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. 2023 May 15;101:skad155. doi: 10.1093/jas/skad155

Metabolomics and bioinformatic analyses to determine the effects of oxygen exposure within longissimus lumborum steak on beef discoloration

Morgan L Denzer 1, Morgan Pfeiffer 2, Gretchen G Mafi 3, Ranjith Ramanathan 4,✉
PMCID: PMC10294556  PMID: 37184234

Abstract

Meat discoloration starts from the interior and spreads to oxymyoglobin layer on the surface. The effects of oxygen exposure within a steak on the metabolome have not been evaluated. Therefore, the objective of this study was to evaluate the impact of oxygen exposure on the metabolome of the longissimus lumborum muscle. Six United States Department of Agriculture (USDA) Low Choice beef strip loins were sliced into steaks (1.91-cm) and packaged in polyvinyl chloride overwrap trays for 3 or 6 d of retail display. The oxygen exposed (OE) surface was the display surface during retail, and the non-oxygen exposed (NOE) surface was the intact interior muscle. The instrumental color was evaluated using a HunterLab MiniScan spectrophotometer. To analyze the NOE surface on days 3 and 6, steaks were sliced parallel to the OE surface to expose the NOE surface. Metmyoglobin reducing ability (MRA) was determined by nitrite-induced metmyoglobin reduction. A gas chromatography–mass spectrometry was used to identify metabolites. The a* values of steaks decreased (P < 0.05) with display time. MRA was greater (P < 0.05) in the NOE surface compared with the OE surface on days 3 and 6. The KEGG pathway analysis indicated the tricarboxylic acid (TCA) cycle, pentose and glucuronate interconversions, phenylalanine, tyrosine, and tryptophan metabolism were influenced by the oxygen exposure. The decrease in abundance of succinate from days 0 to 6 during retail display aligned with a decline in redness during display. Furthermore, citric acid and gluconic acid were indicated as important metabolites affected by oxygen exposure and retail display based on the variable importance in the projection in the PLS-DA plot. Citric acid was lower in the NOE surface than the OE surface on day 6 of retail display, which could relate to the formation of succinate for extended oxidative stability. Greater alpha-tocopherol (P < 0.05) in the NOE surface supported less oxidative changes compared to the OE surface during retail display. These results indicate the presence of oxygen can influence metabolite profile and promote migration of the metmyoglobin layer from interior to surface.

Keywords: meat color, metmyoglobin reducing ability, metabolomics, oxygen exposure

Introduction

Oxygenation of steaks form bright cherry red oxymyoglobin, which is preferred by consumers (Oslon et al., 20119; Mancini et al., 2022). Oxygen binds to the sixth position of the heme group of deoxymyoglobin to form oxymyoglobin. Oxidation of iron within the heme leads to the formation of metmyoglobin, which consumers negatively perceive due to its brown pigmentation (Carpenter et al., 2001). Meat discoloration has been reported to cost the U.S. meat industry approximately $3.7 billion annually (Ramanathan et al., 2022). Metmyoglobin initially forms subsurface below a layer of ­oxymyoglobin and migrates to the surface (Limsupavanich et al., 2004, 2008; King et al., 2023). Research has reported that as time in display increases, the metmyoglobin layer thickens and rises to the surface leading to visible surface discoloration (Ledward, 1970; Ramanathan et al., 2020). More specifically, low oxygen partial pressure between oxygen exposed (OE) surface and non-oxygen exposed (NOE) interior favors metmyoglobin formation (Brown and Mebine, 1969; George and Stratmann, 1952; O’Keeffe and Hood, 1982). However, inherent metmyoglobin reducing systems can limit interior metmyoglobin formation.

After animal harvest, diffusion from the atmosphere is the main source of oxygen for biochemical reactions. In postmortem muscle, oxygen is consumed by mitochondria and oxygen-consuming enzymes (Ramanathan and Mancini, 2018), leading to an oxygen partial pressure gradient within muscle. Therefore, oxygen consumption can influence oxygen penetration and diffusion. Research has reported a thinner layer of oxymyoglobin favors metmyoglobin formation (Limsupavanich et al., 2004, 2008). Oxygen consumption is influenced by postmortem metabolism, including mitochondria (Ke et al., 2017). Specifically, mitochondrial degradation can negatively impact oxygen consumption and lead to reactive oxygen species production (Ke et al., 2017). Furthermore, myoglobin oxidation and lipid oxidation are linked in muscle and occur through reactive oxygen species (Lynch and Faustman, 2000; Faustman et al., 2010). Previous studies reported that lipid oxidation products decrease activity of enzymes involved in metmyoglobin reducing activity (MRA) and mitochondrial function (Ramanathan et al., 2012; Ramanathan et al., 2014; Zhai et al., 2019). Therefore, prolonged oxygen exposure can decrease oxygen consumption and increase myoglobin oxidation. However, understanding the impact of interior metmyoglobin formation and subsequent oxidative changes on the metabolome due to oxygen exposure is limited.

The postmortem metabolome plays a role in color stability. More specifically, glycolytic and tricarboxylic metabolites can regenerate NADH to reduce metmyoglobin via enzymatic-, non-enzymatic-, and electron-transport-mediated pathways (Brown and Snyder, 1969; Arihara et al., 1995; Tang et al., 2005; Denzer et al., 2020). In addition, lactate dehydrogenase (Kim et al., 2006; Kim et al., 2009) and malate dehydrogenase (Mohan et al., 2010) utilize substrates such as malate and lactate to form NADH, respectively (Kim et al., 2006; Kim et al., 2009; Mohan et al., 2010). NADH has been reported as an important electron donor in the reduction of metmyoglobin (Arihara et al., 1995; Brown and Snyder, 1969; Kim et al., 2006; Kim et al., 2009; Mohan et al., 2010). Furthermore, succinate has been described to increase electron transport-mediated metmyoglobin reduction (Tang et al., 2005). While the connection between metabolome changes and color stability of the longissimus muscle during display is well understood, there is limited research on the effects of oxygen exposure on the metabolome of the longissimus muscle. Therefore, the objective of this study was to evaluate the effect of oxygen exposure of the retail surface compared to the interior NOE surface of the longissimus lumborum muscle on the metabolome of the longissimus muscle.

Materials and Methods

Sample collection and preparation

Loins were obtained from a United States Department of Agriculture (USDA) Food Safety Inspection Service-inspected commercial processing plant where humane slaughter practices were followed, according to USDA guidelines. Institutional animal care and use committee approval was not requested.

Six USDA Low Choice strip loins (longissimus lumborum; Institutional Meat Purchasing Specifications #180) were purchased from Creekstone Farms LLC (Arkansas City, KS). Loins were transported to Oklahoma State University Robert Kerr Food and Agricultural Products Center on ice. On day 7 postmortem, pH (Hanna Instruments pH probe Handheld HI 99163; probe FC232; Hanna Instruments, Woonsocket, RI) was analyzed in three random locations across the loin. Loins were sliced into six 1.91-cm steaks. One steak was used for day 0 analysis, and the entire steak was designated as (NOE). Four steaks were randomly selected and paired to package in polyvinyl chloride (15,500–16,275 cm3 O2/m2/24 h at 23 °C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO) overwrap Styrofoam trays for retail display. Packages were assigned to either 3 or 6 d in retail display with continuous light-emitting diode lighting (Philips light-emitting diode lamps, 12 watts, 48 inches, color temperature = 3,500°K, 54 Phillips, China). Instrumental color was measured six times on each package every day of retail display using a HunterLab 4500L MiniScan EZ Spectrophotometer (2.5-cm aperture, illuminant A, and 10̊ standard observer angle, HunterLab Associates, Reston, VA). The remaining steak was the most posterior and was used for proximate analysis.

Proximate composition

Approximately 200 g from the steak was ground after removing external fat with a table-top grinder (Big Bite Grinder, 4.5 mm, fine grind, LEM), and proximate composition was analyzed using a near-infrared spectrophotometer (FoodScan Lab Analyzer, Serial No. 91753206; Foss, NIRsystem Inc.; Slangerupgrade, Denmark).

Oxygen exposure

Steaks were exposed to oxygen during retail display, and the retail surface was considered an OE surface. The interior muscle was considered a NOE surface. Oxygen consumption (OC), MRA, and metabolomic profiling of OE and NOE were determined on day 3 or 6. Day 0 steak immediately after fabrication was used for biochemical and metabolomics analysis (designated as day 0 NOE).

Metmyoglobin reducing activity

OE and NOE samples (2.54 × 2.54 × 0.96 cm) were submerged in 0.3% sodium nitrite for 20 min to evaluate MRA. Preincubation metmyoglobin formed after incubating in nitrite solution was used to determine MRA (Mancini et al., 2008). Samples were blotted dry and read using a HunterLab MiniScan spectrophotometer after submersion. MRA was calculated as a ratio of K/S572 ÷ K/S525, with a greater ratio indicating a greater MRA.

Oxygen consumption

The NOE and OE surfaces (2.54 × 2.54 × 0.96 cm) were exposed to air for 1 h at 4 °C to promote oxymyoglobin formation. Following bloom, samples were packaged in vacuum and incubated at 30 °C for 60 min based on the AMSA Meat Color Guidelines (King et al., 2023). A HunterLab MiniScan spectrophotometer was used to determine reflectance values from 400 to 700 nm at 0, 30, and 60 min of incubation. The changes in oxymyoglobin content were calculated as pre-incubation (K/S610 ÷K/S525)—post-incubation (K/S610 ÷K/S525). A greater change in oxymyoglobin indicates greater OC.

Metabolomic analysis

Analysis and subsequent identification of metabolites occurred at the National Institute of Health West Coast Metabolomics Center, University of California Davis (CA, USA). Samples (approximately 10 mg) from the NOE surface on days 0 and 6 and the OE surface on day 6 were collected, freeze-dried, and stored at −80 °C until analysis. To extract metabolites, 1,000 µL of degassed acetonitrile/isopropanol/water mixture (3:3:2, v/v/v) was added to the samples. Samples were homogenized for 30 s and shaken at 4 °C for 6 min, and centrifuged at 14,000 × g for 2 min. Additionally, carbon 8-carbon 30 fatty acid methyl esters were added to the mixture as an internal standard. Samples were dried with nitrogen gas and derivatized with methyloxolane in pyridine and N-methyl-N(trimethylsilyl)trifluoroacetamide for trimethylsilylation of acidic protons. The remaining details about metabolomic profiling are included in Fiehn et al., (2008). The metabolite features were identified using a database, BinVestigate, developed at the National Institute of Health West Coast Metabolomics Center, University of California Davis.

Statistical analysis

This experiment was a split-plot design with a randomized complete block blocked by loin. MRA and OC were analyzed using a split-plot design. The whole plot factor was retail day, and the sub-plot factor was oxygen exposure. Instrumental color analysis was evaluated as a repeated measure, with the retail day being the repeated measure. The covariance structure for the repeated measures was first-order autoregressive and was selected based on Akaike’s Information Criterion values. The fixed effects were retail day, oxygen exposure, and their interactions. From the Glimmix procedure of SAS (Version 9.4, SAS Institute Inc. Cary, NC), the least square means were calculated and separated with the pdiff option, and significance was considered with a P < 0.05.

Metabolite profiling was evaluated using MetaboAnalyst 5.0. The metabolite peak intensities were normalized by the median, transformed by log, and scaled with Pareto scaling. To compare days 0 and 6 metabolites as well as NOE and OE surfaces, an analysis of variance was completed to indicate the significantly different metabolites between the two groups, with Fisher’s LSD used to separate the abundance of specific metabolites. Significance was considered as a P < 0.05. Supervised projections to latent structure-discriminant analysis (PLS-DA) were used to demonstrate differences in metabolites between the retail display and oxygen exposure. Metabolites were ranked based on their importance in determining differences in oxygen exposure and retail day using the variable importance in the projection (VIP) in PLS-DA. A pathway analysis of MetaboAnalyst 5.0 established pathways impacted by significantly different metabolites.

Results

Proximate composition and retail display color

The proximate composition of the strip loins is included in Table 1. The mean pH was 5.55 on day 0 of display. These results confirm the loins were in the normal postmortem pH range for beef. There was a significant effect of retail display day on the a* values (Table 2). There was a decrease (P < 0.05) in redness with greater retail display time.

Table 1.

Proximate composition and pH of longissimus lumborum muscles (n = 6).

Parameter Mean SEM
Protein 22.53 0.29
Moisture 73.59 0.59
Fat 4.66 0.66
pH 5.55 0.03
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SEM = standard error of mean.

Table 2.

Effects of retail display day on redness of steaks (n = 6) displayed for 6 d.

Retail display day a*
0 35.24a
1 31.41b
2 30.36c
3 29.72cd
4 28.00e
5 28.94d
6 27.12e
SEM = 0.72
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a-eLeast squares means with different letters are significantly different (P < 0.05).

SEM = standard error of mean.

Metmyoglobin reducing ability and oxygen consumption

Metmyoglobin reducing ability and OC were influenced by retail display time and oxygen exposure (Table 3). The OE surface had lower (P < 0.05) MRA than the NOE surface on days 3 and 6 of retail display, while the OC of the OE surface was only lower (P < 0.05) than the NOE on day 3. Overall, oxygen exposure had a negative impact on the MRA of the longissimus muscle in retail display and may be linked to the loss of redness during display. However, in the current research, we are not sure why MRA of NOE did not change with storage. The color stable nature of longissimus might have limited MRA of NOE surface.

Table 3.

Least squares means of oxygen consumption1 and metmyoglobin reducing ability2 (retail display day × oxygen exposure3) of steaks (n = 6)

Parameter Oxygen exposure Retail display day
0 3 6
Oxygen consumption Non-oxygen exposed 0.14a 0.15a 0.12ab
 SEM = 0.02 Oxygen exposed 0.14a 0.09b 0.12a
Metmyoglobin reducing ability Non-oxygen exposed 0.92a 0.94a 0.95a
 SEM = 0.02 Oxygen exposed 0.92a 0.76b 0.75b
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abLeast squares means with different letters are significantly different (P < 0.05).

1Change in oxymyoglobin formation before and after 30 min of incubation determined by the change in K/S ratio of oxymyoglobin (Preincubation K/S610/K/S525—post incubation K/S610/K/S525).

2Metmyoglobin formation after submersion in sodium nitrite solution determined by K/S ratio of metmyoglobin (K/S 572/K/S525), where a greater number indicates greater reduction.

3Exposure of muscle to oxygen as display surface indicated as oxygen exposed or interior of the muscle as non-oxygen exposed.

SEM = standard error of the mean.

Metabolite profiles of OE- and NOE surfaces

From the nontargeted metabolomics, 403 features were identified, and of those, 149 were detected in the metabolite library. The PLS-DA scores plot demonstrates the separation between the metabolite profiles of day 0 NOE, day 6 NOE, and day 6 OE surfaces (Figure 2A). A VIP analysis reported citric acid, gluconic acid, and ribonic acid as the top metabolites influencing the PLS-DA plot separation between the retail days and oxygen exposure (Figure 2B). A heatmap was created to demonstrate the metabolite differences across display time and oxygen exposure (Figure 3).

Figure 2.

Figure 2.

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Partial least-squares discriminant analysis of metabolites present in oxygen (OE) exposed and non-oxygen exposed (NOE) surfaces (A). Important features identified by PLS-DA analysis of oxygen exposure of the longissimus muscle in retail display (B). A variable importance projection (VIP) is a measure of a variable importance in the PLS-DA model. The VIP score indicates the contribution a variable makes to the model. A greater value denotes more importance. D0NOE-LL = longissimus lumborum on day 0 NOE surface, D6NOE-LL = longissimus lumborum on day 6 NOE surface, D0OE-LL = longissimus lumborum on day 0 OE surface.

Figure 3.

Figure 3.

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Heat map demonstrating the patterns in metabolite concentrations based on normalized intensity values across oxygen exposure (OE) and display time. The red color indicates a higher abundance, and the blue color indicates a lower abundance. D0NOE-LL = longissimus lumborum on day 0 non-oxygen exposed surface, D6NOE-LL = longissimus lumborum on day 6 non-oxygen exposed surface, D0OE-LL = longissimus lumborum on day 0 OE surface.

Figure 1.

Figure 1.

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Schematic representation of oxygen penetration, oxygen exposed and non-oxygen exposed surfaces.

There were 30 differently abundant (P < 0.05) metabolites between day 0 NOE surface and day 6 OE surface (Table 4). Twelve metabolites were lower on day 0 compared with day 6, while the remaining 18 were greater on day 0 NOE versus day 6 OE surface (Figure 5). Upregulated metabolites on day 0 included succinic acid, 4-hydroxybutyric acid, creatinine, and alpha-tocopherol. Ribonic acid, citric acid, hypoxanthine, gluconic acid, and xylulose were lower on day 0 OE surface versus day 6 OE surface. Evaluating the effect of retail display on the metabolite profile of the NOE surface indicated 28 metabolites were differently abundant (P < 0.05) between days 0 and 6 of display. Of the 28, 14 metabolites were greater, and 14 metabolites were lower on day 0 compared with day 6. Stearic acid, saccharic acid, and arachidic acid were greater on day 0 vs. day 6 of the NOE surface. Furthermore, metabolites such as xylitol, xylulose, and gluconic acid were lower on day 0 in comparison with day 6. The amino acid metabolism increased during display resulting in greater methionine sulfoxide, serine, and tyrosine on day 6 compared with day 0 for both surfaces.

Table 4.

Metabolites differentially abundant in non-oxygen exposed (NOE) and oxygen exposed (OE) surfaces1 of the longissimus lumborum during days 0 and 6 of display

Functional role Metabolite FDR P-value NOE2 OE Day 6
Day 0 Day 6 Day 0 Day 6 NOE OE
Pentose and glucuronate interconversions Xylulose 0.01 0 Down Up Down Up NS
Xylitol 0.04 0.001 Down Up NS Up Down
Pentose phosphate pathway Gluconic acid 0.04 0.001 Down Up Down Up Down Up
Fatty acid metabolism Stearic acid 0.05 0.003 Up Down Up Down NS
Saccharic acid 0.1 0.012 Up Down Up Down NS
Arachidic acid 0.16 0.03 Up Down Up Down Up Down
Hexadecylglycerol 0.1 0.013 Up Down Up Down NS
1-Monopalmitin 0.09 0.007 Up Down Up Down NS
Glycerol 0.07 0.004 Up Down Up Down NS
TCA cycle Citric acid 0.04 0.001 Down Up Down Up Down Up
Aconitic acid 0.09 0.007 Down Up Down Up NS
Succinic acid 0.15 0.027 Up Down Up Down NS
Amino acid metabolism
Methionine sulfoxide 0.15 0.025 Down Up Down Up NS
Serine 0.15 0.027 Down Up Down Up NS
Tyrosine 0.16 0.034 Down Up Down Up NS
Sugar metabolism Ribose 0.13 0.018 Down Up Down Up NS
Ribonic acid 0.14 0.022 Down Up Down Up Down Up
Ascorbate metabolism Isothreonic acid 0.05 0.003 Down Up Down Up Up Down
Steroid hormone biosynthesis Cholesterol 0.05 0.003 Up Down Up Down NS
Oxidative phosphorylation Creatinine 0.07 0.005 Up Down Up Down Up Down
Purine metabolism Hypoxanthine 0.09 0.008 Down Up Down Up NS
Pyrazine metabolism 2,5-Dihydroxypyrazine 0.09 0.009 Up Down Up Down Up Down
Collagen metabolism Glycylproline 0.1 0.01 Down Up Down Up NS
Antioxidant Alpha-tocopherol 0.1 0.012 NS Up Down Up Down
Neurotransmitter 4-Hydroxybutyric acid 0.1 0.012 NS Up Down Up Down
Propionate metabolism Propylene glycol 0.14 0.021 Up Down Up Down Up Down
Glyoxylate and dicarboxylate metabolism Glycolic acid 0.16 0.033 NS Up Down Up Down
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1Exposure of muscle to oxygen as display surface indicated as oxygen exposed (OE) or interior of the muscle as non-oxygen exposed (NOE).

2Represents comparison between NOE days 0 and 6; NOE day 6 and OE day 6, and NOE day 0 and OE day 6.

Figure 5.

Figure 5.

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Separation of the significantly differentially abundant metabolites for the non-oxygen exposed (NOE) surface on day 0, NOE surface on day 6, and oxygen exposed surface on day 6 of the longissimus muscle during retail display.

Thirteen metabolites were identified to be differently abundant (P < 0.05) in the NOE surface relative to the OE surface on day 6 of retail display (Figure 6). Three metabolites were lower on the NOE surface in contrast to the OE surface. Metabolites such as creatinine, alpha-tocopherol, and 4-hydroxybutyric acid are greater, while gluconic acid, citric acid, and ribonic acid are lower in the NOE surface in comparison with the OE surface.

Figure 6.

Figure 6.

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Box and whisker plots of significantly different metabolites between the non-oxygen exposed (NOE) and oxygen exposed (OE) surfaces of the longissimus muscle during retail display. D0NOE-LL = longissimus lumborum on day 0 NOE surface, D6NOE-LL = longissimus lumborum on day 6 NOE surface, D0OE-LL = longissimus lumborum on day 0 OE surface.

Discussion

Several studies have reported the loss of redness during retail display of the longissimus muscle (Joseph et al., 2012; Abraham et al., 2017). However, limited studies considered biochemical changes occurring on surfaces exposed to oxygen and within the interior of steaks. A previous study noted that MRA was lower for the OE surface compared to the NOE surface of the longissimus muscle (Mancini et al., 2008). In addition, there was a strong correlation between the OE surface MRA and the loss of redness during retail display (Mancini et al., 2008). The longissimus is categorized as a color-stable muscle with greater antioxidant capacity (Joseph et al., 2012) and lower oxidative stress (Ke et al., 2017) compared with less color-stable muscles such as the psoas major muscle. Therefore, the OC of the longissimus muscle may have a limited impact by the presence of oxygen. The results of the current study also indicate color stable nature of longissimus based on the limited effects of oxygen on metabolites such as succinate related to color stability.

Metabolite profiles of OE and NOE surfaces were different with storage time. Free amino acids have been reported to increase in the longissimus muscle during storage (Abraham et al., 2017; Ma et al., 2017; Mitacek et al., 2019). Postmortem proteolysis increases free amino acids such as serine and tyrosine (Feidt et al.,1996). Tyrosine (Subbaraj et al., 2016; Ma et al., 2017) and serine (Subbaraj et al., 2016; Mitacek et al., 2019) have been reported to increase in the longissimus during storage. However, the connection between these amino acids and color stability is unclear. The enzyme methionine sulfoxide reductase has been reported to reduce methionine sulfoxide to methionine (Lee and Gladyshev, 2011). Meanwhile, methionine sulfoxide was indicated to be a potential biomarker of aging and disease, whereas methionine was used to create antioxidant systems in the body (Lee and Gladyshev, 2011). Wu et al. (2016) reported a strong positive correlation between a* values and mitochondrial peptide methionine sulfoxide reductase in the longissimus muscle. Joseph et al. (2012) noted more methionine sulfoxide reductase, and Abraham et al. (2017) reported more methionine in the color-stable longissimus muscle over the color labile psoas major muscle. Furthermore, higher methionine in color-stable was demonstrated in ovine meat than color labile (Subbaraj et al., 2016). Hence, methionine and methionine sulfoxide reductase are indicative of color stability. Therefore, the presence of methionine sulfoxide supports less color stability and more myoglobin oxidation during retail display of the muscle.

Fatty acid metabolism decreased during retail display for both the NOE and OE surfaces. Fatty acids such as stearic acid, arachidic acid, and saccharic acid decreased during ­display. Previous studies also reported changes in fatty acid levels in longissimus steaks with storage time (Abraham et al., 2017; Yu et al., 2020). Fatty acid attached to glycerol can be released during storage time (Mitacek et al., 2019), which would support the changes in fatty acids in the current study. Depletion of creatinine and glycerol during display indicates the shift in metabolism due to the depletion of energy sources during beef longissimus retail display (Mitacek et al., 2019). In support, creatinine content decreased with beef longissimus aging time (Mitacek et al., 2019). Creatinine had greater abundance in the NOE surface than the OE surface on day 6 of display. In a previous live animal study, creatinine production was higher in diseased small ruminant animals than in healthy animals (Bozukluhan et al., 2018). In physiological conditions, creatinine will be broken down to creatine by the kidneys (Bozukluhan et al., 2018). The creatinine metabolites such as methyl guanidine and creatol increase in the presence of oxidants (Abramowitz et al., 2010). Therefore, the presence of oxygen at the retail surface and subsequent oxidative stress may lead to the breakdown and lower content of creatinine in comparison to the interior surface.

Sugar metabolism is increased during display, with ribose and ribonic acid being greater on day 6 compared with day 0 for both surfaces. A previous study also reported increased ribonic acid in longissimus muscle during retail display (Abraham et al., 2017). Ribose content increased with greater postmortem time in chicken, and ribose contributes to flavor from the breakdown of IMP (Aliani et al., 2013). Ribose is a component of ATP and is formed from the breakdown of ATP in postmortem muscle (Dhanoa and Housner, 2007).

The changes in TCA cycle metabolites were more specific to oxygen exposure and storage time. Citric acid and aconitic acid increased during retail display for both surfaces, while succinic acid decreased for both surfaces. Citrate can form succinate through the TCA cycle, with aconitate acting as an intermediate (Krebs and Johnson, 1980). Succinate can reduce metmyoglobin through electron transport-mediated metmyoglobin reduction (Tang et al., 2005) and reverse transport metmyoglobin reduction (Belskie et al., 2015). Therefore, the changes in TCA metabolites may be due to forming succinate via citric and aconitic acid to stabilize the color of the longissimus during retail display. Abraham et al. (2017) reported greater citric acid in the longissimus with greater display time but did not consider the impact of oxygen exposure. As succinate is a part of metmyoglobin reduction (Tang et al., 2005), the accumulation of citric acid at the surface may be due to the increased demands for the reduction of metmyoglobin by producing succinate from citrate. However, oxidative conditions can limit the activity of enzymes involved in MRA and OC. Therefore, a greater abundance of citric acid on the OE surface may relate to the oxidative changes occurring at the surface.

4-Hydroxybutyric acid is a neurotransmitter in the brain and is active in mitochondria (Bourguignon et al., 1988; Gibson and Nyhan, 1989; Caputo et al., 2009). In the mitochondria, oxidation of 4-hydroxybutyric acid can form succinic acid (Gibson and Nyhan, 1989; Caputo et al., 2009). Therefore, greater 4-hydroxybutyric acid in the NOE surface may indicate the use of 4-hydroxybutyric acid on the exposed surface to form succinate. Therefore, the NOE would have more ability to reduce metmyoglobin via MRA.

After death, ATP production continued in beef for 24 h (Matarneh et al., 2017). As ATP production decreases with postmortem time, ADP is broken down to hypoxanthine (Matarneh et al., 2017). Hypoxanthine has been reported to increase with beef longissimus aging (Ma et al., 2017) and postmortem time (Yu et al., 2020) supporting the increase during retail display. Hypoxanthine increases superoxide and decreases the activity of antioxidant enzymes (Mesquita Casagrande et al., 2013; Rodrigues et al., 2014). Therefore, the greater amount of hypoxanthine during retail display could promote radical oxygen species and oxidative stress during retail display on both NOE and OE surfaces.

Glycolate is reported to be present in humans and can form glyoxylate and, subsequently toxalate (Fry and Richardson, 1979; Booth et al., 2006). The reduction of glyoxylate to glycolate occurs by lactate dehydrogenase or glyoxylate reductase via the consumption of NADH in the muscle to prevent the accumulation of glyoxylate, which can be highly reactive (Mdluli et al., 2005; Booth et al., 2006). Glyoxylate addition could increase radical oxygen species leading to the apoptosis of cells by affecting the membrane potential of the mitochondria (Patra et al., 2018). Therefore, greater glycolate may indicate less reactive glyoxylate formation in the NOE portion of the muscle and limited oxidative changes.

Isothreonic acid is a product of ascorbic acid oxidation (Arun et al., 2018). A greater formation of isothreonic acid was reported in mice brain under oxidative stress from greater radical oxygen species (Arun et al., 2018). Furthermore, isothreonic acid content was greater in obese women under oxidative stress (Ruebel et al., 2019). Therefore, the oxidation of ascorbic acid to isothreonic acid may be due to the response and prevention of further oxidative stress on the NOE surface. Furthermore, ascorbic acid was indicated to reduce metmyoglobin in vitro (Vestling, 1942; Tomoda et al.,1978; Tsukahara and Yamamoto, 1983;). Furthermore, ascorbic acid has been reported to extend color stability of beef (Sanchez-Escalante et al., 2001, 2003) and lamb (Andres et al., 2017) during retail display. Therefore, the greater formation of isothreonic acid may indicate the use of ascorbic acid to extend color stability of the NOE surface.

Alpha-tocopherol (vitamin E) is an antioxidant that can increase meat color stability when fed to cattle (Chan et al., 1995). More specifically, dietary vitamin E decreases myoglobin and lipid oxidation in beef (Faustman et al., 1998) due to the deposition of vitamin E in cell membranes when fed to cattle. Furthermore, researchers speculated that the supplementation of tocopherol influenced the mitochondrial proteome and oxidative enzymes and possibly minimized oxidative stress (Zhai et al., 2019). In support, research comparing the longissimus and psoas major reported greater oxidative enzymes in the psoas major, which could be important in the lower color stability and greater oxidative stress of the psoas major (Zhai et al., 2020). Hence, the greater tocopherol content in the NOE surface may be due to limited oxygen presence and reduced oxidative stress in comparison to the OE surface.

Gluconic acid is part of glycolytic metabolism and is formed through the degradation of glucose (Bankar et al., 2009; Kornecki et al., 2020). The increase in gluconic acid on the exposed surface could relate to increased glycolytic metabolites. Xylitol was lower in the NOE surface of the longissimus muscle. Furthermore, xylitol is a product of the pentose phosphate pathway from the consumption of NADH and the breakdown of xylose (Aguer et al., 2017). Past research has indicated a greater xylitol content in mice with overexpression of proteins minimizing oxidative damage (Aguer et al., 2017). Therefore, in the present study, greater xylitol on the OE surface could indicate the response to oxygen-induced oxidative stress during retail display and the use of NADH and xylose to limit damage.

Bioinformatics analysis of differently abundant metabolites to understand the impact of oxygen exposure on muscle biochemistry

Based on the heatmap, retail display time and oxygen exposure influenced the metabolite profile changes. The KEGG pathway impact analysis was completed to evaluate the metabolic pathways associated with the differently abundant metabolites among day 0 NOE, day 6 NOE, and day 6 OE surfaces (Figure 4). The oxygen exposure influenced pentose phosphate pathway, TCA cycle, pentose and glucuronate interconversions, phenylalanine, tyrosine, and tryptophan metabolism. A VIP analysis reported citric acid, gluconic acid, and ribonic acid influencing the PLS-DA plot separation between the retail days and oxygen exposure. Longissimus is a color-stable muscle and has predominant glycolytic fibers. Hence, oxidative stress is relatively lower than color labile muscles such as psoas major muscles. In support, mitochondrial damage was less in longissimus than psoas muscles (Ke et al., 2017). A previous study noted that the pentose phosphate pathway is activated to produce reducing amino acids and nucleotide equivalents during oxidative stress (Stincone et al., 2015). Furthermore, retail storage and oxidative stress influenced tricarboxylic acid cycle metabolites such as citric acid, aconitic acid, and succinic acid. Previous bioinformatic analysis showed the presence of more glycolytic proteins in more color stable muscles (Gagaoua et al., 2020). Oxygen impacts on the oxidative stress of the longissimus are supported by the increase in oxidation of ascorbic acid and formation of isothreonic acid as well as the loss of alpha-tocopherol and 4-hydroxybutyric acid.

Figure 4.

Figure 4.

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KEGG pathway analysis to determine the impact of differently present metabolites on biological processes in longissimus muscle during retail display and oxygen exposure. X-axis represents the impact of the pathway, and the y-axis is the −log(P-value). The dot color is based on the P-value, and the dot size is based on the pathway impact values.

Conclusions

The oxygen exposure during retail display negatively affected color stability compared to the interior of muscle. Specifically, OC and MRA decreased with oxygen exposure indicating the negative effects of oxygen on biochemical attributes of the longissimus muscle. Oxygen exposure influences the metabolome of the longissimus during retail display. The exposed surface represented more changes in response to oxidative stress with the loss of alpha-tocopherol and 4-hydroxybutyric acid and a lower abundance of metabolites that can regenerate NADH and extend color stability, such as citric acid. Therefore, the depletion of metabolites that can be involved in metmyoglobin reduction accelerates the migration of brown later from the interior to the surface and promotes discoloration.

Acknowledgment

This research was supported, in part, by the Agriculture and Food Research Institute Grant 2021-09413 from the USDA National Institute of Food and Agriculture program [Accession Number: 1027928].

Glossary

Abbreviations

NADH

nicotinamide adenine dinucleotide plus hydrogen

TCA

tricarboxylic acid cycle

USDA

United States Department of Agriculture

Contributor Information

Morgan L Denzer, Department of Animal Sciences, Oklahoma State University, Stillwater, OK 74078, USA.

Morgan Pfeiffer, Department of Animal Sciences, Oklahoma State University, Stillwater, OK 74078, USA.

Gretchen G Mafi, Department of Animal Sciences, Oklahoma State University, Stillwater, OK 74078, USA.

Ranjith Ramanathan, Department of Animal Sciences, Oklahoma State University, Stillwater, OK 74078, USA.

Conflict of Interest Statement

The authors declare no conflict of interest.

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