Evaluating the failure to bloom in dark-cutting and lactate-enhanced beef longissimus steaks.

Previous studies have noted lower L* (lightness) values for both dark-cutting beef and normal-pH beef enhanced with lactate. In the current study, absorption-coefficient, scattering-coefficient, CIE L*a*b* values, refractive index of sarcoplasm, and inter-muscle bundle space were evaluated for dark-cutting beef, normal-pH beef enhanced with lactate, normal-pH beef enhanced with water, and normal-pH beef not enhanced with either water or lactate. Compared with non-enhanced loins, lactate-enhancement had lower a*, chroma, oxymyoglobin, reflectance, scattering, and inter-muscle bundle space as well as greater absorption and refractive index. Dark-cutting steaks had lower a*, chroma, oxymyoglobin values, reflectance, and scattering as well as less inter-muscle bundle space compared with lactate-enhanced steaks. Sarcoplasm refractive index values were greater in lactate-enhanced steaks than dark-cutting steaks. The results suggest that changes in muscle structure and optical properties due to either pH or lactate addition can alter muscle darkening and blooming properties.

Muscle-Specific Mitochondrial Functionality and Its Influence on Fresh Beef Color Stability.

Fresh beef color stability is a muscle-specific trait. Mitochondria remain biochemically active in postmortem beef muscles and influence meat color. Although several intrinsic factors governing muscle-specific beef color have been studied extensively, the role of mitochondrial functionality in muscle-dependent color stability is yet to be examined. Therefore, the objective of this study was to examine mitochondrial oxygen consumption rate (OCR), mitochondrial metmyoglobin reducing activity (MMRA), and instrumental color attributes in beef Longissimus lumborum (LL) and Psoas major (PM) during retail display. Using a split-plot design, six (n = 6) beef LL and PM muscles were fabricated into 2.54-cm-thick steaks, packaged in polyvinylchloride overwrap, and randomly assigned to instrumental color measurement for six days and mitochondrial isolation for days 0, 1, 3, or 5 of display at 4 °C. Mitochondria isolated from steaks were used to assess the effects of muscle and display time on OCR and MMRA. The PM steaks were less color-stable (p < 0.05) during display compared with the LL counterparts. For both muscles, OCR decreased during display, albeit the decrease was more rapid in PM than in the LL. Similarly, MMRA decreased during display for the LL and PM. However, this decrease was less (p < 0.05) for mitochondria from LL steaks, which were more resistant to display-mediated effects on OCR and MMRA. These results indicated that the muscle-specific differences in mitochondrial activity may contribute partially to the variations in color stability of beef LL and PM muscles. PRACTICAL APPLICATION: During retail display tenderloin steaks packaged in PVC overwrap discolor quicker than strip loin steaks. This research determines the basis for muscle-specific differences in color stability. The results indicate that mitochondria present in tenderloin lose its functionality faster than strip loin mitochondria. Developing strategies to minimize muscle-specific differences in mitochondrial changes can increase color stability and value of fresh beef.

Lipid-oxidation-induced carboxymyoglobin oxidation.

We evaluated the usefulness of the ratio A503/A581 as a browning index (BI) for estimating brown color formation in solutions containing oxymyoglobin (OxyMb) and carboxymyoglobin (COMb). In split-chamber cuvette analyses with different proportions of metmyoglobin (MetMb), COMb and OxyMb, BI was highly correlated (r = 0.93-0.94) with direct estimation of MetMb. Moreover, A503/A581 was not influenced by different COMb-OxyMb proportions. Second, we investigated 4-hydroxy-2-nonenal (HNE)-induced spectral changes in OxyMb and COMb solutions. At pH 7.4 and 37 degrees C, BI was greater in HNE-treated OxyMb and COMb samples than in aldehyde-free controls (P < 0.05). However, at pH 5.6 and 4 degrees C, HNE-induced browning was more pronounced in COMb than in OxyMb. These results indicated that COMb is susceptible to lipid-oxidation-induced browning in a pH- and temperature-dependent manner.

Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles.

The objective was to characterize the beef psoas major (PM), longissimus lumborum (LL), superficial semimembranosus (SSM), deep semimembranosus (DSM), and semitendinosus (ST) muscles for differences in instrumental and visual color, metmyoglobin-reducing activity (MRA), total reducing activity (TRA), and cytochrome c oxidase activity. The LL and ST had the most color stability and MRA (p < 0.05), the DSM and PM had the least (p < 0.05), and values for the SSM were intermediate. Visual color (r = -0.66) and a and chroma (r = 0.68) were more correlated with MRA than with TRA (r < 0.14 for all measures). This research supports previous reports that color stability among muscles is variable and that MRA is more useful than TRA for explaining the role of reducing activity in muscle-color stability.

Mechanism for lactate-color stabilization in injection-enhanced beef.

In two experiments, the relationship between metmyoglobin (MMb) reduction and lactate to pyruvate conversion with concomitant production of reduced nicotinamide adenine dinucleotide (NADH) via lactic dehydrogenase (LDH) was investigated. In experiment 1, nonenzymatic reduction of horse MMb occurred in a lactate-LDH-NAD system. Exclusion of NAD+, L-lactic acid, or LDH resulted in minimal MMb reduction. Increasing NAD+ and L-lactic acid concentrations increased reduction. In experiment 2, beef strip loins (longissimus lumborum muscle) were injected with combinations of potassium lactate, sodium tripolyphosphate, sodium chloride, and/or sodium acetate. Steaks were packaged in high-oxygen (80% oxygen/20% carbon dioxide) modified-atmosphere packaging and stored for 2-9 days and then placed in a fluorescent-lighted, open-top display case for 5 days at 1 degrees C. Enhancing loins with 2.5% potassium lactate significantly increased LDH activity, NADH concentration, MMb-reducing activity, and subsequent color stability during display. These research results support the hypothesis that enhancing beef with lactate replenishes NADH via increased LDH activity, ultimately resulting in greater meat color stability.

The effects of freeze-thaw and sonication on mitochondrial oxygen consumption, electron transport chain-linked metmyoglobin reduction, lipid oxidation, and oxymyoglobin oxidation.

Mitochondria potentially influence Mb redox stability in meat by (1) decreasing partial oxygen pressure via oxygen consumption, (2) mitochondrial electron transport chain (ETC)-linked reduction of MetMb, and/or (3) oxidation of mitochondrial membrane lipid. The objective of this study was to investigate the effect of freeze-thaw and sonication treatments on mitochondrial oxygen consumption, ETC-dependent MetMb reducing activity, lipid oxidation, and Mb redox stability. Mitochondria were frozen and thawed (-18°C for 2h and 4°C for 0.5h) for 3 cycles, or sonicated for 30s with a sonic dismembrator. State III oxygen consumption rate (OCR) was decreased by both treatments at pH 7.2, and by sonication only at pH 5.6 (P<0.05). There was no effect on state IV OCR (P>0.05). Respiratory control ratio (RCR) was decreased by freeze-thaw and sonication at pH 7.2 and 5.6 (P<0.05). Sonication increased mitochondrial lipid oxidation and MetMb formation (P<0.05); a similar effect was observed in sonicated samples in the presence of ascorbic acid and ferric chloride (P<0.05). Sonication also decreased mitochondrial ETC-dependent MetMb reduction (P<0.05). These results suggested that sonication treatment had the potential to affect Mb stability via mitochondrial lipid oxidation and/or ETC-mediated MetMb reduction, but the effect on myoglobin stability by freeze-thaw treatment was minimal.

Postmortem oxygen consumption by mitochondria and its effects on myoglobin form and stability.

The objective of this study was to assess the morphological integrity and functional potential of mitochondria from postmortem bovine cardiac muscle and evaluate mitochondrial interactions with myoglobin (Mb) in vitro. Electron microscopy revealed that mitochondria maintained structural integrity at 2 h postmortem; prolonged storage resulted in swelling and breakage. At 2 h, 96 h, and 60 days postmortem, the mitochondrial state III oxygen consumption rate (OCR) and respiratory control ratio decreased with time at pH 7.2 and 5.6 (p < 0.05). Mitochondria isolated at 60 days did not exhibit ADP-induced transitions from state IV to state III oxygen consumption. Tissue oxygen consumption also decreased with time postmortem (p < 0.05). Mitochondrial oxygen consumption was inhibited by decreased pH in vitro (p < 0.05). In a closed system, mitochondrial respiration resulted in decreased oxygen partial pressure (pO(2)) and enhanced conversion of oxymyoglobin (OxyMb) to deoxymyoglobin (DeoMb) or metmyoglobin (MetMb). Greater mitochondrial densities caused rapid decreases in pO(2) and favored DeoMb formation at pH 7.2 in closed systems (p < 0.05); there was no effect on MetMb formation (p > 0.05). MetMb formation was inversely proportional to mitochondrial density at pH 5.6 in closed systems. Mitochondrial respiration in open systems resulted in greater MetMb and DeoMb formation at pH 5.6 and pH 7.2, respectively, vs controls (p < 0.05). The greatest MetMb formation was observed with a mitochondrial density of 0.5 mg/mL at both pH values in open systems. Mitochondrial respiration facilitated a shift in Mb form from OxyMb to DeoMb or MetMb, and this was dependent on pH, oxygen availability, and mitochondrial density.

Interactions between mitochondrial lipid oxidation and oxymyoglobin oxidation and the effects of vitamin E.

Off-flavor and discoloration of meat products result from lipid oxidation and myoglobin (Mb) oxidation, respectively, and these two processes appear to be interrelated. The objective of this study was to investigate their potential interaction in mitochondria and the effects of mitochondrial alpha-tocopherol concentrations on lipid oxidation and metmyoglobin (MetMb) formation in vitro. The addition of ascorbic acid and ferric chloride (AA-Fe(3+)) increased ovine and bovine mitochondrial lipid oxidation when compared with their controls (p < 0.05); MetMb formation also increased with increased lipid oxidation relative to controls (p < 0.05). Reactions containing Mb and mitochondria with greater alpha-tocopherol concentrations demonstrated less lipid oxidation and MetMb formation than mitochondria with lower alpha-tocopherol concentrations. Greater mitochondrial alpha-tocopherol concentration was also correlated with increased mitochondrial oxygen consumption in vitro and with a more pronounced effect at pH 7.2 than at pH 5.6. Relative to controls, succinate addition to bovine mitochondria resulted in increased concentrations of ubiquinol 10 and alpha-tocopherol and decreased lipid and Mb oxidation (p < 0.05). Mitochondrial lipid oxidation was closely related to MetMb formation; both processes were inhibited by alpha-tocopherol in a concentration-dependent manner.

Mitochondrial reduction of metmyoglobin: dependence on the electron transport chain.

Reduction of ferric myoglobin (metmyoglobin, MetMb) to its ferrous form is important for maintaining fresh meat color because only reduced myoglobin can bind oxygen to form the consumer-preferred cherry red color in fresh meat. The objective of this study was to characterize an apparent mitochondria electron transport chain (ETC)-linked pathway for MetMb reduction in vitro. MetMb was reduced in the presence of mitochondria and succinate (p < 0.05); mitochondria or succinate alone did not facilitate MetMb reduction relative to controls (p > 0.05). Flushing samples with oxygen greatly decreased MetMb reduction, while flushing with argon increased MetMb reduction when compared with controls (p < 0.05). ETC inhibitors were used to localize the site where electrons became available for MetMb reduction. MetMb reduction was increased by rotenone addition and decreased by malonic acid (p < 0.05); the reduction was completely abolished by additions of antimycin A or myxothiazol when compared with controls (p < 0.05). These results suggest that electrons become available for MetMb reduction at a site(s) between complex III and IV. Mitochondrial ETC-linked MetMb reduction increased with increased mitochondrial density and succinate concentration (p < 0.05); the greatest MetMb reduction was observed at pH 7.2 and 37 degrees C, and ETC-linked MetMb reducing activity decreased with time postmortem (p < 0.05). These results indicate that ETC-linked MetMb reduction exists but would be minimally active in postmortem muscles.

Covalent binding of 4-hydroxy-2-nonenal to lactate dehydrogenase decreases NADH formation and metmyoglobin reducing activity.

Lactate dehydrogenase (LDH) activity can regenerate NADH, which is a critical component in metmyoglobin reduction. However, limited research has determined the effects of lipid oxidation products on LDH activity. The overall objective of this study was to determine the effects of 4-hydroxy-2-nonenal (HNE) on LDH activity. LDH was reacted with HNE at pH 5.6 and 7.4, and LDH activity was measured as NADH formation following the addition of lactate and NAD. The effects of HNE on NADH-dependent metmyoglobin reduction also were analyzed. Mass spectrometric examination revealed that HNE adducts to LDH at both pH 5.6 and 7.4. More specifically, HNE binds with cysteine and histidine residues of LDH at pH 5.6 and 7.4. Covalent binding of HNE decreased NADH formation and metmyoglobin reduction (P < 0.05). These results indicate that secondary lipid oxidation products can inactivate enzymes involved in metmyoglobin reduction and have the potential to increase beef discoloration.

Effects of pyruvate on lipid oxidation and ground beef color.

UNLABELLED: Our overall objective was to better understand the effects of added pyruvate on enhanced beef color stability. The 2 possible mechanisms assessed were the role of pyruvate in lipid oxidation and direct interaction between pyruvate and beef myoglobin. Microsomes were incubated with pyruvate at pH 5.6, 25 °C, and lipid oxidation was measured hourly for 3 h. Bovine oxymyoglobin at pH 5.6 was incubated with pyruvate and used to quantify both redox stability (metmyoglobin formation) and pyruvate-myoglobin adduction using mass spectrometry analysis. Surface color and lipid oxidation were measured on ground beef patties stored for 6 d in polyvinyl chloride over-wrap (PVC) or high oxygen. Addition of pyruvate to microsomes decreased lipid oxidation compared with controls (P < 0.05). Conversely, no effect on myoglobin was observed (no changes in redox stability and no peaks corresponding to pyruvate were observed; P > 0.05). However, pyruvate increased color stability and decreased lipid oxidation of ground beef patties packaged in PVC and high oxygen. Pyruvate decreased nitric oxide metmyoglobin-reducing capacity and oxygen consumption of patties compared with controls (P < 0.05). This research suggests that pyruvate may improve beef color stability primarily through its antioxidant effect on lipids. PRACTICAL APPLICATION: Discoloration of meat often results in significant revenue loss. This study suggests that pyruvate can improve the color stability of patties packaged in high oxygen and PVC primarily through its antioxidant effect on lipids.

Redox instability and hemin loss of mutant sperm whale myoglobins induced by 4-hydroxynonenal in vitro.

The effects of 4-hydroxy-2-nonenal (HNE) on redox stability of Oxy- and Deoxy- wild-type (WT) and recombinant sperm whale myoglobins (P88H/Q152H, L29F, H97A, and H64F) and hemin loss from Met-myoglobin (Mb) were investigated. HNE induced greater redox instability in WT and mutant Mbs compared to controls (p < 0.05). The extent of HNE-induced OxyMb oxidation was lesser in L29F (p < 0.05) and greater in H97A and P88H/Q152H than in WT (p < 0.05). H64F DeoxyMb was more redox stable than WT DeoxyMb in the presence of HNE (p < 0.05). HNE alkylation occurred exclusively on histidine residues, and histidine 48 was alkylated in all sperm whale myoglobins. HNE alkylation accelerated the protoporphyrin moiety loss only in H97A. Met- forms of WT and L29F but not Deoxy- or Oxy- forms released hemin during storage. Primary structure strongly influenced Mb redox stability in the presence of reactive secondary lipid oxidation products.

Species-specific myoglobin oxidation.

The effect of the lipid oxidation product, 4-hydroxy-2-nonenal (HNE), on oxidation of oxymyoglobin (OxyMb) from seven different meat-producing species was investigated. Relative to controls, HNE increased OxyMb oxidation within all species (p < 0.05) at both 25 and 4 °C, pH 5.6. The relative effect of HNE was greater for myoglobins (Mbs) that contained 12 ± 1 histidine (His) residues than for those that contained 9 His residues (p < 0.05); HNE efficacy in all species except chicken and turkey decreased with time. Mono-HNE adducts were detected in all species except chicken and turkey. In general, HNE alkylation increased the Mbs' ability to accelerate lipid oxidation in a microsome model. However, neither an HNE nor a Mb species dependent effect was observed. Results suggested that microsome model system associated lipid oxidation overshadowed HNE and species effects on OxyMb oxidation observed in lipid-free systems.

Effects of lactate on bovine heart mitochondria-mediated metmyoglobin reduction.

Our objective was to determine the combined effects of lactate, LDH, and NAD on metmyoglobin reduction in mitochondria isolated from bovine cardiac muscle. Mitochondria were reacted with various combinations of lactate, LDH, NAD, and mitochondrial inhibitors, and oxygen consumption was measured using a Clark oxygen electrode. Mitochondria (3 mg/mL) and bovine metmyoglobin (0.15 mM) also were reacted with substrates/enzymes/inhibitors to determine mitochondria-mediated metmyoglobin reduction in vitro. Combining lactate-LDH-NAD with isolated mitochondria increased oxygen consumption as well as metmyoglobin reduction compared with those of either control mitochondria (without lactate) or mitochondria with added lactate, at pH 5.6 and 7.4 (p < 0.05). The addition of mitochondrial and LDH inhibitors to lactate-LDH-NAD decreased oxygen consumption and metmyoglobin reduction (p < 0.05). NADH formed from lactate-LDH-NAD can be used for nonenzymatic (via the electron transport chain) and enzymatic (NADH-dependent metmyoglobin reductase) metmyoglobin reduction.

Effects of lactate on beef heart mitochondrial oxygen consumption and muscle darkening.

The mechanism of lactate-induced beef color darkening is unclear. Our objective was to evaluate the ability of mitochondria isolated from bovine cardiac muscle to utilize lactate as a fuel for respiration. Addition of lactate (4, 8, and 16 mM) to isolated bovine cardiac mitochondria resulted in state IV oxygen consumption at pH 7.2 and 25 degrees C measured using a Clark oxygen electrode. Combining mitochondria with lactate, LDH, and NAD increased state IV oxygen consumption compared with that of lactate alone (p < 0.05). Moreover, oxygen consumption resulting from the addition of lactate-LDH-NAD (0.2 mM each) was comparable to oxygen consumption resulting from the direct addition of NADH (0.2 mM) to mitochondria at pH 7.2. Rotenone reduced (p < 0.05) lactate-mediated darkening in bovine cardiac muscle homogenates. Lactate-induced beef color darkening may be due to increased oxygen consumption by mitochondria, which out-competes myoglobin for oxygen and results in dark colored muscle.

Mass spectrometric characterization and redox instability of turkey and chicken myoglobins as induced by unsaturated aldehydes.

Turkey and chicken myoglobins (Mbs) were isolated, purified, and characterized using electrospray ionization mass spectrometry (ESI-MS), and the effect of unsaturated aldehydes (nonenal and hexenal) on their redox stability was investigated in vitro. The deconvoluted spectra from ESI-MS exhibited a molecular mass of 17291 Da for both turkey and chicken Mbs. Significant homogeneity in the fragmentation pattern of both Mbs was indicated by ESI-MS/MS. Both turkey and chicken oxymyoglobins (OxyMbs) were more prone to oxidation at pH 5.8 than at pH 7.4. Metmyoglobin formation was greater in the presence of unsaturated aldehydes than controls (P < 0.05). The results demonstrated that both turkey and chicken Mbs have identical molecular mass and that the effects of alpha,beta-unsaturated aldehydes on their redox stability are consistent with those of mammalian livestock Mbs.