Meat quality traits and proteome profile of woody broiler breast (pectoralis major) meat.

Woody breast meat has recently become prevalent in the broiler industry in both the United States and European Union. Recent publications have described the meat quality characteristics of woody breast meat as having hardened areas and pale ridge-like bulges at both the caudal and cranial regions of the breast. The present study investigated the meat quality (pH, color, cooking loss, and shear force) and protein quality characteristics (protein and salt-soluble protein content) in woody breast meat as compared to normal breast meat. In addition, the differences in the muscle proteome profiles of woody and normal breast meat were characterized. Results indicated that woody breast meat had a greater average pH (P < 0.0001) and cooking loss (P = 0.001) than normal breast meat, but woody breast meat did not differ in shear force (P > 0.05) in comparison to normal breast meat samples. The L*, a*, and b* values of woody breast fillets were greater than normal breast fillets (P < 0.0001 to L*; P = 0.002 to a*; P = 0.016 to b*). The woody breast meat had more fat (P < 0.0001) and moisture (P < 0.021) and less protein (P < 0.0001) and salt-soluble protein (P < 0.0001) when compared with normal breast fillets. Whole muscle proteome analysis indicated 8 proteins that were differentially expressed (P < 0.05) between normal and woody breast meat samples. The differences in muscle proteome between normal and woody breast meat indicated an increased oxidative stress in woody breast meat when compared to normal meat. In addition, the abundance of some glycolytic enzymes, which are critical to the regeneration of adenosine triphosphate (ATP) in postmortem muscles, was lower in woody breast meat than in normal breast meat. Proteomic differences provide additional information on the biochemical pathways and genetic variations that lead to woody breast meat. Further research should be conducted to elucidate the genetic and nutritional contributions to the proliferation of woody breast meat in the United States.

Proteomic approach to characterize biochemistry of meat quality defects.

Proteomics can be used to characterize quality defects including pale, soft, and exudative (PSE) meat (pork and poultry), woody broiler breast meat, reddish catfish fillets, meat toughness, and beef myoglobin oxidation. PSE broiler meat was characterized by 15 proteins that differed in abundance in comparison to normal broiler breast meat, and eight proteins were differentially expressed in woody breast meat in comparison to normal breast meat. Hemoglobin was the only protein that was differentially expressed between red and normal catfish fillets. However, inducing low oxygen and/or heat stress conditions to catfish fillets did not lead to the production of red fillets. Proteomic data provided information pertaining to the protein differences that exist in meat quality defects. However, these data need to be evaluated in conjunction with information pertaining to genetics, nutrition, environment of the live animal, muscle to meat conversion, meat quality analyses and sensory attributes to understand causality, protein biomarkers, and ultimately how to prevent quality defects.

2013 Early Career Achievement Award--Proteomics of muscle- and species-specificity in meat color stability.

Meat color is the most important quality trait influencing consumer purchase decisions. The interinfluential interactions between myoglobin and biomolecules govern color stability in meat. The advances in proteomics, such as high throughput analytical tools in mass spectrometry, 2-dimensional electrophoresis, and bioinformatics, offer themselves as robust techniques to characterize the proteome basis of muscle- and species-specific meat color phenomena. Differential abundance of chaperones and antioxidant proteins contributes to muscle-specific color stability in beef; the greater abundance of chaperones and antioxidant proteins in color-stable Longissimus lumborum than in color-labile Psoas major protects myoglobin and contributes to superior color stability of beef Longissimus steaks. Lipid oxidation-induced myoglobin oxidation is more critical to beef color than pork color due to the inherent differences in myoglobin chemistry; the number of nucleophilic histidine residues adducted by reactive aldehydes is greater in beef myoglobin than in pork myoglobin. Preferential adduction of secondary products of lipid oxidation to beef myoglobin accelerates metmyoglobin formation at a greater degree than in its pork counterpart. Mass spectrometric investigations revealed that although cherry-red carboxymyoglobin is more stable than oxymyoglobin, both redox forms undergo lipid oxidation-induced oxidation in model systems. The accuracy of mass spectrometry to detect the molecular mass of proteins has been applied to differentiate myoglobins from closely related meat animals, such as goats and sheep or emu and ostrich. In addition, this approach indicated that turkey myoglobin is 350 Da greater in molecular mass than beef myoglobin, and the unique biochemistry of turkey myoglobin could be responsible for its greater thermostability in model systems as well as the pink color defect observed in fully cooked uncured turkey products.

Lipid oxidation-induced oxidation in emu and ostrich myoglobins.

Emu and ostrich are ratites gaining increasing popularity as sources of low-fat meats. Secondary products of lipid oxidation, such as 4-hydroxy-2-nonenal (HNE), compromise myoglobin redox stability in a species-specific manner. However, the molecular basis of lipid oxidation-induced oxidation in ratite myoglobins has not been investigated. Therefore, our objective was to characterize lipid oxidation-induced oxidation in ratite myoglobins, in comparison with beef myoglobin. At physiological condition (pH7.4, 37 °C) HNE accelerated (P<0.05) oxidation of emu, ostrich, and beef oxymyoglobins. Autoxidation and HNE-induced oxidation were greater (P<0.05) in ostrich oxymyoglobin than in emu and beef oxymyoglobins. Mass spectrometric analyses revealed that HNE formed mono-adduct with both emu and ostrich myoglobins after 6h of incubation. Tandem mass spectrometry demonstrated that HNE adducted histidine 36 in ostrich myoglobin, whereas histidines 34 and 36 were adducted in emu myoglobin. The results indicate that primary structure of ratite myoglobins influences their redox stability in the presence of prooxidants.

Temperature- and pH-dependent effect of lactate on in vitro redox stability of red meat myoglobins.

Our objective was to evaluate the influence of lactate on in vitro redox stability and thermostability of beef, horse, pork, and sheep myoglobins. Lactate (200 mM) had no effect (P>0.05) on redox stability at physiological (pH7.4, 37°C) and meat (pH 5.6, 4°C) conditions. However, lactate increased (P<0.05) metmyoglobin formation at a condition simulating stressed live skeletal muscle (pH 6.5, 37°C). The redox stability of myoglobins at stressed live skeletal muscle and meat conditions was species-specific (P<0.05). Myoglobin thermostability at 71°C was lower (P<0.05) in the presence of lactate compared with controls and was influenced (P<0.05) by species. The results of the present study indicate that the effects of lactate on myoglobin are temperature and pH dependent. The observed lack of influence of lactate on myoglobin redox stability at meat condition suggests that the color stability of lactate-enhanced fresh meat is not due to direct interactions between the ingredient and the heme protein.

Bovine mitochondrial oxygen consumption effects on oxymyoglobin in the presence of lactate as a substrate for respiration.

Research suggests that NADH formed from lactate addition can increase mitochondrial oxygen consumption. However, limited research has assessed the subsequent effects of lactate-mediated mitochondrial oxygen consumption on oxymyoglobin. Therefore, our objective was to assess the effects of bovine mitochondrial oxygen consumption on oxymyoglobin in the presence of lactate, LDH, and NAD. Isolated beef cardiac and skeletal muscle mitochondria (n=5) were reacted with lactate (40 mM), LDH (100 units), and NAD (0.02 mM) to initiate oxygen consumption. Myoglobin redox state was measured to assess the effects of oxygen consumption on oxymyoglobin. The addition of lactate-LDH-NAD to mitochondria increased (p<0.05) both oxygen consumption and conversion of oxymyoglobin to deoxymyoglobin compared with control mitochondria without substrates. The addition of antimycin A to mitochondria decreased oxygen consumption and deoxymyoglobin formation (p<0.05). The results suggest that increased oxygen consumption can influence myoglobin redox state and this effect might be partially responsible for the darkening effect in lactate enhanced beef.

Effects of 4-hydroxy-2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction.

The effects of 4-hydroxy-2-nonenal (HNE) on mitochondria isolated from bovine hearts (n=5) were assessed using ultrastructure, oxygen consumption, membrane permeability, HNE binding, and metmyoglobin reduction in vitro. Pre-incubation (pH 5.6 and 7.4 at 25°C) of mitochondria with HNE decreased oxygen consumption compared with samples without HNE (P<0.05). Electron microscopy revealed that HNE-treated mitochondria were swollen and had increased membrane permeability at pH 7.4, compared with ethanol controls. Conversely, mitochondria incubated with HNE at pH 5.6 had decreased volume and permeability. Fluorescence studies indicate that HNE binds to the membrane of mitochondria isolated from bovine cardiac muscle (at pH 5.6 and 7.4). HNE-treated mitochondria at both pH 5.6 and 7.4 had lower metmyoglobin reduction and NADH dependent metmyoglobin reductase activity compared with control mitochondria without HNE (P<0.05). In addition to covalent binding with myoglobin, HNE may influence beef color stability by interacting with mitochondria.

Effects of succinate on ground beef color and premature browning.

The objective of this experiment was to determine the effects of succinate on raw and cooked ground beef color. Chubs (n=10) were divided in half and assigned to either succinate (final w/w concentration of 2.5%) or distilled water. Patties (n=14 per chub half) were assigned to initial day 0 color and each of 6 treatment combinations, created by crossing 3 packaging types (vacuum, high-oxygen/80% O(2), and PVC) with 2 storage times (days 1 and 3). After storage, patties were cooked to either 66 °C or 71 °C. Succinate increased (P<0.05) ground beef pH and metmyoglobin reducing activity but had no effect (P>0.05) on raw a* and chroma values. Moreover, succinate decreased (P<0.05) raw L* values, lipid oxidation, and premature browning for patties packaged in PVC and high-oxygen. Succinate may increase cooked patty redness via its influence on meat pH.

Chitosan inhibits premature browning in ground beef.

Our objective was to evaluate the effect of chitosan on premature browning in refrigerated ground beef patties stored in different packaging systems. Ground beef patties (15% fat) with chitosan (1% w/w) or without chitosan (control) were individually packaged either in vacuum (VP), aerobic packaging (AP), carbon monoxide modified atmosphere packaging (LO-OX; 0.4% CO+19.6% CO(2)+80% N(2)), or high-oxygen modified atmosphere packaging (HI-OX; 80% O(2)+20% CO(2)), and stored for 0, 1, or 3 days at 1°C. At the conclusion of storage, raw surface redness was evaluated, patties were cooked to internal end-point temperatures of either 66°C or 71°C, and internal cooked color was measured. The incorporation of chitosan increased (P<0.05) the interior redness of patties stored in AP, VP, and LO-OX, but not in HI-OX. The results of the present study suggest that the incorporation of 1% chitosan minimizes premature browning in ground beef patties stored under AP, VP, and LO-OX.

Packaging-specific influence of chitosan on color stability and lipid oxidation in refrigerated ground beef.

We examined the influence of chitosan on lipid oxidation and color stability of ground beef stored in different modified atmosphere packaging (MAP) systems. Ground beef patties with chitosan (1%) or without chitosan (control) were packaged either in high-oxygen MAP (HIOX; 80% O(2)+20% CO(2)), carbon monoxide MAP (CO; 0.4% CO+19.6% CO(2)+80% N(2)), vacuum (VP), or aerobic packaging (PVC) and stored at 1 °C. Chitosan increased (P<0.05) redness of patties stored in PVC and CO, whereas it had no effect (P>0.05) in HIOX. Chitosan patties demonstrated lower (P<0.05) lipid oxidation than controls in all packaging. Control patties in PVC and HIOX exhibited greater (P<0.05) lipid oxidation than those in VP and CO, whereas chitosan patties in different packaging systems were not different (P>0.05) from each other. Our findings suggested that antioxidant effects of chitosan on ground beef are packaging-specific.

Amino acid sequence of myoglobin from emu (Dromaius novaehollandiae) skeletal muscle.

The objective of the present study was to characterize the primary structure of emu myoglobin (Mb). Emu Mb was isolated from Iliofibularis muscle employing gel-filtration chromatography. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was employed to determine the exact molecular mass of emu Mb in comparison with horse Mb, and Edman degradation was utilized to characterize the amino acid sequence. The molecular mass of emu Mb was 17,380 Da and was close to those reported for ratite and poultry myoglobins. Similar to myoglobins from meat-producing livestock and birds, emu Mb has 153 amino acids. Emu Mb contains 9 histidines. Proximal and distal histidines, responsible for coordinating oxygen-binding property of Mb, are conserved in emu. Emu Mb shared more than 90% homology with ratite and chicken myoglobins, whereas it demonstrated only less than 70% sequence similarity with ruminant myoglobins.

Characterization of bison (Bison bison) myoglobin.

Bison is an alternate meat species gaining increased popularity in North America. Although previous investigations reported that bison meat discolors faster than beef, the molecular basis of this observation has not been investigated. Therefore, the objective of the present study was to determine the redox stability, thermostability, and primary structure of bison myoglobin (Mb), in comparison with beef Mb. Purified bison and beef myoglobins were analyzed for autoxidation, lipid oxidation-induced oxidation, and thermostability. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was utilized for determining the exact molecular mass of bison Mb, whereas Edman degradation was employed to determine the amino acid sequence. Bison and beef myoglobins behaved similarly in autoxidation, lipid oxidation-induced oxidation, and thermostability. The observed molecular mass of bison and beef myoglobins was 16,949 Da, and the primary structure of bison Mb shared 100% similarity with beef and yak myoglobins. Noticeably, the amino acid sequence of bison Mb was different from other ruminant myoglobins, such as water-buffalo, sheep, goat, and red-deer. The present study is the first to report the primary structure of bison Mb. Same primary structure and similar biochemical attributes of bison and beef myoglobins suggested that the observed rapid discoloration in bison meat could not be attributed to biochemistry of bison Mb.

Color-stabilizing effect of lactate on ground beef is packaging-dependent.

Previous research on lactate-induced color stability in ground beef did not address the potential influence of packaging. The objective of the present study was to examine the effects of lactate on the color stability of ground beef patties stored in different modified atmosphere packaging (MAP) systems. Ground beef patties with either 2.5% potassium lactate or no lactate were packaged in vacuum (VP), high-oxygen MAP (HIOX; 80% O(2)+20% CO(2)), carbon monoxide MAP (CO; 0.4% CO+19.6% CO(2)+80% N(2)), or aerobic packaging (PVC) and stored for 0, 2, or 4 days at 2 degrees C. Lactate-treated patties were darker (P<0.05; lower L * values) than control patties. Surface redness (a * values) was greater (P<0.05) for lactate patties than the controls when stored in PVC, HIOX, and VP. However, lactate's effects on a * values were not evident when packaged in CO (P>0.05). The color-stabilizing effect of CO could have masked lactate's effect on surface redness. While lactate patties in PVC and VP demonstrated lower (P<0.05) discoloration than controls, no differences (P>0.05) existed between controls and lactate samples in CO and HIOX. Our results indicated that the effects of lactate on ground beef color are dependent on packaging.

Effects of lactate and modified atmospheric packaging on premature browning in cooked ground beef patties.

Our objectives were to determine the effects of lactate and modified atmosphere packaging on raw surface color, lipid oxidation, and internal cooked color of ground beef patties. Eight chubs (85% lean) were divided in half and each half was either assigned to the control (no lactate) or mixed with 2.5% lactate (w/w). Following treatment, patties were prepared and packaged in either vacuum, PVC (atmospheric oxygen level), high-oxygen (80% O(2)+20% CO(2)), or 0.4% CO (30% CO(2)+69.6% N(2)) and stored for 0, 2, or 4days at 2 degrees C. After storage, raw surface color and lipid oxidation were measured and patties were cooked to either 66 degrees C or 71 degrees C. Lactate improved (p<0.05) color stability of PVC, high-oxygen, and vacuum packaged raw patties, but had no effect (p>0.05) on the a * values and visual color scores of patties in 0.4% CO. Lactate decreased (p<0.05) lipid oxidation in all packaging atmospheres. Nevertheless, high-oxygen and PVC-packaged patties had more (p<0.05) lipid oxidation than patties in CO and vacuum. Lactate had no effect (p>0.05) on premature browning, whereas patties packaged in high-oxygen demonstrated premature browning. Conversely, cooked patties in 0.4% CO and vacuum were more red (p<0.05) than both high-oxygen and PVC-packaged patties. Although lactate improved raw color stability, it did not minimize premature browning in cooked ground beef patties.

Mass spectrometric investigations on lactate adduction to equine myoglobin.

Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15mM) were incubated with sodium lactate (200mM) at 4 degrees C, pH 5.6 in 50mM sodium citrate buffer or at 37 degrees C, pH 7.4 in 50mM sodium phosphate buffer, simulating typical meat storage and physiological conditions, respectively. Controls consisted of myoglobin plus a volume of deionized water equivalent to that used to deliver the lactate treatments. No peaks corresponding to lactate-Mb adducts could be detected in the mass spectra of samples incubated up to 360min at pH 7.4, 37 degrees C or 8days at pH 5.6 and 4 degrees C. Our results suggest that lactate did not form covalent adducts with equine oxy- and carboxy-myoglobin.

Effect of carbon monoxide packaging and lactate enhancement on the color stability of beef steaks stored at 1°C for 9 days.

Our objective was to assess the effects of lactate enhancement in combination with different packaging systems on beef longissimus lumborum and psoas major steak color. Strip loins and tenderloins (n=16) were assigned to one of four injection treatments (non-injected control, water-injected control, 1.25%, and 2.5% lactate in the finished product). Steaks were individually packaged in either vacuum, high-oxygen (80% O(2)/20% CO(2)), or 0.4% CO (30% CO(2)/69.6% N(2)) and stored for either 0, 5, or 9 days at 1°C. The L(∗) and a(∗) values of both the longissimus and psoas responded similarly to lactate, which at 2.5% darkened steaks (P<0.05) packaged in all atmospheres and improved (P<0.05) the redness of steaks packaged in high-oxygen. Packaging steaks in CO did not counteract the darkening effects of lactate. Nevertheless, CO improved (P<0.05) color stability compared with high-oxygen packaging.

Effect of lactate-enhancement, modified atmosphere packaging, and muscle source on the internal cooked colour of beef steaks.

Earlier studies on lactate-mediated colour stability in beef did not address the possible influence on cooked colour. Our objective was to examine the effect of lactate-enhancement, muscle source, and modified atmosphere packaging (MAP) on the internal cooked colour of beef steaks. Longissimus lumborum (LL) and Psoas major (PM) muscles from 16 (n=16) beef carcasses (USDA Select) were randomly assigned to 4 enhancement treatments (non-injected control, distilled water-enhanced control, 1.25% and 2.5% lactate), and fabricated into 2.54-cm steaks. Steaks were individually packaged in either vacuum (VP), high-oxygen MAP (HIOX; 80% O(2)+20% CO(2)), or carbon monoxide MAP (CO; 0.4% CO+19.6% CO(2)+80% N(2)), and stored for 0, 5, or 9 days at 1°C. At the end of storage, surface and internal colour (visual and instrumental) was measured on raw steaks. Steaks were cooked to an internal temperature of 71°C, and internal cooked colour (visual and instrumental) was evaluated. Lactate-enhancement at 2.5% level resulted in darker (P<0.05) cooked interiors than other treatments. Interior cooked redness decreased (P<0.05) during storage for steaks in VP and HIOX, whereas it was stable for steaks in CO. Our findings indicated that the beef industry could utilise a combination of lactate-enhancement and CO MAP to minimise premature browning in whole-muscle beef steaks.

Primary structure of goat myoglobin.

Color stability attributes of goat meat are different from those of sheep meat, possibly due to species-specific differences in myoglobin (Mb) biochemistry. An examination of post-genomic era protein databases revealed that the primary structure of goat Mb has not been determined. Therefore, our objective was to characterize the primary structure of goat Mb. Goat Mb was isolated from cardiac muscles employing ammonium sulfate precipitation and gel-filtration chromatography, and Edman degradation was utilized to determine the amino acid sequence. Sequence analyses of intact Mb as well as tryptic- and cyanogen bromide-peptides yielded the complete primary structure of goat Mb, which shared 98.7% similarity with sheep Mb. Similar to other livestock myoglobins goat Mb has 153 residues. Comparison of the sequences of goat and sheep myoglobins revealed two amino acid substitutions - THRgoat8GLNsheep and GLYgoat52GLUsheep. Goat Mb contains 12 histidine residues. As observed in other meat-producing livestock species, distal and proximal histidines, responsible for stabilizing the heme group and coordinating oxygen-binding, are conserved in goat Mb.

Mass spectrometric evidence for aldehyde adduction in carboxymyoglobin.

Lipid oxidation generates secondary oxidation products, which compromise myoglobin (Mb) redox stability. Although lipid oxidation-induced discoloration in carboxymyoglobin (COMb) is documented, the molecular basis for interactions between COMb and lipid oxidation products has not been investigated. Our objective was to characterize the adduction of 4-hydroxy-2-nonenal (HNE), a reactive lipid oxidation product, with COMb, utilizing mass spectrometry. Equine COMb and equine OxyMb (0.15mM) were incubated with HNE (1.0mM) at pH 7.4, 37°C (physiological condition) for 6h, and at pH 5.6, 4°C (typical meat storage condition) for 7days. The samples were analyzed in Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). MS spectra revealed that HNE formed mono-, di-, and tri-adducts with COMb at physiological conditions, whereas mono-, di-, tri-, and tetra-adducts were detected in OxyMb. This observation suggested a lower reactivity of COMb towards HNE at physiological conditions, compared to OxyMb. In contrast, at meat storage conditions, HNE formed mono- and di-adducts with both COMb and OxyMb, thus revealing a similar trend for aldehyde adduction in the cherry-red colored Mb redox forms. The present study is the first to report HNE adduction in COMb, and proteomic investigations are underway to determine the sites of HNE adduction in COMb.