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New alternatives for improving and assessing the color of dark–cutting beef – a review

ABSTRACT

Myoglobin (Mb) is a sarcoplasmic heme protein present in muscle cells, which acts as a short–term oxygen (O2) reserve in the muscle tissue. After slaughtering and exsanguination, Mb is the major pigment that provides the red color in meat. The concentration of Mb together with its redox state are two pivotal factors that determine meat color. The elevated pH of dark–cutting beef can affect both physical and biochemical properties resulting in decreased oxygenation. The darkening observed in high ultimate pH (pHu) beef concerns meat processors as color is the initial attribute that impacts on the purchase. Thus, any atypical meat color (i.e., loss of brightness) reduces consumer interest in the product. Several studies have demonstrated that immunological castration is effective in preventing both aggressive behavior and undesirable dark–cutting of bull meat. However, little information is available on the effects of processing techniques that limit the oxidation of ferrous iron (Fe2+), Mb or promote metmyoglobin (MMb) reduction in dark–cutting beef. Because of the importance of color to fresh beef marketability, this review aimed at overviewing the significance of pHu in beef color and color stability and to discuss new alternatives for improving and assessing the beef color of dark–cutting beef, especially in Nellore bulls and their crossbreds, which are widely used in beef cattle production in Brazil.

MMb reductase enzyme; high pHu; ultimate pH; meat quality; bull

Introduction

Brazil is one of the world’s largest beef producers (Associação Brasileira das Indústrias Exportadoras de Carnes – ABIEC, 2019), producing a total of 8.23 million tons in carcass–weight equivalent in 2019 (Instituto Brasileiro de Geografia e Estatística – IBGE, 2019, 2020). Of this total, 80.9 % was destined for domestic consumption, providing an annual consumption of approximately 42 kg per capita (ABIEC, 2019). Bos indicus genetics, mainly the Nellore breed, have a considerable share of the Brazilian cattle herd (Mueller et al., 2019Mueller, L.F.; Balieiro, J.C.C.; Ferrinho, A.M.; Martins, T.S.; Corte, R.R.P.S.; Amorim, T.R.; Furlan, J.J.M.; Baldi, F.; Pereira, A.S.C. 2019. Gender status effect on carcass and meat quality traits of feedlot Angus × Nellore cattle. Journal of Animal Science 90: 1078–1089.), in which males represent 59.1 % of the total cattle slaughtered in Brazil during 2019 (IBGE, 2019, 2020).

The irritable behavior of bulls as well as other biological and environmental elements may impair proper muscle acidification until achieving the ultimate pH (pHu) of 5.3 –5.8 (Ponnampalam et al., 2017Ponnampalam, E.N.; Hopkins, D.L.; Bruce, H.; Li, D.; Baldi, G.; Bekhit, A.E.D. 2017. Causes and contributing factors to “dark cutting” meat: current trends and future directions; a review. Comprehensive Reviews in Food Science and Food Safety 16: 400–430.), which is considered the normal pHu range (Ouali et al., 2006Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58.; Contreras–Castillo et al., 2016Contreras–Castillo, C.J.; Lomiwes, D.; Wu, G.; Frost, D.; Farouk, M.M. 2016. The effect of electrical stimulation on post mortem myofibrillar protein degradation and small heat shock protein kinetics in bull beef. Meat Science 113: 65–72.). Indeed, an ultimate pH above 5.8 is detrimental as it enhances the darkening of beef, which is known as dark–cutting beef (Tang et al., 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.; McKeith et al., 2016McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.).

Dark–cutting beef results either from reduced myofibrillar shrinkage (i.e., lower light scattering) (Hughes, 2019) or from higher myoglobin (Mb) content (Ponnampalam et al., 2017Ponnampalam, E.N.; Hopkins, D.L.; Bruce, H.; Li, D.; Baldi, G.; Bekhit, A.E.D. 2017. Causes and contributing factors to “dark cutting” meat: current trends and future directions; a review. Comprehensive Reviews in Food Science and Food Safety 16: 400–430.). A high pHu then inhibits the Mb oxidation from developing a brown layer of metmyoglobin (MMb) due to improved metmyoglobin reducing activity (MRA) and consequent stability of the surface dark red color (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.).

Darkening is also attributable to increased mitochondrial oxygen consumption rate (OCR), which depletes the cellular oxygen (O2) content and is amplified in high pHu muscles (Tang et al., 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.). Accordingly, higher pHu favors the formation of the purplish deoxymyoglobin (DMb) at the expense of the bright red oxymyoglobin (OMb) on the surface of the muscle (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). The negative consequence of this undesirable effect, dark cutting beef, is less marketable (Suman et al., 2014Suman, S.P.; Hunt, M.C.; Nair, M.N.; Rentfrow, G. 2014. Improving beef color stability: practical strategies and underlying mechanisms. Meat Science 98: 490–504.).

Since dark cutting beef has a negative impact on the fresh beef market and consumer acceptance, an understanding of the biochemical reactions and pathways is necessary in order to mitigate this effect. Thus, the objectives of this review were to provide an overview of the significance of pHu on beef color and color stability and to discuss new alternatives aimed at improving and assessing the beef color of dark–cutting beef, especially for Nellore bulls and their crossbreds, which are widely used in beef cattle production in Brazil.

Beef ultimate pH

After slaughtering, the pHu is reached after stabilization of the drop in pH. Although pH measurements at 24 h postmortem may be conducted on group beef muscles (Li et al., 2014Li, P.; Wang, T.; Mao, Y.; Zhang, Y.; Niu, L.; Liang, R.; Zhu, L.; Luo, X. 2014. Effect of Ultimate pH on postmortem myofibrillar protein degradation and meat quality characteristics of Chinese yellow crossbreed cattle. The Scientific World Journal 2014: 1-8.; Ponnampalam et al., 2017Ponnampalam, E.N.; Hopkins, D.L.; Bruce, H.; Li, D.; Baldi, G.; Bekhit, A.E.D. 2017. Causes and contributing factors to “dark cutting” meat: current trends and future directions; a review. Comprehensive Reviews in Food Science and Food Safety 16: 400–430.; Pulford et al., 2008Pulford, D.J.; Vazquez, S.F.; Frost, D.F.; Fraser–Smith, E.; Dobbie, P.; Rosenvold, K. 2008. The intracellular distribution of small heat shock proteins in post-mortem beef is determined by ultimate pH. Meat Science 79: 623–630.; Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.), pHu seems to stabilize only after 48 h postmortem in longissimus lumborum muscles. In fact, a decline in pHu values was observed when measured repeatedly at 24 h and at 48 h postmortem; and this continuous drop profile until 48 h postmortem was presented by Ouali et al. (2006)Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58. and Mlynek et al. (2012)Mlynek, K.; Janiuk, I.; Dzido, A. 2012. Effect of growth intensity of bulls on the microstructure of musculus longissimus lumborum and meat quality. Acta Veterinaria Brno 81: 127–131..

There is a global incidence of beef muscle with pHu higher than the normal threshold established by experts and the beef industry. For instance, Mach et al. (2008)Mach, N.; Bach, A.; Velarde, A.; Devant, M. 2008. Association between animal, transportation, slaughterhouse practices, and meat pH in beef. Meat Science 78: 232–238. reported an incidence of 13.9 % within pH > 5.8, 24 h postmortem in Spain. According to Contreras–Barón (Personal communication), most of the cattle slaughtered in Brazil has shown pH above 5.8 48 h postmortem, which, to a certain extent, may impact the exporting of beef from Brazil. Nevertheless, there are still no records on the incidence of abnormal pH in meat in the Brazilian slaughter system.

To achieve a broader understanding on the influence of the high pHu on beef color, an initial overview on how pH drops to a determined pHu is required. Furthermore, the factors that can lead to a high pHu muscle and the definition of a pHu range to establish a cut–off standard are of pivotal importance.

Biochemical basis of ultimate pH

Living muscle has a neutral pH (7.2 – 7.4). After animal exsanguination, the delivery of O2 to muscle cells is interrupted. The depletion of O2 impairs the ability of cells to metabolize glucose aerobically by the tricarboxylic acid cycle and the respiratory chain. Glucose generated from muscle glycogen must be metabolized by glycolysis to generate lactate and H+ together with adenosine triphosphate (ATP), whose hydrolysis boosts the H+ ion concentration and thereby decreases the intracellular pH (England et al., 2016England, E.M.; Matarneh, S.K.; Oliver, E.M.; Apaoblaza, A.; Scheffler, T.L.; Shi, H.; Gerrard, D.E. 2016. Excess glycogen does not resolve high ultimate pH of oxidative muscle. Meat Science 114: 95–102.; Scheffler et al., 2015Scheffler, T.L.; Matarneh, S.K.; England, E.M.; Gerrard, D.E. 2015. Mitochondria influence postmortem metabolism and pH in an in vitro model. Meat Science 110: 118–125.).

The pH drop profile does not present continuous behavior, but this profile passes through phases of transient pH stability until reaching the pHu (Ouali et al., 2006Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58.). Ouali et al. (2006)Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58. observed the gradual pH drop in two 19–month–old Charolais bulls’ longissimus muscle over the 0 – 10 h post–slaughter period. The authors associated these pH drop profiles with the phospholipid–dependent inversion of polarity in cellular membranes. This phenomenon involves the electronegative phosphatidylserine groups switching to the external leaflet of the membrane, while the electropositive phosphatidylcholine and phosphatidylethanolamine groups change to the internal leaflet when apoptosis occurs in muscle cells. This switching causes transient partial neutralization of protons formed from glucose by glycolysis (Ouali et al., 2006Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58.).

Muscle glycolytic potential is the molar sum of all substrates of glycolysis (glycogen, glucose and lactate). Many researchers have demonstrated the negative correlation between the glycolytic potential and pHu (Holdstock et al., 2014Holdstock, J.; Aalhus, J.L.; Uttaro, B.A.; López–Campos, O.; Larsen, I.L.; Bruce H.L. 2014. The impact of ultimate pH on muscle characteristics and sensory attributes of the longissimus thoracis within the dark cutting (Canada B4) beef carcass grade. Meat Science 98: 842–849.; McKeith et al., 2016McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.; Wulf et al., 2002Wulf, D.M.; Emnett, R.S.; Leheska, J.M.; Moeller, S.J. 2002. Relationships among glycolytic potential, dark cutting (dark, firm, and dry) beef, and cooked beef palatability. Journal of Animal Science 80: 1895–1903.). However, the type of fiber in the muscle may be a major determinant, as described in section 2.2.

Biological and environmental effects on pHu dropping

Several pre–harvest elements may determine the extension of pH drop. Physiological factors include the types of the myofiber and the muscle, as well as the testosterone level (Fink et al., 2018Fink, J.; Schoenfeld, B.J.; Nakazato, K. 2018. The role of hormones in muscle hypertrophy. Physician and Sportsmedicine 46: 129–134.). Environmental factors involve the type of finishing, diet, and pre–slaughter stress, such as physical exercise, inadequate handling, and fighting between animals immediately prior to slaughter (Dunne et al., 2011Dunne, P.G.; Monahan, F.J.; Moloney, A.P. 2011. Current perspectives on the darker beef often reported from extensively-managed cattle: does physical activity play a significant role? Livestock Science 142: 1–22, 2011.; Ponnampalam et al., 2017Ponnampalam, E.N.; Hopkins, D.L.; Bruce, H.; Li, D.; Baldi, G.; Bekhit, A.E.D. 2017. Causes and contributing factors to “dark cutting” meat: current trends and future directions; a review. Comprehensive Reviews in Food Science and Food Safety 16: 400–430.).

Ultimate pH may vary according to the type of fiber that comprises a muscle. As stated by Patten et al. (2008)Patten, L.E.; Hodgen, J.M.; Stelzleni, A.M.; Calkins, C.R.; Johnson, D.D.; Gwartney, B.L. 2008. Chemical properties of cow and beef muscles: benchmarking the differences and similarities. Journal of Animal Science 86: 1904-1916. and England et al. (2014)England, E.M.; Matarneh, S.K.; Scheffler, T.L.; Wachet, C.; Gerrard, D.E. 2014. pH inactivation of phosphofructokinase arrests postmortem glycolysis. Meat Science 98: 850–857., glycolytic fibers, such as type IIB, present a fast glycolysis rate, which is able to produce a high concentration of ATP – substrate to H+ production. Greater concentration of glycogen on glycolytic fibers favors the acidification that provides sufficient amount of substrate. Therefore, in glycolytic muscles, the pH reduction is inversely correlated to the initial concentration of glycogen in the muscle (Lawrence et al., 2012Lawrence, L.J.; Fowler, V.R.; Novakofski, J.E. 2012. Growth of Farm Animals. 3ed. CABI, Wallingford, UK.).

Ultimate pH closer to the typical range (5.4 – 5.8) inhibits the activity of the glycolysis–regulatory enzyme phosphofructokinase, which ends the metabolic process, and then the pH drop in glycolytic fibers (England et al., 2014England, E.M.; Matarneh, S.K.; Scheffler, T.L.; Wachet, C.; Gerrard, D.E. 2014. pH inactivation of phosphofructokinase arrests postmortem glycolysis. Meat Science 98: 850–857.). Thus, glycolysis is active until the medium reaches an acidic pHu. Conversely, oxidative fibers, such as type I, seem to have a shorter glycolysis pathway before reaching the necessary pH to inhibit the phosphofructokinase activity. Higher pHu in oxidative fibers may occur under two conditions: reduced glycogen content in the cell with low final ATP production, and a slow rate of glycolysis, which does not produce H+ ions given the extent to which the enzyme is inhibited at pH close to 5.8, even when there is an excess of glycogen (England et al., 2016England, E.M.; Matarneh, S.K.; Oliver, E.M.; Apaoblaza, A.; Scheffler, T.L.; Shi, H.; Gerrard, D.E. 2016. Excess glycogen does not resolve high ultimate pH of oxidative muscle. Meat Science 114: 95–102.).

As reviewed by Seideman et al. (1982)Seideman, S.C.; Cross, H.R.; Oltjen, R.R.; Schanbacher, B.D. 1982. Utilization of the intact male for red meat production: a review. Journal of Animal Science 55: 826–840. and reported by Wȩglarz (2010)Wȩglarz, A. 2010. Meat quality defined based on pH and colour depending on cattle category and slaughter season. Czech Journal of Animal Science 55: 548–556., bulls have been classically known to produce beef with higher pHu. The bull’s excitable temperament encompasses both aggressive and sexual activities, and it is linked to the testosterone level (Seideman et al., 1982Seideman, S.C.; Cross, H.R.; Oltjen, R.R.; Schanbacher, B.D. 1982. Utilization of the intact male for red meat production: a review. Journal of Animal Science 55: 826–840.). Testosterone stimulates the animal, which depletes the muscle content of glycogen prior to slaughter, thus interrupting the proper muscle acidification.

On the other hand, reports have shown that different categories of animal temperament do not result in different pHu values (King et al., 2006King, D.A.; Schuehle P.C.E.; Randel, R.D.; Welsh, T.H.Jr.; Oliphint, R.A.; Baird, B.E.; Curley Jr., K.O.; Vann, R.C.; Hale, D.S.; Savell, J.W. 2006. Influence of animal temperament and stress responsiveness on the carcass quality and beef tenderness of feedlot cattle. Meat Science 74: 546–556.). However, the degree of pre–slaughter stress should be taken into account because when stressful conditions are minimized or avoided, bulls do not produce elevated pHu. Even though the longissimus dorsi muscles from bulls have appeared darker compared to steers, DeVol et al. (1985)DeVol, D.L.; Vanderwert, W.; Bechtel, P.J.; McKeith, F.K. 1985. Comparison of M. longissimus dorsi pigment concentration from implanted and control Angus and Limousin bulls and steers. Meat Science 14: 165–173. found no correlation between the excitatory effect of testosterone on muscle color because there was no pHu >5.8 (5.66 for bulls and 5.58 for steers) nor any correlation between these pHu values. Therefore, this result was associated with pre–slaughter stress.

Extensive exercise and/or grass feeding is reported to enhance significant changes in muscle fiber composition, as these factors increase the proportion of the oxidative fibers (MyHC–IIa) at the expense of the glycolytic type (MyHC–IIx) (Gagaoua et al., 2017Gagaoua, M.; Monteils, V.; Couvreur, S.; Picard, B. 2017. Identification of biomarkers associated with the rearing practices, carcass characteristics, and beef quality: an integrative approach. Journal of Agricultural and Food Chemistry 65: 8264-8278.). As previously described, oxidative fibers have a slower rate and limited extent of pH drop and, subsequently, greater production of dark meat. Although the high pHu after pasture has been associated with lower intracellular glycogen content and decreased glycolytic potential the shift towards oxidative fibers reveals that the lower energetic potential is not the preponderant factor in dropping the pH (Apaoblaza et al., 2020Apaoblaza, A.; Gerrard, S.D.; Matarneh, S.K.; Wicks, J.C.; Kirkpatrick, L.; England, E.M.; Scheffler, T.L.; Duckett, S.K.; Shia, H.; Silva, S.L.; Grant, A.L.; Gerrard, D.E. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Science 161: 107996.; Picard and Gagaoua, 2020Picard, B.; Gagaoua, M. 2020. Muscle fiber properties in cattle and their relationships with meat qualities: an overview. Journal of Agricultural and Food Chemistry 68: 6021-6039.).

The nutrient restriction seems to increase the proportion of oxidative fiber type in the muscle and deplete the intracellular glycogen concentration (Bray et al., 1989Bray, A.R.; Graafhuis, A.E.; Chrystall, B.B. 1989. The cumulative effect of nutritional, shearing and pre-slaughter washing stresses on the quality of lamb meat. Meat Science 25: 59–67.; Kandeepan et al., 2009Kandeepan, G.; Anjaneyulu, A.S.R.; Rao, V.K.; Pal, U.K.; Mondal, P.K.; Das, C.K. 2009. Feeding regimens affecting meat quality characteristics. MESO 11: 241-249.). Both conditions lead to less H+ ions produced during the rigor mortis process (i.e., high pHu meat).

Understanding how pre–slaughter circumstances impact on the achievement of muscles with normal pHu and the economic consequences of postmortem biochemical reactions is a means of fomenting good animal welfare and ethical issues.

Animal stress may be avoided by a handler’s careful perception of animal behavior prior to and/or during slaughter, especially if there are signs of fear, pain, and distress (Grandin, 2010Grandin, T. 2010. Auditing animal welfare at slaughter plants. Meat Science 86: 56-65., 2019Grandin, T. 2019. Slaughter plants: behavior and welfare assessment. p. 153-162. In: Choe, J.C., ed. Encyclopedia of animal behavior. 2ed. Academic Press, San Diego, CA, USA.). According to Bomzon (2011)Bomzon, A. 2011. Pain and stress in cattle: a personal perspective. Israel Journal of Veterinary Medicine 66: 12–20., cattle may disguise signs of pain so as not to demonstrate weakness in front of a possible predator. However, changes in cattle mobility, behavior and/or appearance may indicate poor welfare, such as lameness, falling during handling, vocalization, apparent sclera (the white of the eye), struggling, agitation and slipping in the stunning box, and body lesion (Bomzon, 2011Bomzon, A. 2011. Pain and stress in cattle: a personal perspective. Israel Journal of Veterinary Medicine 66: 12–20.; Grandin, 2010Grandin, T. 2010. Auditing animal welfare at slaughter plants. Meat Science 86: 56-65., 2019Grandin, T. 2019. Slaughter plants: behavior and welfare assessment. p. 153-162. In: Choe, J.C., ed. Encyclopedia of animal behavior. 2ed. Academic Press, San Diego, CA, USA.).

Vocalization above a certain level is a clear indicator of pain or stress and can be perceived by the handler while moving cattle or holding them restrained during slaughter. However, certain bulls in a herd, normally vocalize at the lairage even in the absence of stressful conditions. Vocalizing animals show increased blood concentration of the stress biomarker cortisol, lactate, and glucose, indicating glycogenolysis (Grandin, 2019Grandin, T. 2019. Slaughter plants: behavior and welfare assessment. p. 153-162. In: Choe, J.C., ed. Encyclopedia of animal behavior. 2ed. Academic Press, San Diego, CA, USA.). The neuropeptide P level also increases when painful procedures are inflicted (Coitzee et al., 2008Coitzee, J.F.; Lubbers, B.V.; Toerber, S.E.; Gehring, R.; Thomson, D.U.; White, B.J.; Apley, M.D. 2008. Plasma concentration of substance P and cortisol in beef calves after castration and simulated castration. American Journal of Veterinary Research 69: 751−752.).

The frequent and gentle animal handling on the farm as well as the previous experience of moving into a veterinary restraint encourage cattle to become accustomed to the presence of humans and to walking to the slaughter box at the abattoir (Probst et al, 2013; Grandin, 2019Grandin, T. 2019. Slaughter plants: behavior and welfare assessment. p. 153-162. In: Choe, J.C., ed. Encyclopedia of animal behavior. 2ed. Academic Press, San Diego, CA, USA.). When this approach is adopted, animals can be cajoled into a calmer and fearless behavior right up to the slaughter procedure itself.

Ultimate pH ranges

Meat pHu ranges from 5.3 to over 7.0, as reported by several authors (Abril et al., 2001Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78.; Contreras–Castillo et al., 2016Contreras–Castillo, C.J.; Lomiwes, D.; Wu, G.; Frost, D.; Farouk, M.M. 2016. The effect of electrical stimulation on post mortem myofibrillar protein degradation and small heat shock protein kinetics in bull beef. Meat Science 113: 65–72.; Hunt and Hedrick, 1977Hunt, M.C.; Hedrick, H.B. 1977. Chemical, physical and sensory characteristics of bovine muscle from four quality groups. Journal of Food Science 42: 716–720.; Lawrie, 1958Lawrie, R.A. 1958. Physiological stress in relation to dark-cutting beef. Journal of the Science of Food and Agriculture 9: 721–727.; McKeith et al., 2016McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.; Pulford et al., 2008Pulford, D.J.; Vazquez, S.F.; Frost, D.F.; Fraser–Smith, E.; Dobbie, P.; Rosenvold, K. 2008. The intracellular distribution of small heat shock proteins in post-mortem beef is determined by ultimate pH. Meat Science 79: 623–630.). Nevertheless, grouping pHu into ranges is a challenging task and there is no universal consensus, which results in several classifications and cut–off values.

Studies have focused on the meat tenderness of longissimus muscles, and a number of authors have grouped pHu into three ranges: normal (< 5.8) intermediate (5.9 – 6.2) and high (> 6.20) (Lomiwes et al., 2014Lomiwes, D.; Hurst, SM.; Dobbie, P.; Frost, DA.; Hurst, R.D.; Young, O.A.; Farouk, M.M. 2014. The protection of bovine skeletal myofibrils from proteolytic damage post mortem by small heat shock proteins. Meat Science 97: 548–557.; Wu et al., 2014, Wu, G; Farouk, M.M.; Clerens, S.; Rosenvold, K. 2014. Effect of beef ultimate pH and large structural protein changes with aging on meat tenderness. Meat Science 98: 637–645., Contreras–Castillo et al., 2016)Contreras–Castillo, C.J.; Lomiwes, D.; Wu, G.; Frost, D.; Farouk, M.M. 2016. The effect of electrical stimulation on post mortem myofibrillar protein degradation and small heat shock protein kinetics in bull beef. Meat Science 113: 65–72.. This can be attributable to the meat tenderness observed in the three groups, in which the intermediate range is tougher than the other ones (Lomiwes, et al., 2013; Pulford et al., 2008)Pulford, D.J.; Vazquez, S.F.; Frost, D.F.; Fraser–Smith, E.; Dobbie, P.; Rosenvold, K. 2008. The intracellular distribution of small heat shock proteins in post-mortem beef is determined by ultimate pH. Meat Science 79: 623–630.. However, these ranges which assess meat tenderness cannot be applied to meat color studies.

As regards meat color, different classifications are proposed to group beef muscles according to their values of pHu. For instance, Viljoen et al. (2002)Viljoen, H.F.; De Kock, H.L.; Webb, E.C. 2002. Consumer acceptability of dark, firm and dry (DFD) and normal pH beef steaks. Meat Science 61: 181–185. and Park et al. (2007)Park, B.Y.; Lee, J.M.; Hwang, I.H. 2007. Effect of postmortem metabolic rate on meat color. Asian-Australasian Journal of Animal Science 20: 598–604. considered pHu > 5.8 as a single group to be studied. On the other hand, McKeith et al. (2016)McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173. split high pHu carcasses into four classes for longissimus thoracis muscles, according to their dark color: shady (pHu: 6.1 ± 0.03), mild (6.4 ± 0.03), moderate (6.6 ± 0.03), and severe (6.9 ± 0.03). Holdstock et al. (2014)Holdstock, J.; Aalhus, J.L.; Uttaro, B.A.; López–Campos, O.; Larsen, I.L.; Bruce H.L. 2014. The impact of ultimate pH on muscle characteristics and sensory attributes of the longissimus thoracis within the dark cutting (Canada B4) beef carcass grade. Meat Science 98: 842–849. used pHu 6.0 as the threshold for classifying beef muscles as normal (pHu < 6.0) and typically dark (pHu > 6.0).

One approach to reducing the human effect on pHu range classification is based on cluster analysis (Abril et al., 2001Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78.). This statistical technique involves analyzing the experimental data collected on the basis of their proximity – forming a group of samples with similar characteristics. This tool can show that data from the parameters assessed from three pHu muscle ranges can behave as two groups of pHu, which increases the credibility of data analysis. Abril et al. (2001)Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78. applied cluster analysis to all the color coordinates and divided the samples into two groups according to their pHu: pHu < 6.1 and pHu > 6.1. This difference was also observed in the reflectance spectra.

Dark–cutting beef and color stability

A typical denomination for beef darkening is dark–cutting beef. Certain authors have also referred it as DFD beef (Dark, Firm and Dry). Since the term firm might be interpreted as tough, it is important to note that not every high pHu muscle is tough. Because of enzymatic action by calpain proteases, meats with high pHu (especially above 6.3) can be as tender as those with normal pHu (Lomiwes et al., 2013Lomiwes, D.; Farouk, M.M.; Frost, D.A.; Dobbie, P.M.; Young, O.A. 2013. Small heat shock proteins and toughness in intermediate pHu beef. Meat Science 95: 472–479.). Therefore, the “dark–cutting” term seems more appropriate for studies focused on color.

Researchers have reported that pHu > 5.8 may result in dark meat (Abril et al., 2001Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78.; Ashmore et al., 1972Ashmore, C.R.; Parker, W.; Doerr, L. 1972. Respiration of mitochondria isolated from dark-cutting beef: postmortem changes. Journal of Animal Science 34: 46–48.; Holdstock et al., 2014Holdstock, J.; Aalhus, J.L.; Uttaro, B.A.; López–Campos, O.; Larsen, I.L.; Bruce H.L. 2014. The impact of ultimate pH on muscle characteristics and sensory attributes of the longissimus thoracis within the dark cutting (Canada B4) beef carcass grade. Meat Science 98: 842–849.; Hunt and Hedrick, 1977Hunt, M.C.; Hedrick, H.B. 1977. Chemical, physical and sensory characteristics of bovine muscle from four quality groups. Journal of Food Science 42: 716–720.; Page et al., 2001Page, J.K.; Wulf, D.M.; Schwotzer, T.R. 2001. A survey of beef muscle color and pH. Journal of Animal Science 79: 678–687.; Stackhouse et al., 2016)Stackhouse, R.J.; Apple, J.K.; Yancey, J.W.S.; Keys, C.A.; Johnson, T.M.; Mehall, L.N. 2016. Postrigor citric acid enhancement can alter cooked color but not fresh color of dark-cutting beef. Journal of Animal Science 94: 1738–1754.. Because of the global incidence of dark–cutting beef and its importance to the quality of meat, a broad consideration of the causal and consequential aspects involved may reveal gaps in the literature that require further investigation.

Moore et al. (2012)Moore, M.C.; Gray, G.D.; Hale, D.S.; Kerth, C.R.; Griffin, D.B.; Savell, J.W.; Raines, C.R.; Belk, K.E.; Woerner, D.R.; Tatum, J.D.; Igo, J.L.; Van Overbeke, D.L.; Mafi, G.G.; Lawrence, T.E.; Delmore, R.J. Jr; Christensen, L.M.; Shackelford, S.D.; King, D.A.; Wheeler, T.L.; Meadows, L.R.; O’Connor, M.E. 2012. National Beef Quality Audit–2011: In-plant survey of targeted carcass characteristics related to quality, quantity, value, and marketing of fed steers and heifers. Journal of Animal Science 90: 5152–5160. reported that 3.2 % of the carcasses evaluated in the 2011 United States’ National Beef Quality Audit were dark. The Beef Cattle Research Council – BCRC (BCRC, 2018) reported the incidence of dark cuts in Canada has been increasing: 0.84 % in 1998/99, 1.28 % in 2010/11, and 1.64 % in 2016/17. In relation to the incidence of pHu higher than 5.8 in the Brazilian beef industry, Rosa et al. (2016)Rosa, A.; Fonseca, R.; Balieiro, J.C.; Poletid, M.D.; Domenech–Péreze, K.; Farnetanib, B.; Elera, J. 2016. Incidence of DFD meat on Brazilian beef cuts. Meat Science 112: 132-136. studied 485 F1 crossbred animals (Nellore × South African Simmental) and according to the data, 4.53 % of the animals (n = 22) presented pH > 5.8 and the meat color was darker than that of animals with normal pHu. Although the percentage was not so high, given the volume of beef carcasses marketed worldwide, a reduced percentage of the occurrence of undesirable color represents serious loss for the meat industry, as stated by Hughes et al. (2014a).

As reported by Page et al. (2001)Page, J.K.; Wulf, D.M.; Schwotzer, T.R. 2001. A survey of beef muscle color and pH. Journal of Animal Science 79: 678–687., dark–cutting beef is highly correlated with pHu (r = 0.80). Authors showed that 91.7 % of the muscles with pHu ≥ 5.87 were classified as dark cutters, following USDA grading standards, whereas in muscles with normal pHu, the proportion was 0.6 %. Nonetheless, an atypical darkening may occur in normal pHu muscles. Holdstock et al. (2014)Holdstock, J.; Aalhus, J.L.; Uttaro, B.A.; López–Campos, O.; Larsen, I.L.; Bruce H.L. 2014. The impact of ultimate pH on muscle characteristics and sensory attributes of the longissimus thoracis within the dark cutting (Canada B4) beef carcass grade. Meat Science 98: 842–849. reported three groups of longissimus thoracis beef muscles: a) Canada AA carcass muscles (Japanese Meat Grading Association – JMGA – with a score of 3.50 and muscle pH of 5.57, n = 10); b) Canada B4 carcass muscles, grouped into 1) atypical dark cutters (JMGA of 6.80 and muscle pH of 5.83, n = 10), and 2) typical dark cutters (JMGA of 7.75 and muscle pH of 6.62, n = 10). JMGA has a range from 1 (lightest color) to 8 (dark cutter). Both AA muscles and atypical dark cutter presented high glycosidic potential, which facilitates a proper pH drop to normal pHu. Darkening can be associated with both physical (Jerez–Timaure et al., 2019Jerez–Timaure, N.; Gallo, C.; Ramírez–Reveco, A.; Greif, G.; Strobel, P.; Pedro, A.V.F.; Morera, F.J. 2019. Early differential gene expression in beef Longissimus thoracis muscles from carcasses with normal (<5.8) and high (>5.9) ultimate pH. Meat Science 153: 117-125.) and biochemical effects and/or reactions (Wills et al., 2017Wills, K.M.; Mitacek, R.M.; Mafi, G.G.; Vanoverbeke, D.L.; Jaroni, D.; Jadeja, R.; Ramanathan, R. 2017. Improving the lean muscle color of dark-cutting beef by aging, antioxidant-enhancement, and modified atmospheric packaging. Journal of Animal Science 95: 5378-5387.).

Effects of dark–cutting on the beef physical properties

Meat color perceived by the consumer is the result of the combination of the light reflected and absorbed by or scattered on the surface of the meat. Redness attributes tend to be strongly associated with the Mb pigment, while brightness (light intensity) is related to the structural attributes of the muscle, which together determine the light reflected (Hughes et al., 2014b). Ultimate pH is one of the most important intrinsic factors in myofibril structure (Abril et al., 2001Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78.), which is demonstrated by the expressive correlation between the increase in muscle pH and extracellular space (Ouali et al., 2006Ouali, A.; Herrera–Mendez, C.H.; Coulis, G.; Becila, S.; Boudjellal, A.; Aubry, L.; Sentandreu, M.A. 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74: 44–58.).

Dark–cutting beef with high pHu undergoes minimal shrinkage in the myofibril structure. This alteration in the meat microstructure leads to an increase in hydration capacity by muscle proteins, also defined as water holding capacity (WHC). The greater content of water inside the muscle structure reduces the light scattered by the muscle fibers, which makes the meat surface look darker (Hughes et al., 2019Hughes, J.; Clarke, F.; Li, Y.; Purslow, P.; Warner, R. 2019. Differences in light scattering between pale and dark beef longissimus thoracis muscles are primarily caused by differences in the myofilament lattice, myofibril and muscle fibre vtransverse spacings. Meat Science 149: 96-106.).

Myofibrillar structural darkness has a great impact on beef marketability. However, the most significant attribute in purchasing is meat redness (Venturini et al., 2014Venturini, A.C., Faria, J.A.F., Olinda, R.A., Contreras–Castillo, C.J., 2014. Shelf life of fresh beef stored in master packages with carbon monoxide and high levels of carbon dioxide. Packaging Technology and Science 27: 29-35.). The intensity of redness is dependent on the presence, concentration and chemical state of the central pigment of beef tissue: myoglobin (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.).

Influencing factors on myoglobin concentration in dark–cutting development

Similar to the determination of the pHu value, Mb concentration may be affected by both intrinsic (biological) and extrinsic elements. Endogenous components comprise bovine genetics, animal age, testosterone concentration, muscle type, and fiber involved. A noteworthy environmental influence on beef Mb concentration is the type of cattle, which dictates the frequency and extent of physical exercise.

King et al. (2010)King, D.A.; Shackelford, S.D.; Kuehn, L.A.; Kemp, C.M., Rodriguez; A.B., Thallman, R.M.; Wheeler, T.L. 2010. Contribution of genetic influences to animal-to-animal variation in myoglobin content and beef lean color stability. Journal of Animal Science 88: 1160–1167. demonstrated that genetic plays a role in Mb content in longissimus thoracis muscles. Seven steer breeds were investigated: Angus, Charolais, Gelbvie, Hereford, Limousin, Red Angus, and Simmental. Simmental showed the greatest concentration of Mb (3.71 mg g1), whereas Charolais and Limousin had significantly lower Mb values (2.77 and 2.72 mg g1, respectively).

DeVol et al. (1985)DeVol, D.L.; Vanderwert, W.; Bechtel, P.J.; McKeith, F.K. 1985. Comparison of M. longissimus dorsi pigment concentration from implanted and control Angus and Limousin bulls and steers. Meat Science 14: 165–173. demonstrated the effect of age on the darkening of longissimus dorsi muscles from bulls and steers from Limousin and Angus genotypes. The authors reported that the muscles of bulls were visually darker (p < 0.01) with 3.25 mg g1of Mb content in fresh muscle, whereas the muscles of steers had 2.90 mg g1of Mb (p < 0.01). As animals get older, the amount of Mb increases but the affinity of O2 for Mb decreases; thus, they need to synthesize more Mb to stock O2. This explains why meat from older animals looks darker than meat from younger animals.

Although stress can significantly trigger the dark–cutting, the high proportion of oxidative fiber types within muscles is the main factor that determines the susceptibility of animals to triggering this effect (Ponnampalam et al., 2017Ponnampalam, E.N.; Hopkins, D.L.; Bruce, H.; Li, D.; Baldi, G.; Bekhit, A.E.D. 2017. Causes and contributing factors to “dark cutting” meat: current trends and future directions; a review. Comprehensive Reviews in Food Science and Food Safety 16: 400–430.). Based on the activity of myosin ATPase, fiber types within muscles can be classified as red, intermediate, and white fibers (Voisinet et al., 1997Voisinet, B.D., Grandin, T., O’Connor, S.F., Tatum, J.D., Deesing, M.J., 1997. Bos indicus-cross feedlot cattle with excitable temperaments have tougher meat and a higher incidence of borderline dark cutters. Meat Science 46: 367-377.).

Red fibers, involved in oxidative metabolism, have higher Mb contents and capillary blood flow than white ones, which maintain the O2 supply to cells. These two characteristics allow red fiber to oxidize glucose via aerobic pathways, which is corroborated by the larger number of mitochondria and tricarboxylic acid enzymes in muscle cells (Choi and Kim, 2009Choi, Y.M.; Kim, B.C. 2009. Muscle fiber characteristics, myofibrillar protein isoforms, and meat quality. Livestock Science 122: 105–118.). Moreover, the increased content of Mb in oxidative fiber has been correlated with higher cytochrome c oxidase expression, which is present in the mitochondria and discernibly impacts on color stability (Apaoblaza et al., 2020Apaoblaza, A.; Gerrard, S.D.; Matarneh, S.K.; Wicks, J.C.; Kirkpatrick, L.; England, E.M.; Scheffler, T.L.; Duckett, S.K.; Shia, H.; Silva, S.L.; Grant, A.L.; Gerrard, D.E. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Science 161: 107996.).

Intermediate fibers have an oxidative–glycolytic metabolism and are intermediate between red and white fibers which have a glycolytic metabolism (Choi and Kim, 2009Choi, Y.M.; Kim, B.C. 2009. Muscle fiber characteristics, myofibrillar protein isoforms, and meat quality. Livestock Science 122: 105–118.; Zerouala and Stickland, 1991Zerouala, A.C.; Stickland, N.C. 1991. Cattle at risk for dark-cutting beef have a higher proportion of oxidative muscle fibres. Meat Science 29: 263–270.). In other words, it is the ratio of red and intermediate fibers to white fibers that matters: muscles with high oxidative metabolic activity, where there is a predominance of β and α–red fibers, have a high risk of dark–cutting because their fibers tend to have greater affinity with circulating adrenaline which depletes glycogen more easily with the same level of activity or stress compared to an animal with α–white muscle fibers (McGilchrist et al., 2012McGilchrist, P.; Alston, C.L.; Gardner, G.E.; Thomson, K.L.; Pethick, D.W. 2012. Beef carcasses with larger eye muscle areas, lower ossification scores and improved nutrition have a lower incidence of dark cutting. Meat Science 92: 474-480.).

White fibers have a glycolytic metabolism and the least Mb concentration compared to the other two fibers because of the reduced O2 demand by cells. The lower abundance is related to the reduced stress undergone by glycolytic muscles, which are used for fast bursts of energy (Choi and Kim, 2009Choi, Y.M.; Kim, B.C. 2009. Muscle fiber characteristics, myofibrillar protein isoforms, and meat quality. Livestock Science 122: 105–118.; Wicks et al., 2019Wicks, J.; Beline, M.; Gomez, J.F.M.; Luzardo, S.; Silva, S.L.; Gerrard, D. 2019. Muscle energy metabolism, growth, and meat quality in beef cattle. Agriculture 9: 1–10.).

In general, muscles with high oxidative metabolic activity, where there is more than 40 % of slow oxidative red fibers, such as psoas major muscles, exhibit higher Mb concentration (4.66 ± 0.31 mg g1), higher content of mitochondria and thus more intense respiration, and are characterized as muscles with less color stability. In contrast, muscles comprising more than 40 % of fast glycolytic white fibers, such as longissimus lumborum, have lower Mb concentration (3.97 ± 0.12 mg g1), lower concentration of mitochondria and thus high color stability (Kirchofer et al., 2002Kirchofer, K.S.; Calkins, C.B.; Gwartney, B.L. 2002. Fiber-type composition of muscles of the beef chuck and round. Journal of Animal Science 80: 2872-2878.; Salim et al., 2019Salim, A.P.A.A.; Suman, S.P.; Canto, A.C.V.C.S.; Costa–Lima, B.R.C.; Viana, F.M.; Monteiro, M.L.G.; Silva, T.J.P.; Conte–Junior, C.A. 2019. Muscle-specific color stability in fresh beef from grain-finished Bos indicus cattle. Asian-Australasian Journal of Animal Science 32: 1036–1043.).

Endogenous testosterone and steroidal compounds, such as trenbolone acetate, supplemented in diet or implanted in the animal trigger the growth of muscle fibers (Johnson and Chung, 2007Johnson, B.J.; Chung, K.Y. 2007. Alterations in the physiology of growth of cattle with growth-enhancing compounds. Veterinary Clinics of North America - Food Animal Practice 23: 321–332.). Fink et al. (2018)Fink, J.; Schoenfeld, B.J.; Nakazato, K. 2018. The role of hormones in muscle hypertrophy. Physician and Sportsmedicine 46: 129–134. reported larger fiber cross–sectional area in bull muscles than in neutered males and females. Moreover, testosterone may also stimulate the prenatal enlargement and proliferation of the muscle cells, although postnatal cell formation is inhibited (Johnson and Chung, 2007Johnson, B.J.; Chung, K.Y. 2007. Alterations in the physiology of growth of cattle with growth-enhancing compounds. Veterinary Clinics of North America - Food Animal Practice 23: 321–332.; Wicks et al., 2019Wicks, J.; Beline, M.; Gomez, J.F.M.; Luzardo, S.; Silva, S.L.; Gerrard, D. 2019. Muscle energy metabolism, growth, and meat quality in beef cattle. Agriculture 9: 1–10.).

Nevertheless, hypertrophic pressure enlarges preferentially type IIB glycolytic fibers rather than type I oxidative fibers. Therefore, anabolic hormones and their analogues tend to generate muscles proportionally more glycolytic and thus lighter because of the lower Mb content (Wicks et al., 2019Wicks, J.; Beline, M.; Gomez, J.F.M.; Luzardo, S.; Silva, S.L.; Gerrard, D. 2019. Muscle energy metabolism, growth, and meat quality in beef cattle. Agriculture 9: 1–10.). Vaughn et al. (2019)Vaughn, M.A.; Lancaster, P.A.; Roden, K.C.; Sharman, E.D.; Krehbiel, C.R.; Horn, G.W.; Starkey, J.D. 2019. Effect of stocker management program on beef cattle skeletal muscle growth characteristics, satellite cell activity, and paracrine signaling impact on preadipocyte differentiation. Journal of Animal Science and Technology 61: 260-271. found the growth of the glycolytic muscle longissimus was related to the extent of the proliferative capacity of satellite cells, which are muscle stem cells that continue to induce postnatal hypertrophy in myofibrils. Therefore, the darker color attributable to non–castrated cattle is likely related to the high pHu–dependent change in muscle microstructure rather than higher intracellular Mb concentration.

The shifting in the muscle fibers stimulated towards greater proportion in response to pasture–finishing boosts intracellular Mb concentration as a consequence (Dunne et al., 2011Dunne, P.G.; Monahan, F.J.; Moloney, A.P. 2011. Current perspectives on the darker beef often reported from extensively-managed cattle: does physical activity play a significant role? Livestock Science 142: 1–22, 2011.; Picard and Gagaoua, 2020Picard, B.; Gagaoua, M. 2020. Muscle fiber properties in cattle and their relationships with meat qualities: an overview. Journal of Agricultural and Food Chemistry 68: 6021-6039.). This conversion is also stimulated by long–term physical exercise, as observed by Dunne et al. (2005)Dunne, P.G.; O’Mara, F.P.; Monahan, F.J.; French, P.; Moloney, A.P. 2005. Colour of muscle from 18-month-old steers given long-term daily exercise. Meat Science 71: 219–229. as well as by the grass–fed nutritional plane (Apaoblaza et al., 2020Apaoblaza, A.; Gerrard, S.D.; Matarneh, S.K.; Wicks, J.C.; Kirkpatrick, L.; England, E.M.; Scheffler, T.L.; Duckett, S.K.; Shia, H.; Silva, S.L.; Grant, A.L.; Gerrard, D.E. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Science 161: 107996.; Wicks et al. 2019Wicks, J.; Beline, M.; Gomez, J.F.M.; Luzardo, S.; Silva, S.L.; Gerrard, D. 2019. Muscle energy metabolism, growth, and meat quality in beef cattle. Agriculture 9: 1–10.). The resultant increase in Mb content is a response to the high demand of O2 by the mitochondrial oxidative metabolism. As observed by Apaoblaza et al. (2020)Apaoblaza, A.; Gerrard, S.D.; Matarneh, S.K.; Wicks, J.C.; Kirkpatrick, L.; England, E.M.; Scheffler, T.L.; Duckett, S.K.; Shia, H.; Silva, S.L.; Grant, A.L.; Gerrard, D.E. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Science 161: 107996., even glycolytic muscles such as longissimus dorsi, show greater Mb concentration after grass feeding compared to grain feeding, with significant darkening (lower instrumental lightness – L* by Commission Internationale de l’Eclairage (CIE) in the longissimus muscle).

Myoglobin structure and ligands

Structurally, Mb is composed of a polypeptide chain and a group of Fe2 protoporphyrin called heme. Heme consists of a tetrapyrrolic ring with conjugated double bonds, which binds coordinately to an Fe2 (American Meat Science Association –– AMSA, 2012). The resonant electronic distribution of these conjugated bonds can absorb visible light and enhance the Mb color. Hematinic iron forms six–coordinate bonds. Four of these bonds have heme pyrrole nitrogen molecules in the same plane. The fifth coordination involves the interaction between iron and proximal histidine, located at position 93 (His93) (Møller and Skibsted, 2006Møller, J.K.S.; Skibsted, L.H. 2006. Myoglobins: the link between discoloration and lipid oxidation in muscle and meat. Quimica Nova 29: 1270-1278.). The sixth site of heme is available for reversible binding with a small ligand, such as O2, nitric oxide (NO) and carbon monoxide (CO). Fifth and sixth bonds are perpendicular to the heme ring. Hematinic iron may bind to O2 as a result of Mb’s hydrophobic pocket, which is proper to the heme location. Inside this pocket, the accessibility of the heme group to solvent is highly restricted, which protects iron from oxidation. Bound to iron, oxygen forms a hydrogen bond to distal histidine (His64), which improves its bond (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.).

The three states of Mb present in the fresh muscle interchange simultaneously under natural conditions – in the retail store or at home, and the conversion takes place in dynamic equilibrium. A higher proportion of one pigment is dependent on intrinsic and environmental factors, such as meat pHu, MRA, lipid oxidation, gas composition, temperature, and microbial growth (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.).

Biochemical basis of color stability

The superficial dynamic equilibrium between DMb, OMb, and MMb over a period of time defines muscle color stability, which can be impaired depending on internal and external conditions favoring the prevalence of one form on the muscle surface (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.).

Functionality of mitochondria plays a critical role in the intensity and stability of color from the postmortem aging muscle (Ma et al., 2017Ma, D.; Kim, Y.H.B.; Cooper, B.R.; Oh, J.H.; Chun, H.; Choe, J.; Schoonmaker, J.P.; Ajuwon, K.M.; Min, B. 2017. Metabolomics profiling to determine the effect of postmortem aging on color and lipid oxidative stabilities of different bovine muscles. Journal of Agricultural and Food Chemistry 65: 6708-6716.). Respiratory consumption of O2 generates reactive oxygen species (ROS) as by-products. Lipids and proteins are the main targets and are involved in the oxidation of Fe2–Mb to Fe3–Mb and thus increase muscle MMb content (Tang et al., 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.). Nevertheless, the mitochondria also possess the ability to convert MMb back to DMb (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.).

High OCR in the initial periods of the postmortem is deleterious to the development of the surface bright red color since mitochondrial respiration outcompetes Mb for O2 and ultimately results in dark colored muscle (Suman et al., 2014Suman, S.P.; Hunt, M.C.; Nair, M.N.; Rentfrow, G. 2014. Improving beef color stability: practical strategies and underlying mechanisms. Meat Science 98: 490–504.). Over time, there is an improvement in color development because of the decrease in OCR. The depletion of substrates for OC, such as lactate, succinate, and the reduced state of nicotinamide adenine dinucleotide (NADH), during storage time makes O2 diffuse more rapidly through the tissue in order to bind to Mb, which improves color development and stability (MacDougall, 1982MacDougall, D.B. 1982. Changes in the colour and opacity of meat. Food Chemistry 9: 75–88.; Mancini and Ramanathan, 2014Mancini, R.A.; Ramanathan, R. 2014. Effects of postmortem storage time on color and mitochondria in beef. Meat Science 98: 65–70.).

The improvement in the superficial color of meat aged in the short–term can be attributed to a decrease in the OCR of substrates and enzyme activity in the mitochondria (Ramanathan et al., 2013Ramanathan, R.; Mancini, R.A.; Joseph, P.; Suman, S.P. 2013. Bovine mitochondrial oxygen consumption effects on oxymyoglobin in the presence of lactate as a substrate for respiration. Meat Science 93: 893–897.; Seyfert et al., 2006Seyfert, M.; Mancini, R.A.; Hunt, M.C.; Tang, J.; Faustman, C.; Garcia, M. 2006. Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles. Journal of Agricultural and Food Chemistry 54: 8919–8925.). On the other hand, long–term aging reduces the potential for oxygenation (blooming) by decreasing mitochondria–mediated MRA, when aged beef is subsequently displayed under retail light conditions (Mancini and Ramanathan, 2014Mancini, R.A.; Ramanathan, R. 2014. Effects of postmortem storage time on color and mitochondria in beef. Meat Science 98: 65–70.).

Cytochrome c oxidase (complex IV) has been reported to be essential to OCR (Grabež et al., 2015Grabež, V.; Kathri, M.; Phung, V.; Moe, K.M.; Slinde, E.; Skaugen, M.; Saarem, K.; Egelandsdal, B. 2015. Protein expression and oxygen consumption rate of early postmortem mitochondria relate to meat tenderness. Journal of Animal Science 93: 1967–1979.; Seyfert et al., 2006Seyfert, M.; Mancini, R.A.; Hunt, M.C.; Tang, J.; Faustman, C.; Garcia, M. 2006. Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles. Journal of Agricultural and Food Chemistry 54: 8919–8925.). This enzyme is located on the inner mitochondrial membrane and comprises the electron–transport chain along with complexes I (NADH oxidoreductase), II (succinate–Q reductase), and III (coenzyme Q–cytochrome c oxidoreductase) (Arihara et al., 1995Arihara, K.; Cassens, R.G.; Greaser, M.L.; Luchansky, J.B.; 1995. Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle. Meat Science 39: 205–213.).

Under oxidative condition, MMb generation occurs beneath the surface, between the superficial OMb and inner DMb layers. In the intermediate layer, the insufficient availability of O2 to oxygenate all available DMb creates a suitable condition for ROS molecules to initiate O2 oxidation, which can propagate this chain reaction (O’Keeffe and Hood, 1982O’Keeffe, M.; Hood, D.E. 1982. Biochemical factors influencing metmyoglobin formation on beef from muscles of differing colour stability. Meat Science 7: 209–228.). MMb can be formed precisely in the intermediate layer; the subsurface layer of MMb increases and moves towards the surface as the OMb layer on the surface becomes thinner and is replaced by the MMb layer (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.).

Oxidation requires the prior deoxygenation of OMb so that MMb can be obtained. This precondition is due to the high resonant structure of OMb providing it with great oxidative stability, which makes OMb oxidation thermodynamically unfavorable (Faustman and Cassens, 1990Faustman, C.; Cassens, R.G.; 1990. The biochemical basis for discoloration in fresh meat: a review. Journal of Muscle Foods 1: 217–43.). Once formed, MMb can be endogenously reduced to DMb either by enzymatic or non–enzymatic reactions and favors maintenance of ferrous forms of Mb in meat (Faustman et al., 2010Faustman, C.; Sun, Q.; Mancini, R.; Suman, S.P. 2010. Myoglobin and lipid oxidation interactions: mechanistic bases and control. Meat Science 86: 86-94.). However, the enzymatic system is considered prominent.

Metmyoglobin reducing activity is an intrinsic cellular ability that will reduce MMb to DMb, which can be oxygenated to OMb (Ramanathan et al., 2013Ramanathan, R.; Mancini, R.A.; Joseph, P.; Suman, S.P. 2013. Bovine mitochondrial oxygen consumption effects on oxymyoglobin in the presence of lactate as a substrate for respiration. Meat Science 93: 893–897.). Because of this reversible characteristic, MRA seems to be critically important to maintaining or prolonging surface color stability. The increase in MMb proportion throughout retail time implies a decrease in MRA (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.).

Enzymatic MRA involves the NADH–cytochrome b5 MMb reductase enzyme and is based on the transference of two electrons from an NADH–coenzyme to a ferricytochrome b5, thus reducing it to ferrocytochrome b5. As an intermediate, ferricytochrome b5 non–enzymatically reduces MMb to DMb, regenerating the content of oxidized cytochrome b5 and improving meat color (Arihara et al., 1995Arihara, K.; Cassens, R.G.; Greaser, M.L.; Luchansky, J.B.; 1995. Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle. Meat Science 39: 205–213.; Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.; Zhai et al., 2019Zhai, C.; Peckham, K.; Belk, K.E.; Ramanathan, R.; Nair, M.N. 2019. Carbon chain length of lipid oxidation products influence lactate dehydrogenase and NADH-dependent metmyoglobin reductase activity. Journal of Agricultural and Food Chemistry 67: 13327–13332.).

In non–enzymatic MMb reduction, an electron from DMb is transferred to MMb by an artificial electron carrier, such as NADH or NAPDH in the presence of ethylene c acidiaminetetraacetid (EDTA), cytochrome c, methylene blue, ascorbate, uridine diphosphate sugar (UDP–sugar), vitamin E, psychrotrophic bacteria and other systems (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.). In addition to the enzymatic process, non–enzymatic MMb reduction replenishes NADH through the added oxidized form of nicotinamide adenine dinucleotide (NAD+) and Krebs cycle intermediate substrates. However, non–enzymatic processes depend on the O2 intracellular content, which is inhibited under vacuum conditions (Ramanathan and Mancini, 2018Ramanathan, R.; Mancini, R.A. 2018. Role of mitochondria in beef color: a review. Meat and Muscle Biology 2: 309-320.).

Denzer et al. (2020)Denzer, M.; Mowery, C.; Comstock, H.; Maheswarappa, N.B.; Mafi, G.G.; VanOverebeke, D.L.; Ramanathan, R. 2020. Characterization of the cofactors involved in nonenzymatic metmyoglobin/methemoglobin reduction in vitro. Meat and Muscle Biology 4: 1-10. studied how these electron donors and carriers act in non–enzymatic MMb reduction in vitro with equine muscle MMb at meat pH (5.2, 5.6, 6.0, and 6.4) and storage temperatures (4 and 25 ºC). The authors used ascorbate and nicotinamide adenine dinucleotide, in a reduced form (NADH) as electron donors and methylene blue and cytochrome c as cofactors. Methylene blue in the presence of NADH or ascorbate was more reduced at 4 ºC than at 25 ºC, with ascorbate and NADH. Higher pH increased methemoglobin reduction with ascorbate and cytochrome c. These non–enzymatic data corroborated the greater enzymatic MMb reduction in high pHu range.

Despite Denzer et al. (2020)Denzer, M.; Mowery, C.; Comstock, H.; Maheswarappa, N.B.; Mafi, G.G.; VanOverebeke, D.L.; Ramanathan, R. 2020. Characterization of the cofactors involved in nonenzymatic metmyoglobin/methemoglobin reduction in vitro. Meat and Muscle Biology 4: 1-10. obtaining results from equine muscle MMb, Bechtold et al. (2019)Bechtold, E.; Suman, S.; Mohanty, S.; Mazumder, S.; Krishnan, S.; Nerimetla, R. 2019. Species-specificity in metmyoglobin reduction. Journal of Animal Science 97: 83-84. found that the non–enzymatic MMb reduction was higher in bovine samples than in equine counterparts.

Metmyoglobin reducing activity and NADH can be gradually depleted throughout storage/display time (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). Kim et al. (2009)Kim, Y.H.; Keeton, J.T.; Smith S.B.; Berghman, L.R.; Savell, J.W. 2009. Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles. Meat Science 83: 376–382. observed a pattern for the decrease in lactate dehydrogenase (LDH) activity, NADH concentration and the percentage of MRA during the retail time of longissimus lumborum muscle packaged under fluorescent light for seven days covered with a polyvinylchloride (PVC) film. Furthermore, MMb may also be reduced via electrons from the mitochondrial electron–transport chain to form DMb (Belskie et al., 2015Belskie, K.M.; Van Buiten, C.B.; Ramanathan, R.; Mancini, R.A. 2015. Reverse electron transport effects on NADH formation and metmyoglobin reduction. Meat Science 105: 89–92.; Tang et al., 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.).

Canto et al. (2016)Canto, A.C.V.C.S.; Costa–Lima, B.R.C.; Monteiro, M.L.G.; Viana, F.M.; Silva, T.J.P.; Suman, S.P.; Conte–Junior, C.A. 2016. Color attributes and oxidative stability of longissimus lumborum and psoas major muscles from Nellore bulls. Meat Science 121: 19–26. evaluated MRA in Nellore longissimus lumborum and psoas major packaged on polystyrene trays with O2–permeable film and stored refrigerated for nine days. The authors observed a progressive drop during all retail periods for both muscles. Longissimus lumborum showed more MRA than psoas major throughout the storage period. Similarly, other authors have reported an inverse correlation between MRA and meat discoloration (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.; Ledward, 1985Ledward, D.A. 1985. Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Science 15: 149–171.; Mancini et al., 2008Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2008. Effects of data expression, sample location, and oxygen partial pressure on initial nitric oxide metmyoglobin formation and metmyoglobin-reducing-activity measurement in beef muscle. Meat Science 79: 244–251.; Reddy and Carpenter, 1991Reddy, L.M.; Carpenter, C.E. 1991. Determination of metmyoglobin reductase activity in bovine skeletal muscles. Journal of Food Science 56: 1161–1164.; Seyfert et al., 2006Seyfert, M.; Mancini, R.A.; Hunt, M.C.; Tang, J.; Faustman, C.; Garcia, M. 2006. Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles. Journal of Agricultural and Food Chemistry 54: 8919–8925.).

Higher MMb reductase activity may explain the greater color stability shown by certain muscles, such as longissimus lumborum compared to psoas major, as observed by Canto et al. (2016)Canto, A.C.V.C.S.; Costa–Lima, B.R.C.; Monteiro, M.L.G.; Viana, F.M.; Silva, T.J.P.; Suman, S.P.; Conte–Junior, C.A. 2016. Color attributes and oxidative stability of longissimus lumborum and psoas major muscles from Nellore bulls. Meat Science 121: 19–26. and Salim et al. (2019)Salim, A.P.A.A.; Suman, S.P.; Canto, A.C.V.C.S.; Costa–Lima, B.R.C.; Viana, F.M.; Monteiro, M.L.G.; Silva, T.J.P.; Conte–Junior, C.A. 2019. Muscle-specific color stability in fresh beef from grain-finished Bos indicus cattle. Asian-Australasian Journal of Animal Science 32: 1036–1043..

Several beef muscles were sorted by Renerre (1990)Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630. according to their oxidation stability: longissimus dorsi, obliquus externus and tensor fasciae latae were the most stable muscles. semi–membranosus had intermediate stability, while gluteus medius, supra–spinatus, psoas major and diaphragma medialis were the most prone to oxidation. Renerre´s classification agrees with previous studies (Ledward, 1971Ledward, D.A. 1971. Metmyoglobin formation in beef muscles as influenced by water content and anatomical location. Journal of Food Science 61: 138-140.; O’Keeffe and Hood, 1980–81).

To date, the relationship between MRA and discoloration is not consensual. Although Kim et al. (2009)Kim, Y.H.; Keeton, J.T.; Smith S.B.; Berghman, L.R.; Savell, J.W. 2009. Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles. Meat Science 83: 376–382. found a decrease in MRA; these authors did not find an increase in surface MMb accumulation (%) in beef steaks of longissimus lumborum stored for seven days at 1 ºC. According to Sammel et al. (2002)Sammel, L.M.; Hunt, M.C.; Kropf, D.H.; Hachmeister, K.A.; Johnson, D.E. 2002. Comparison of assays for metmyoglobin reducing ability in beef inside and outside semimembranosus muscle. Journal of Food Science 67: 978–984., the lack of uniformity in the MRA methodologies could explain these contradictory findings.

Color stability of dark–cutting beef

Higher postmortem muscle pH is the most important factor that can prolong mitochondrial functionality (Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.). Previous reports state that the mitochondria from dark–cutting beef has progressively greater O2 consumption and higher level of MMb reductase activity as the pH of dark–cutting meat increases (McKeith et al., 2016McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.; Wu et al., 2020Wu, S.; Luo, X.; Yang, X.; Hopkins, D.L.; Mao, Y.; Zhang, Y. 2020. Understanding the development of color and color stability of dark cutting beef based on mitochondrial proteomics. Meat Science 163: 108046.).

High pHu carcasses are reported to have greater mitochondrial OCR due to a higher activity in the enzyme cytochrome c oxidase (complex IV), which oxidizes O2 into water, resulting in less surface OMb and a darker color (Bendall and Taylor, 1972Bendall, J.R.; Taylor, A.A. 1972. Consumption of oxygen by the muscles of beef animals and related species. II. Consumption of oxygen by post-rigor muscle. Journal of the Science of Food and Agriculture 23: 707–719.; Tang et al. 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.; Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.). According to Bendall and Taylor (1972)Bendall, J.R.; Taylor, A.A. 1972. Consumption of oxygen by the muscles of beef animals and related species. II. Consumption of oxygen by post-rigor muscle. Journal of the Science of Food and Agriculture 23: 707–719., mitochondrial O2 consumption is approximately 50 – 75 % faster at pH 7.2 than at pH 5.8. As a result, the conversion to the DMb redox form is favored at high pHu, giving the meat a purplish color.

Muscle structure also contributes to color development by affecting O2 influx through the tissue, facilitating interaction between gas and Mb (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). A closed structure observed in high pHu muscles makes it difficult to diffuse O2 into the swollen muscle fibers due to the higher WHC (Hughes et al., 2014b; Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). In addition, glycolytic fibers often have a smaller diameter, which is detrimental to O2 diffusion (Choi and Kim, 2009Choi, Y.M.; Kim, B.C. 2009. Muscle fiber characteristics, myofibrillar protein isoforms, and meat quality. Livestock Science 122: 105–118.; Kirchofer et al., 2002Kirchofer, K.S.; Calkins, C.B.; Gwartney, B.L. 2002. Fiber-type composition of muscles of the beef chuck and round. Journal of Animal Science 80: 2872-2878.).

The decrease in O2 concentration within the myofibrils favors the formation of DMb and the color of fresh beef turns to dark purple (Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.). Since the O2 depth depends on the O2 concentration in the medium, oxygenation by means of air–exposition (approximately 20 % O2) would not result in a thick OMb layer on dark–cutting beef surfaces.

In addition to the O2 concentration, oxygenation also depends on the time and the temperature of exposition, blooming takes place more efficiently when meat is exposed to O2 at 0 – 2 ºC for, at least, 30 min. Higher temperatures increase mitochondrial activity and thus the O2 uptake, which reduces the O2 available to bind to Mb (Bendall and Taylor, 1972Bendall, J.R.; Taylor, A.A. 1972. Consumption of oxygen by the muscles of beef animals and related species. II. Consumption of oxygen by post-rigor muscle. Journal of the Science of Food and Agriculture 23: 707–719.; Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.).

The opposite phenomenon is observed in high pHu meat compared to MRA. These muscles show a protective effect on the oxidation of Mb. As a result, dark–cutting beef is resistant to forming the brownish layer of MMb over time (Lu et al., 2020Lu, X.; Cornforth, D.P.; Carpenter, C.E.; Zhu, L.; Luo, X. 2020. Effect of oxygen concentration in modified atmosphere packaging on color changes of the M. longissimus thoraces et lumborum from dark cutting beef carcasses. Meat Science 161: 107999.). In contrast to OCR, the protective effect is endogenous and as regards the content of substrates, such as NADH, the MMb reductase activity decays over time.

Therefore, at a certain point, the oxidative pressure by fluorescent illumination, the amount of ROS produced in mitochondrial respiration, or other factors will promote Mb oxidation that will be noticeable on the surface of the muscle (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.; Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.; Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.) which leads to lower discoloration over the display time because of the accumulation of MMb on the meat surface (Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.). In short, low O2 tension, pHu up to 5.8, high temperature, concomitant lipid oxidation and loss of MMb–reducing activity are amongst the factors that influence iron oxidation (Bekhit and Faustman, 2005Bekhit, A.E.D.; Faustman, C. 2005. Metmyoglobin reducing activity. Meat Science 71: 407–439.; Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). To prevent oxidation of DMb to MMb, residual O2 within vacuum packaging should be avoided. At low O2 concentrations (< 7 mm Hg) DMb is susceptible to oxidation by ROS, forming MMb. At O2 concentrations > 7 mm Hg, O2 competes with the peroxide radicals for the DMb, inhibiting the formation of MMb (AMSA, 2012).

The role of pH in the rate of MMb formation has been the focus of research for quite some time. Chemically, iron–catalyzed oxidation has been reported to be more active under acidic conditions (George and Stratmann, 1952George, P.; Stratmann, C. J. 1952. The oxidation of myoglobin to metmyoglobin by oxygen. I. The Biochemical Journal 51: 103–108.). Enzymatically, Echevarne et al. (1990)Echevarne, C.; Renerre, M.; Labas, R. 1990. Metmyoglobin reductase activity in bovine muscles. Meat Science 27: 161–172. showed that MMb reductase activity increases as a function of the medium pH, achieving its maximum activity at pH 7.3. Ramanathan et al. (2012)Ramanathan, R.; Mancini, R.A.; Suman, S.P.; Cantino, M.E. 2012. Effects of 4-hydroxy -2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction. Meat Science 90: 564–571. reported that a mitochondrial NADH–dependent reductase reduced more MMb in the control group at pH 7.4 than at pH 5.6 (p < 0.05). The unbalanced equilibria among DMb, OMb, and MMb in the high pHu muscles interfere with the visual and instrumentally measured surface color. Table 1 summarizes the data on the effect of high pHu on color attributes (instrumental, visual and color stability).

Table 1
– Summary of studies on the effects of high ultimate pH on meat color.

The most used instrumental colorimetric system to characterize color or evaluate color changes is the Commission Internationale de l’Eclairage (CIE) L*a*b*, where L* measures lightness (0: black, 100: white), a* measures redness (–60: green, +60: red), and b* measures yellowness (–60: blue, +60 yellow). Another system available is the HunterLab, such as the RGB (red, green and blue) models.

Redness (a*) is reduced at a higher pHu due to a decreased accumulation in OMb on the muscle surface because of more intense cellular respiration (Bendall and Taylor, 1972Bendall, J.R.; Taylor, A.A. 1972. Consumption of oxygen by the muscles of beef animals and related species. II. Consumption of oxygen by post-rigor muscle. Journal of the Science of Food and Agriculture 23: 707–719.; Tang et al., 2005Tang, J.; Faustman, C.; Hoagland, T.A.; Mancini, R.A.; Seyfert, M.; Hunt, M.C. 2005. Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability. Journal of Agricultural and Food Chemistry 53: 1223–1230.). Page et al. (2001)Page, J.K.; Wulf, D.M.; Schwotzer, T.R. 2001. A survey of beef muscle color and pH. Journal of Animal Science 79: 678–687. found that muscle pHu was more correlated with a* and b* values than with L* values. The authors stated that pHu affected the muscle color by changing the hue angle. Reduction in hue angle values moves color attributes closer to the +a* axis (red) and farther from the +b* axis (yellow). It was concluded that the closer the hue angle was to 0º and thus to b* 0, the less the muscle was discolored. A number of authors have observed this decrease in angle and chroma as the pHu increased (Mckeith et al., 2016McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.; Stackhouse et al., 2016Stackhouse, R.J.; Apple, J.K.; Yancey, J.W.S.; Keys, C.A.; Johnson, T.M.; Mehall, L.N. 2016. Postrigor citric acid enhancement can alter cooked color but not fresh color of dark-cutting beef. Journal of Animal Science 94: 1738–1754.; Wȩglarz, 2010Wȩglarz, A. 2010. Meat quality defined based on pH and colour depending on cattle category and slaughter season. Czech Journal of Animal Science 55: 548–556.; Abril et al., 2001Abril, M.; Campo, M.M.; Önenç, A.; Sañudo, C.; Albertı́, P.; Negueruela, A.I. 2001. Beef colour evolution as a function of ultimate pH. Meat Science 58: 69–78.).

Improving the color of dark–cutting beef

Currently, vacuum–packaged meat is becoming more common because of its convenience for processors and consumers. However, vacuum–packaged muscles are dark and purplish in color due to the DMb layer on the surface, which can be easily oxygenated after adequate exposition to O2. A high DMb concentration is also observed in muscle immediately after cutting, since O2 penetration does not persist through the entire meat (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.). The absence of OMb on a steak immediately after cutting is particularly useful for OCR analysis to measure O2 intake by mitochondria. The higher O2 partial pressure found in high oxygen modified atmosphere packaging (HiOx MAP) increases the depth of penetration of O2 through the muscle microstructure, which results in a thicker OMb layer on the muscle surface. Therefore, packaging dark–cutting beef in HiOx MAP is a strategy which can induce a bright red surface on the muscle despite the high pHu (McMillin, 2008McMillin, K.W. 2008. Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat Science 80: 43–65.; Renerre, 1990Renerre, M. 1990. Factors involved in the discoloration of beef meat. International Journal of Food Science and Technology 25: 613–630.). Moreover, HiOx retards uprising in the MMb layer on the muscle surface (Mancini and Hunt, 2005Mancini, R.A.; Hunt, M.C. 2005. Current research in meat color. Meat Science 71: 100–121.), whereas steaks packaged with O2 permeable films can maintain the cherry red color for hours or a few days. Thus, beef with HiOx MAP can be displayed for 6 – 10 days only (McMillin, 2008McMillin, K.W. 2008. Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat Science 80: 43–65.), because of microbial spoilage (Sun and Holley, 2012Sun, X.D.; Holley, R.A. 2012. Antimicrobial and antioxidative strategies to reduce pathogens and extend the shelf life of fresh red meats. Comprehensive Reviews in Food Science and Food Safety 11: 340–354.).

Another strategy for improving the quality of dark–cutting meat is the use of carbon monoxide modified atmosphere packaging (CO–MAP). Beef with high pHu was shown to be lighter and redder in CO–MAP, but resulted in a higher surface MMb content than beef in HiOx MAP, which indicates that the relationship between MRA and MMb content in CO–MAP was different compared to that found in HiOx MAP (Zhang et al., 2018Zhang, Y.; Qin, L.; Mao, Y.; Hopkins, D.L.; Han, G.; Zhu, L.; Luo, X. 2018. Carbon monoxide packaging shows the same color improvement for dark cutting beef as high oxygen packaging. Meat Science 137: 153-159.).

The use of carbon dioxide (CO2) can inhibit the growth of food pathogens and Gram–negative aerobic bacteria, since they are more sensitive to CO2 compared to Gram–positive bacteria (Daniels et al., 1985Daniels, J.A.; Krishnamurthi, R.; Rizvi, S.S.H. 1985. A review of effects of carbon dioxide on microbial growth and food quality. Journal of Food Protection 48: 532–537.). A higher proportion of CO2 can be used to package meat and prevent aerobic deterioration with an increase in microbial control (Sun and Holley, 2012Sun, X.D.; Holley, R.A. 2012. Antimicrobial and antioxidative strategies to reduce pathogens and extend the shelf life of fresh red meats. Comprehensive Reviews in Food Science and Food Safety 11: 340–354.).

Although the use of HiOx MAP increases consumer acceptance of meat color (O’Sullivan et al., 2015O’Sullivan, M.G.; Le Floch, S.; Kerry, J.P. 2015. Resting of MAP (modified atmosphere packed) beef steaks prior to cooking and effects on consumer quality. Meat Science 101: 13–18.), this modified atmosphere has been associated with some loss in overall meat quality because of the presence of off–odors and off–flavors attributable to lipid oxidation (Jayasingh et al., 2002Jayasingh, P.; Cornforth, D.P.; Brennand, C.P.; Carpenter, C.E.; Whittier, D.R. 2002. Sensory evaluation of ground beef stored in high-oxygen modified atmosphere packaging. Journal of Food Science 67: 5–8.). Seyfert et al. (2006)Seyfert, M.; Mancini, R.A.; Hunt, M.C.; Tang, J.; Faustman, C.; Garcia, M. 2006. Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles. Journal of Agricultural and Food Chemistry 54: 8919–8925. also found an increase in thiobarbituric acid reactive substance (TBARS) values of beef packaged in HiOx MAP after storage and display.

According to Ramanathan et al. (2012)Ramanathan, R.; Mancini, R.A.; Suman, S.P.; Cantino, M.E. 2012. Effects of 4-hydroxy -2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction. Meat Science 90: 564–571. and Suman et al. (2014)Suman, S.P.; Hunt, M.C.; Nair, M.N.; Rentfrow, G. 2014. Improving beef color stability: practical strategies and underlying mechanisms. Meat Science 98: 490–504., beef Mb is greatly susceptible to nucleophilic attack by ROS and aldehydes generated from peroxidation, such as 4–hydroxynonenal, which increases the proportion of MMb (Fe3) on the beef surface. However, fresh steaks from Nellore bulls showed extremely low TBARS values in both HiOx MAP and CO–MAP (Santos et al., 2016Santos, P.R.; Contreras–Castillo, C.J.; Venturini, A.C. 2016. Color stability of Bos indicus bull steaks in modified atmosphere packaging (MAP). Scientia Agropecuaria 7: 401 – 408.). Fiber IIB exhibit glycolytic metabolism and pasture–fed zebu steers (longissimus lumborum muscles), and contains natural antioxidants, which may explain these results (Canto et al., 2016Canto, A.C.V.C.S.; Costa–Lima, B.R.C.; Monteiro, M.L.G.; Viana, F.M.; Silva, T.J.P.; Suman, S.P.; Conte–Junior, C.A. 2016. Color attributes and oxidative stability of longissimus lumborum and psoas major muscles from Nellore bulls. Meat Science 121: 19–26.).

Research indicates that the addition of various glycolytic and tricarboxylic acid metabolites, such as succinate, lactate, and malate can regenerate NADH through NADH–dependent reducing systems to enhance MMb reduction and the color stability of whole–muscle beef cuts (Table 2).

Table 2
– Summary of results from studies that have examined the relationship between injection–enhanced beef with various Krebs cycle intermediates and the color stability.

Furthermore, the ability of pyruvate and succinate to minimize lipid oxidation has been reported (Ramanathan et al., 2011). Kim et al. (2006)Kim, Y.H.; Hunt, M.C.; Mancini, R.A.; Seyfert, M.; Loughin, T.M.; Kropf, D.H.; Smith, J.S., 2006. Mechanism for lactate-color stabilization in injection-enhanced beef. Journal of Agricultural Food Chemistry 54: 7856-7862. reported that color stabilization by lactate enhancement is related to MRA, by NADH replacement via LDH, reducing NAD+ both enzymatically and non–enzymatically. However, NADH can also promote mitochondrial O2 consumption resulting in darkened muscle because of the lowering of Mb oxygenation (Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.). Lactate also results in color stabilization of cooked beef (Knock et al., 2006Knock, R.C.; Seyfert, M.; Hunt, M.C.; Dikeman, M.E.; Mancini, R.A.; Unruh, J.A.; Higgins J.J.; Monderen, R.A. 2006. Effects of potassium lactate, sodium chloride, sodium tripolyphosphate, and sodium acetate on colour, colour stability, and oxidative properties of injection-enhanced beef rib steaks. Meat Science 74: 312–318.). In addition, the application of solutions containing lactate improve the meat’s juiciness, tenderness, taste, shelf life, and yield (Lawrence et al., 2003Lawrence, T.E.; Dikeman, M.E.; Hunt, M.C.; Kastner, C.L.; Johnson, D.E. 2003. Effects of calcium salts on beef longissimus quality. Meat Science 64: 299-308.).

The addition of potassium lactate increased the color stability of steaks packaged in HiOx MAP (Kim et al., 2006Kim, Y.H.; Hunt, M.C.; Mancini, R.A.; Seyfert, M.; Loughin, T.M.; Kropf, D.H.; Smith, J.S., 2006. Mechanism for lactate-color stabilization in injection-enhanced beef. Journal of Agricultural Food Chemistry 54: 7856-7862.). Additional research indicated the effect of lactate enhancement on beef steaks is packaging–dependent (Suman et al., 2009Suman, S.P.; Mancini, R.A.; Ramanathan, R.; Konda, M.R. 2009. Effect of lactate-enhancement, modified atmosphere packaging, and muscle source on the internal cooked colour of beef steaks. Meat Science 81: 664–670.): lactate can be utilized for improving the color stability of beef steaks in HiOx MAP, but has no effect on the beef color stored in vacuum packaging and CO–MAP. Many advances have been made in the field of active packaging, such as the detection of microbial spoilage and safety within different MAP systems, to which dark–cutting has increased susceptibility (Holman et al., 2018Holman, B.W.B.; Kerry, J.P.; Hopkins, D.L. 2018. Meat packaging solutions to current industry challenges: a review. Meat Science 144: 159-168.). Sensors for real–time monitoring of beef freshness and quality can be used as an attractive and effective tool for assessing the microbial quality of packaged fresh meat (Kuswandi and Nurfawaidi, 2017Kuswandi, B.; Nurfawaidi, A. 2017. On-package dual sensors label based on pH indicators for real-time monitoring of beef freshness. Food Control 82: 91-100.; Shukla et al., 2015Shukla, V.; Kandeepan, G.; Vishnuraj, M.R. 2015. Development of on-package indicator sensor for real-time monitoring of buffalo meat quality during refrigeration storage. Food Analytical Methods 8: 1591–1597.).

Color methods applied in dark–cutting beef fresh muscle

Because of the difference in pHu, certain research methods for evaluating meat color in high pHu beef samples require adaptations or a new approach to obtain reliable data. Herein, we discussed a number of methods employed in color stability research and compare them with standard methods applicable to normal pHu beef muscles. It is important to note that these methods are in–line with the Meat Color Measurement Guidelines issued by AMSA (2012): Mb quantification, MRA, and OCR.

There are two protocols for quantifying total Mb of fresh and cooked meat in AMSA’s guidelines (AMSA, 2012): the first based on the isobestic spectrophotometric point and the second, by reducing all Mb forms to DMb. Both strategies can be used for meat with high pHu. However, the extraction of the pigment with neutral buffer (40 mM potassium phosphate, pH 6.8) is hampered in high pH meat, which results in incomplete total pigment extraction and, thus, in a pink color remaining in the centrifuge pellet after one extraction. In order to improve the pigment extraction, an acidic buffer adapted from Poel’s Mb quantification method can be deployed (DeDuve, 1948DeDuve, C. 1948. A spectrophotometric method for the simultaneous determination of myoglobin and hemoglobin in extracts of human muscle. Acta Chemica Scandinavica 2: 264–289.; Hunt and Hedrick, 1977Hunt, M.C.; Hedrick, H.B. 1977. Chemical, physical and sensory characteristics of bovine muscle from four quality groups. Journal of Food Science 42: 716–720.). The use of an acidic buffer (0.01 N, Hunt and Hedrick (1977)Hunt, M.C.; Hedrick, H.B. 1977. Chemical, physical and sensory characteristics of bovine muscle from four quality groups. Journal of Food Science 42: 716–720., or 800 mM, McKeith et al. (2016)McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173., sodium acetate, pH 4.5) compensates for the native higher pH of the muscle, also observed in pre–rigor muscle (abdominal muscle from biopsy) as shown by DeDuve (1948)DeDuve, C. 1948. A spectrophotometric method for the simultaneous determination of myoglobin and hemoglobin in extracts of human muscle. Acta Chemica Scandinavica 2: 264–289.. The repetition of the extraction step ensures the removal of Mb from the tissue, as followed by Hunt and Hedrick (1977)Hunt, M.C.; Hedrick, H.B. 1977. Chemical, physical and sensory characteristics of bovine muscle from four quality groups. Journal of Food Science 42: 716–720. and McKeith et al. (2016)McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173..

The MRA method is based on the complete oxidation of Mb in sodium nitrite solution (0.3 % for 20 min at 25 ºC) with subsequent Mb reduction under vacuum conditions at 20 – 30 ºC for 2 h. The estimation of the proportion of each Mb redox form is achieved by scanning samples with a Hunter Miniscan colorimeter with settings previously described that had been calibrated through the O2 impermeable film of a vacuum bag. Complete oxidation may be estimated by the ratio of specific wavelengths (572 nm/525 nm), being the proportion of each Mb redox form confirmed by the 630 nm/580 nm ratio.

Another protocol for estimating the MMb redox form on the surface of the samples is based on the creation of reference standards for 100 % MMb and DMb. Thus, the proportion of surface MMb is obtained by dividing the difference between MMb for 100 % DMb and for samples by the difference between MMb for 100 % DMb and 100 % MMb. However, Mb does not oxidize completely in pHu meat > 5.8, and does not result in 100 % MMb samples prior to vacuum–reduction. There is no method in the literature for achieving total Mb oxidation. Therefore, the initial MMb formation is used by a number of authors, such as McKeith et al. (2016)McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173.. The initial MMb formation represents the proportion of surface MMb formed after Mb oxidation in sodium nitrite solution.

Instead of forcing Mb oxidation/reduction, as seen in MRA analysis, OCR methodology is based on the assessment of the Mb oxygenation/deoxygenation stimulated. In OCR protocol, described by Madhavi and Carpenter (1993)Madhavi, D.L.; Carpenter, C.E. 1993. Aging and processing affect color, metmyoglobin reductase and oxygen consumption of beef muscles. Journal of Food Science 58: 939–942., muscle samples are air–oxygenated for 2 h at 2 ºC before vacuum–packaging (reading 1) and subsequent incubation for 20 – 30 min at 25 – 30 ºC (reading 2). Two readings assess the surface OMb proportion in order to evaluate mitochondrial O2 consumption during thermal incubation.

McKeith et al. (2016)McKeith, R.O.; King, D.A.; Grayson, A.L.; Shackelford, S.D.; Gehring, K.B.; Savell, J.W.; Wheeler T.L. 2016. Mitochondrial abundance and efficiency contribute to lean color of dark cutting beef. Meat Science 116: 165–173. observed that performing OCR in high pHu, beef dark–cutting samples had lower initial blooming (before incubation) than normal pHu samples, failing to achieve 100 % of OMb on the surface of the muscle – as was also observed by Krzywicki (1979)Krzywicki, K. 1979. Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef. Meat Science 3: 1–10.. Therefore, similar to the behavior of MRA in high pHu samples, the evaluation of the proportion of the deoxygenated OMb may not be reached. Therefore, the use of the proportion of OMb formed during the blooming step is an interesting alternative for comparing OCR between samples.

Calculating MMb, OMb, and DMb proportions on the beef surface has been conducted by Krzywick (1979). However, assessment of the Mb states in high pHu muscle is challenging when disabling a full conversion of these forms, further to this pHu range changing muscle structure and WHC. English et al. (2016)English, A.R.; Wills, K.M.; Harsh, B.N.; Mafi, G.G.; Vanoverbeke, D.L.; Ramanathan, R. 2016. Effects of aging on the fundamental color chemistry of dark-cutting beef. Journal of Animal Science 94: 4040–4048. estimated DC beef pigments by their reflectance values and demonstrated that using the K/S ratios at isobestic points were useful. K/S improves pigment proportion quantification by making data more linear accounting for absorptive (K) and scattering (S) color properties; and its formula is (1–R)2/(2R), where R is the reflectance obtained using a spectrophotometer (AMSA, 2012).

Although Ramanathan et al. (2010)Ramanathan, R.; Mancini, R.A.; Naveena, B.M.; Konda, M.K.R. 2010. Effects of lactate-enhancement on surface reflectance and absorbance properties of beef longissimus steaks. Meat Science 84: 219–226. have not worked with high pHu, the authors showed that lactate–enhancement in beef had changed the overall percentage reflectance. Nonetheless, 525 nm remained the isobestic point for MMb, OMb, and DMb, while 572, 610, and 473 nm also remained isobestic for MMb, OMb, and DMb, respectively. Therefore, these wavelengths are still useful for calculating surface pigment in high pHu beef using the following formulae: K/S 572 ÷ K/S 525 (MMb), K/S 610 ÷ K/S 525 (OMb), and K/S 473 ÷ K/S 525 (DMb) (AMSA, 2012).

Novelties in meat color studies of dark–cutting beef

Molecular profiling techniques including metabolomic and proteomic, have been increasingly applied as targeted or non–targeted approaches to exploring biochemical changes in postmortem muscle and its influence on meat quality characteristics (Ma et al., 2020Ma, D.; Yu, Q.; Hedrick, V.E.; Cooper, B.R.; Sobreira, T.J.P.; Oh, J.H.; Chun, H.; Kim, Y.H.B. 2020. Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes. Meat Science 166: 108-140.). Mass spectrometry–based metabolomic and proteomic studies have been carried out to evaluate modifications in the metabolite and protein profile as proteins and metabolites are directly involved in molecular mechanisms related to color stability, lipid oxidation, WHC and tenderness in fresh meats (Kim et al., 2016Kim, Y.H.B.; Kemp, R.; Samuelsson, L.M. 2016. Effects of dry-aging on meat quality attributes and metabolite profiles of beef loins. Meat Science 111: 168-176.; Li et al., 2018Li, M.; Li, Z.; Li, X.; Xin, J.; Wang, Y.; Li, G.; Wu, L.; Shen, Q. W.; Zhang, D. 2018. Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability. Food Chemistry 240: 104-111.; Ma et al., 2017Ma, D.; Kim, Y.H.B.; Cooper, B.R.; Oh, J.H.; Chun, H.; Choe, J.; Schoonmaker, J.P.; Ajuwon, K.M.; Min, B. 2017. Metabolomics profiling to determine the effect of postmortem aging on color and lipid oxidative stabilities of different bovine muscles. Journal of Agricultural and Food Chemistry 65: 6708-6716.; Subbaraj et al., 2016Subbaraj, A.K.; Kim, Y.H.B.; Fraser, K.; Farouk, M.M. 2016. A hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) based metabolomics study on colour stability of ovine meat. Meat Science 117: 163-172.).

On the subject of meat color, a proteomic study indicated that the sarcoplasmic proteome of color–stable longissimus lumborum beef muscle has higher levels of soluble antioxidant proteins (thioredoxin, peroxiredoxin–2, and peptide methionine sulfoxide reductase) and chaperones (heat–shock protein–27 kDa) compared to the color–labile psoas major muscle (Joseph et al., 2012Joseph, P.; Suman, S.P.; Rentfrow, G.; Li, S.; Beach, C.M. 2012. Proteomics of muscle specific beef color stability. Journal of Agricultural Food Chemistry 60: 3196-3203.). In turn, Mato et al. (2019)Mato, A.; Rodríguez–Vázquez, R.; López–Pedrouso, M.; Bravo, S.; Franco, D.; Zapata, C. 2019. The first evidence of global meat phosphoproteome changes in response to pre-slaughter stress. BMC Genomics 20: 1-15. assessed phosphoproteomic differences between dark–cutting and normal beef in response to pre–slaughter stress. These authors found that protein phosphorylation levels were three times higher in dark–cutting beef compared to normal beef. This effect was mainly observed in proteins with biological functions related to structural–contractile properties, actin polymerization, stress response, metabolism and electron transport chain.

As postmortem glycolysis and pH decline in muscle are associated with protein phosphorylation, a previous gel–based phosphoproteomic study hypothesized that phosphorylation modifies the Mb structure and its susceptibility to oxidation, thereby influencing meat color stability (Li et al., 2018Li, M.; Li, Z.; Li, X.; Xin, J.; Wang, Y.; Li, G.; Wu, L.; Shen, Q. W.; Zhang, D. 2018. Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability. Food Chemistry 240: 104-111.). This study identified that the phosphorylation of color stability–related proteins regulates the activity of glycolytic enzymes, thus influencing meat discoloration.

In metabolomic studies, Ma et al. (2017)Ma, D.; Kim, Y.H.B.; Cooper, B.R.; Oh, J.H.; Chun, H.; Choe, J.; Schoonmaker, J.P.; Ajuwon, K.M.; Min, B. 2017. Metabolomics profiling to determine the effect of postmortem aging on color and lipid oxidative stabilities of different bovine muscles. Journal of Agricultural and Food Chemistry 65: 6708-6716. stated that metabolic pathways influencing both color and lipid oxidative stability in beef are dependent on the muscle type as well as the postmortem aging period. In fact, psoas major muscle was more susceptible to discoloration, lower free radical scavenging activity, higher non–heme iron content and lipid oxidation compared to semimembranosus and longissimus lumborum muscles, which were more stable. These authors reported that metabolites such as the NAD/NADH ratio, acyl carnitines, free amino acids, nucleotides, nucleosides, and glucuronides play an important role in the oxidative stabilization of beef muscles. These metabolites can be potential biomarkers for further validation studies.

To identify differentially abundant metabolites related to color stability from longissimus lumborum and psoas major muscle, a gas chromatography–mass spectrometry (GC–MS) based non–targeted metabolomic approach was used by Abraham et al. (2017)Abraham, A.; Dillwith, J.W.; Mafi, G.G.; Van Overbeke, D.L.; Ramanathan, R. 2017. Metabolite profile differences between beef longissimus and psoas muscles during display. Meat and Muscle Biology 1: 18–26.. The Longissimus lumborum muscle had higher levels of pyruvic acid, glucose 6–phosphate, fructose and citric acid compared to psoas major muscle. Additionally, key regulatory metabolites can increase MRA and mitochondrial activity. The malonic acid levels were higher in psoas major muscle when compared to the values found for longissimus lumborum. In fact, malonic acid is a complex II inhibitor recognized by promoting NADH oxidation, thus negatively affecting meat color (Abraham et al., 2017Abraham, A.; Dillwith, J.W.; Mafi, G.G.; Van Overbeke, D.L.; Ramanathan, R. 2017. Metabolite profile differences between beef longissimus and psoas muscles during display. Meat and Muscle Biology 1: 18–26.; Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.).

In loins obtained from lamb carcasses and exposed to different storage conditions and display times, the metabolite profile was studied by a hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS)–based metabolomic approach. The study identified metabolites including malic acid, NADH, and guanosine levels as being significantly higher in color–stable samples than in color–labile samples during aging (Subbaraj et al., 2016Subbaraj, A.K.; Kim, Y.H.B.; Fraser, K.; Farouk, M.M. 2016. A hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) based metabolomics study on colour stability of ovine meat. Meat Science 117: 163-172.).

Moreover, as regards the differences in MRA and OCR, the influence of sarcoplasmic proteome and metabolome on the differential color stability of beef has been recently reported (Ramanathan et al., 2019Ramanathan, R.; Nair, M.N.; Hunt, M.C.; Suman, S.P. 2019. Mitochondrial functionality and beef colour: a review of recent research. South African Journal of Animal Science 49: 9-19.; Mancini et al., 2018Mancini, R.A.; Belskie, K.; Suman, S.P.; Ramanathan, R. 2018. Muscle-specific mitochondrial functionality and its influence on fresh beef color stability. Journal of Food Science 83: 2077-2082.). These results indicated that muscle–specific differences in mitochondrial activity may partially contribute to variations in the color stability of longissimus lumborum and psoas major beef muscles.

Final Remarks

Dark–cutting beef has been cataloged as a multifactorial phenomenon dependent on ante and postmortem factors. Although not the exclusive factor, high pHu was the major influencing element to produce dark–cuts and their color stability, even though there was, at the time, no definitive cut–off pH for determining a carcass as high pHu.

Animal genotype was observed as being one factor among several others that affect pHu drop and beef color color stability. However, there was little information available regarding an important breed in Brazilian herds: Nellore bulls and their crossbreeds.

The significant color stability found in the muscles with high pHu was outshone by the darker appearance which, closely associated with the increased mitochondrial OCR, reduces beef marketability. Post–slaughter interventions, such as improvements in the use of organic acid salts through multi–needle injection followed by HiOx MAP either limited the oxidation of Fe2–Mb or promoted reductions in MMb.

Future studies should focus on the use of accurate techniques to quantify tissue OCR and MRA and their relationships to color stability of fresh dark–cutting meat, such as the –omics techniques, especially in Nellore bulls in order to substantiate the effect of animal phenotype on beef color.

Acknowledgments

This review was supported by the São Paulo Research Foundation (FAPESP), Thematic Project 17/26667-2 and Post-Doctoral Fellowship 19/18346-7.

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Edited by

Edited by: Paulo Cesar Sentelhas

Publication Dates

  • Publication in this collection
    18 Jan 2021
  • Date of issue
    2022

History

  • Received
    25 Mar 2020
  • Accepted
    12 July 2020
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