from alterations in the PGYM gene coding regions independent of pre-slaughter stress mediated glycogen depletion and 2) whether an additive effect of altered gene regulation coupled with pre-slaughter stress drives muscle darkening need to be examined.
Besides, defective glycogen metabolism, rate limiting enzymes in other pathways such as mitochondrial TCA and oxidative phosphorylation, fatty acid metabolism and muscle structural organisation have also been identified differentially abundant in typical dark and atypical dark-cutting muscles compared with normal-pH beef.For example, a recent integromics metaanalysis at the proteome level identified 130 protein biomarkers and highlighted oxidation-reduction and TCA cycle as key pathways regulating muscle darkening in beef (Gagaoua et al. 2021).Furthermore, mitochondrial proteomics analysis of dark-cutting muscles showed evidence of enhanced mitochondrial bioenergetics and biogenesis suggesting over-proliferation of mitochondria in dark-cutting muscles (Kiyimba et al. 2021).More specifically proteins involved in mitochondrial dynamics (fusion and fission; Mfn-1 and À2, FIS1 and MTFP1), transport of pre-synthesized mitochondrial proteins from the cytosol into the mitochondria (TOM22, TOM40 and TMEM109), fatty acid oxidation (CPT1), and calcium homeostasis (CAMK2A) are over expressed in dark-cutting muscles.Previously, Wu et al. (2020) reported that mitochondrial protein glutaryl-CoA dehydrogenase (GCDH) was associated with meat colour parameters.Therefore, the overabundance of mitochondrial respiratory enzymes coupled with several mitochondrial dynamics proteins highlight the impact of defective glycogen metabolism on overall energy balance in dark-cutting muscles.
Mitochondrial division is a metabolic stress response to fluctuating energy levels whereby the number of mitochondria in cells is regulated by changes in metabolic demand (Kelly and Scarpulla 2004).Although the cross-talks between mitochondrial division processes and muscle darkening have not been fully investigated, enhanced mitochondrial biogenesis can promote meat darkening via increased mitochondrial respiration (McKeith et al. 2016;Ramanathan et al. 2020Tang et al. 2005;Tang et al. 2005;Kiyimba et al. 2021).Thus, the greater levels of mitochondrial respiratory, TCA and mitochondrial dynamics enzymes observed in dark-cutting muscles could be a reflection of an energy adaptive mechanism wherein alternative pathways independent of muscle glycolysis (fatty-and amino-acid oxidation), become more pronounced to generate the energy substrates necessary to fuel muscle ATP demand.
Interestingly, differential abundance of energy-sensing enzymes including adenosine monophosphate protein kinase (AMPK) and Sirtuin proteins (NADH deacetylases) have not been fully evaluated in darkcutting compared with normal muscles.Previously, Apaoblaza et al. (2015) examining the glycolytic potential and activity of AMPK in steer carcases of normal (<5.8) and high pH (>5.9) at 24 h post-mortem found lower activity of AMPK in high pH muscles.The lower activity of AMPK and the static protein expression in dark-cutting muscles suggest that energy adaptive mechanisms could be mediated via alternative pathways independent of AMPK catalysed reactions.However, to date, no study has been conducted to examine these possibilities.Therefore, future research focusing on substrate preference mechanism in line with energy balance could further our understanding of the molecular aspects governing muscle darkening.

Use of metabolomics and integrated omics approach to characterise dark and atypical dark cutting beef
In support of proteomics studies, metabolomics characterisation of dark-cutting muscles mostly via non-targeted gas chromatography-mass spectrometry (GC-MS) approach has clearly shown low abundances of glycolytic metabolites and overabundance of mitochondrial metabolites (TCA cycle), fatty acid and nucleotide metabolites in typical and atypical dark-muscles compared with normal-pH muscles (Ramanathan et al. 2020;Cônsolo et al. 2021;Ijaz et al. 2022;Ijaz et al. 2022;Kiyimba et al. 2023).However, the exhaustive identification of biochemical and molecular markers associated with muscle darkening from a single omics tool is still limited.Thus, utilising integrative approaches could provide a more comprehensive evaluation of the molecular drivers of muscle darkening.
Data mining by integrating significant changes in expression profiles of published proteomics and metabolomics data from recent studies (Kiyimba et al. 2021(Kiyimba et al. , 2022(Kiyimba et al. , 2023)), revealed a pH dependent gradation in the down-regulation of glycogenolytic enzymes and metabolites, mitochondrial and muscle structural proteins (Figure 1).Furthermore, relatively large changes are observed in protein and metabolites profiles between dark-at high pH vs. normal beef compared with dark-cutting at slightly elevated muscle pH vs. normal-pH beef.Additionally, there are unique changes in expression especially at protein level, while majority of the metabolites reported differentially abundant at high pH muscles especially amino acid metabolites were not different in atypical darkcutting muscles (Ijaz et al. 2022;Ijaz et al. 2022;Kiyimba et al. 2023).This suggests that the changes in protein expression might contribute more to differences in colour and pH decline between atypical and typical dark-cutting phenotypes rather than metabolite changes.Interestingly, there is a greater abundance of muscle contractile protein in atypical dark-cutting (Figure 2) compared with high pH dark-cutting muscle.
Muscle contractile processes can drive carbohydrate metabolism to meet the ATP demand required for muscle contractile processes.Thus, the greater abundance of muscle contractile proteins could provide evidence to support the low pH reported in atypical dark-cutting muscles.However, this contradicts the role muscle pH decline plays in muscle colour regulation mainly through influence on muscle's reflectance capacity by allowing muscle proteins to hold more water and reduce the muscle's capacity to scatter light (Lawrie 1958;MacDougall 1982).Therefore, these observations show that muscle pH decline in atypical compared with typical dark-cutting muscles might not be the main driver of muscle darkening.Although studies are yet to be conducted on the level of stress in animals that produce typical compared with atypical dark-cutters, the extent of stress can induce changes in ATP synthesis and might also alter supply of gluconeogenesis precursors.Hence, future studies focusing on substrate preference and utilisation in atypical and typical dark-cutting muscles may provide a better understanding of muscle colour regulation at the different pH levels.

Conclusion
Desirable meat colour and colour stability are related to numerous inter-related genetic, structural, chemical and physiological factors.Antemortem and post-mortem conditions significantly impact meat colour, especially the rate and extent of pH decline post-mortem.In this regard, the classical abnormal meat quality conditions, such as pale, soft and exudative (PSE) and dark, firm and dry (DFD), have been well-studied.The emergence of novel omics techniques, however, has further advanced our understanding, especially expanding our knowledge of meat colour chemistry to the involvement of postmortem bioenergetics (e.g.energy metabolism and glycolytic potential), mitochondrial dynamics and oxygen competition between respiratory enzymes and myoglobin.In particular, recent studies have demonstrated the incidence of dark-cutting beef post-mortem can be attributed to the combination of lower glycolytic potential, less glycolytic metabolites and enzymes, and dysregulated energy metabolism.Further research is still needed to determine the multi-pathway cross-talks in the aetiology of dark-cutting beef with a particular emphasis on the potential inter-relationships between mitochondrial dynamics, defective glycogen metabolism, oxidative phosphorylation, fatty acid metabolism, muscle structural organisation and dark-cutting muscles.

Figure 1 .
Figure 1.STRING database analysis of differentially abundant mitochondrial proteins in dark-cutting vs. normal-pH beef.Mitochondrial protein expression changes induced by the dark-cutting condition in subnetworks identified were compared with normal-pH beef.Red and green colours represent up-regulated and down-regulated proteins (p 0.05) in dark-cutting beef mitochondrial proteome relative to normal-pH beef.Proteins in clustered circles exhibit protein-protein interactions, while those outside the clusters show no protein-protein interactions.Adapted from Journal of Proteomics, 2022, 265:104637 https://doi.org/10.1016/j.jprot.2022.104637

Figure 2 .
Figure2.STRING database analysis of proteins up-and down-regulated in dark-coloured beef at slightly elevated pH vs. normal-pH beef.The proteins with significant changes in protein abundance between dark-coloured beef at slightly elevated pH vs. normal-pH samples were used to query potential protein-protein interactions in the string database as described in the methods.Protein interactions were confirmed with connections, while non-interacting proteins had no connections between them.The red colour represents overabundant proteins and the green colour represents less abundant proteins in dark-coloured beef at slightly elevated pH vs. normal-pH beef.The size of the circle represents fold change in abundance, while the arrowheads indicate the target of the protein interactions.Adapted from Journal of Animal Science, Journal of Animal Science, 2023, 101, 1-12 https://doi.org/10.1093/jas/skac376