Reduction of metmyoglobin by inorganic disulfide species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.

Effects of iron-catalyzed oxidation and methemoglobin oxidation systems on endogenous enzyme activity and myofibrillar protein degradation in yak meat.

In this study, the effects of different oxidation intensities on the degradation of myofibrillar protein by endogenous enzymes in iron-catalyzed oxidizing (IOS) and metmyoglobin oxidizing system (MOS) were compared. The results showed that carbonyl content and endogenous enzyme activities (caspase-3, caspase-6 and calpain-1) increased significantly and the total sulfhydryl content decreased significantly with H2O2 concentration in both oxidation systems. Meanwhile, the rate of carbonyl formation and the inhibition of endogenous enzymes activities of IOS were significantly lower than MOS for the same oxidation intensity. In addition, IOS and MOS mainly produced myosin light chains degradation products of 20-25 kDa and 20-17 kDa. At the same oxidation intensity, MOS of myofibrillar protein significantly enhanced the degradation of troponin-T and desmin by caspase-3/-6 compared with IOS, while inhibiting the degradation of troponin-T by calpain-1. In conclusion, MOS inhibited endogenous enzyme degradation in vitro more than IOS during post-slaughter maturation of yak meat.

Muscle fiber composition affects the postmortem redox characteristics of yak beef.

Yak infraspinatus (IS), longissimus thoracis (LT), and semitendinosus (ST) muscles were used to explore relationships between myofiber types and postmortem redox characteristics. IS mainly consisted of myofiber type Ⅰ (49.2%), LT has a vast majority of type IIa (72.7%), while ST possessed a similar percentage of type IIa (44.7%) and IIb (51.5%). Compared with LT and ST, IS exhibited higher initial H2O2, myoglobin (T-Mb), and metmyoglobin (MetMb) contents that provoked severe protein oxidation despite higher endogenous antioxidative capacity (e.g., glutathione antioxidative system). Three muscles showed specificities in myofiber type composition and redox characteristics, which were strongly correlated. Specifically, type Ⅰ myofiber positively correlated with H2O2, T-Mb, and multiple antioxidase activity/content while negatively correlated with lipid peroxidation, MetMb, and lactic dehydrogenase activity; also, they tended to employ more SFAs in more intermuscular fat assembly. Overall, muscle-specific myofiber type/redox attributes required differentiated processing to prevent meat oxidation and produce standardized products.

Intracellular imaging of metmyoglobin and oxygen using new dual purpose probe EYFP-Myoglobin-mCherry.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

Antioxidant, Antimicrobial and Metmyoglobin Reducing Activity of Artichoke (Cynara scolymus) Powder Extract-Added Minced Meat during Frozen Storage.

The present study aimed to investigate the bioactive compounds in artichoke (Cynara scolymus) powder, having antioxidant and antimicrobial activity, and to determine the effectiveness of artichoke (C. scolymus) powder extract within the minced meat. C. scolymus was extracted using two different methods. The method incorporating high phenolic and flavonoid content levels was used in other analyses and the phenolic and flavonoid contents in C. scolymus extract was determined using LC-QTOF-MS. Antioxidant, antimicrobial, and metmyoglobin (metMb) reducing activities and pH values of the extract-added minced meat samples were measured for 10 days during storage. DPPH, FRAP, and ABTS were used in the antioxidant analyses. The antimicrobial activity of C. scolymus extract was evaluated on five different food pathogens by using the disc diffusion method. The most resistant bacterium was found to be Listeria monocytogenes (18.05 mm ± 0.24). The amount of metMb was measured in the minced meat sample that was added to the extract during storage (p < 0.05). MetMb formation and pH value on the sixth day of storage were found to be at lower levels than in the control group. In conclusion, C. scolymus exhibited a good antimicrobial and antioxidant effect and can be used in storing and packaging the food products, especially the meat and meat products.

Strategies to limit meat wastage: Focus on meat discoloration.

Limiting meat waste is a significant factor that can help meet future needs to provide high-quality animal protein while maximizing the utilization of natural resources. Fresh meat waste occurs during production, processing, distribution, and marketing to various points of consumption. Consumers' expectation for muscle food quality is often associated with its appearance, and a bright-red color of red meat is an indicator of freshness and wholesomeness. Meat discoloration is a natural process resulting from interactions between the physical structure of meat and the oxidation of the ferrous forms of myoglobin. Understanding the biochemical processes that influence discoloration such as oxygen consumption, metmyoglobin reducing activity, lipid oxidation, and microbial growth help to develop innovative strategies to limit meat waste. The focus of this chapter is to discuss the factors involved in meat discoloration and any other color deviations that may lead to discounted pricing and/or meat loss. The impact of meat waste, economic loss, the role of packaging, and the application of high-throughput techniques to understand the biochemical basis of meat discoloration are also discussed.

Changes in color, myoglobin, and lipid oxidation in beef patties treated by dielectric barrier discharge cold plasma during storage.

The objective of this work was to investigate the changes in color, metmyoglobin reducing activity (MRA), and lipid oxidation (TBARS) and their relationships in beef patties treated by dielectric barrier discharge cold plasma (DBD-CP) at different voltages and frequencies during storage at 4 °C. DBD-CP treatment, including treatment voltages and frequencies, caused the decrease of a⁎ value and MRA (P < 0.05), promoted the accumulation of metmyoglobin (P < 0.05), and significantly increased TBARS values (P < 0.05). It was the first time revealed that the effect of treatment voltage on the quality of beef patty was greater than that of frequency. In addition, there were significant and strong positive correlations between a⁎ value and MRA and negative correlations between a⁎ value, metmyoglobin (MMb)% and TBARS. Taken together, Data demonstrated that DBD-CP treatment can significantly affect meat redness, Mb contents, MRA, and lipid oxidation in ground beef during storage, and accelerate discoloration and lipid oxidation of meat.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Plant extracts effectiveness to extend bison meat shelf life.

The effectiveness of plant extracts (0.05% rosemary and 0.08% oregano) to extend shelf life of bison strip loin steaks in terms of color stability and consumer acceptability was studied. Steaks treated with oregano presented lower oxygen consumption, higher metmyoglobin-reducing activity (MRA), decreased lipid oxidation, and provided a stable red color with less discoloration during the retail display period than the control and rosemary treated steaks (P < 0.05). Results from consumer sensory evaluation indicated that treated steaks under study were not significantly different from the control (P > 0.05) based on palatability attributes and acceptability. However, rosemary treated steaks were more desirable and palatable than their oregano counterparts (P < 0.05). Overall, plant extracts, particularly oregano, can improve color stability of bison steaks due to its antioxidants properties and ability to increase MRA capacity in fresh bison meat without posing any negative impact on its sensory attributes. PRACTICAL APPLICATION: This study will provide valuable information to the bison meat industry on how to offer a more consistent and acceptable product (in terms of palatability and color) to consumers using plant-based natural antioxidants, without diminishing the palatability of their products. This technology can offer two more days of shelf life in retail overwrap packaging, consequently, opening the possibility for the bison industry to expand its market with a potential to reduce retail losses due to poor color stability and early browning (that is, stock out, markdowns, and waste due to expired display life).

Effects of quality grade and intramuscular location on beef semitendinosus muscle fiber characteristics, NADH content, and color stability.

The objective of this study was to determine the impact of quality grade and steak location on color stability of semitendinosus (ST) steaks during a 9-d refrigerated study. Twenty-one ST muscles (12 Choice and 9 Select) were purchased from a commercial beef packing plant and fabricated into twelve 2.54-cm thick steaks per muscle. Steaks 1, 6, and 12 were designated for immunohistochemistry while remaining steak locations of proximal (steaks 2 to 4), middle (5 to 8), and distal (9 to 11) were randomly assigned to 0, 4, or 9 d of simulated retail display. Surface color attributes of day-9 steaks were recorded daily by a visual color panel and spectrophotometer. On days 0, 4, and 9 of display, steaks were analyzed for metmyoglobin reducing ability (MRA) and oxygen consumption (OC). Grade × day of display (DOD) interactions were detected for L*, a*, surface oxymyoglobin (OMb) and metmyoglobin (MMb) percentages, and visual panel surface redness and discoloration scores (P ≤ 0.02); however, no Grade × DOD interactions were observed for MRA or OC (P > 0.17). There were location main effect (LOC) × DOD interactions for L*, a*, surface MMb, visual panel surface redness and discoloration, and MRA (P ≤ 0.02). Distal steaks had lower L* values compared with the other locations (P < 0.01), which coincided with steaks being rated visually darker red (P < 0.01). Proximal steaks had greater a* values and had less surface discoloration than middle steaks (P < 0.05), which had an increased percentage of surface MMb (P ≤ 0.04). Distal and proximal steaks had increased MRA compared with middle steaks on days 0 and 4 (P < 0.05), and distal steaks had greater OC than the other locations throughout display (P < 0.01). There were fewer type I fibers at the proximal end with a greater percentage located at the middle and distal ends, and an increased percentage of type IIX fibers at the middle and proximal locations (P ≤ 0.01). Less type IIA fibers were detected at the middle LOC compared with the other two locations (P < 0.10). Larger type I, IIA, and IIX fibers were located at the proximal and middle locations compared with the distal LOC (P < 0.01). ST color and color-stability characteristics were influenced by DOD and LOC, which may partially be explained by differences in fiber types among locations.

Combined effects of gamma irradiation and aging on tenderness and quality of beef from Nellore cattle.

Combined effects of gamma irradiation (0, 3, 6 and 9 kGy) and aging (1 and 14 days) on quality attributes of vacuum-packaged beef from Nellore cattle were evaluated. The meat water holding capacity was affected by irradiation, increasing (p < 0.05) purge and cooking loss regardless of the dose used. Irradiation negatively affected myofibrillar fragmentation, but samples irradiated at 9 kGy had (p < 0.05) higher soluble collagen and lower shear force values. The meat metmyoglobin reducing activity was reduced (p < 0.05) by the irradiation process, inducing the metmyoglobin accumulation with increasing dose applied. Samples irradiated at 9 kGy presented (p < 0.05) higher lipid oxidation and lower oxymyoglobin proportion and color redness and chroma values. It was concluded that irradiation at 9 kGy combined with aging can be used as an effective tool for improving the tenderness of Nellore beef, but resulted in a discoloration of the beef.

Effect of oxygen concentration in modified atmosphere packaging on color changes of the M. longissimus thoraces et lumborum from dark cutting beef carcasses.

The objective of this study was to investigate the effect of modified atmosphere packaging (MAP) with different oxygen concentrations on color changes of dark-cutting beef steaks from M. longissimus thoraces et lumborum. Four normal-pH (mean pH = 5.52) and four dark-cutting beef carcasses (mean pH = 6.36) were obtained from a commercial abattoir at 48 h post-mortem. The gas compositions in MAP were as follows: (i) 20% O2/20% CO2/60% N2, (ii) 40% O2/20% CO2/40% N2, (iii) 60% O2/20% CO2/20% N2 and (iv) 80% O2/20% CO2. Steaks were stored in the dark at 2 °C for 14 days. Surface color of dark-cutting beef steaks was improved (P < .05) by 60%O2-MAP and 80%O2-MAP after 4 days storage, to a level similar to the initial normal-pH steaks. Steaks in 60%O2-MAP and 80%O2-MAP had lower metmyoglobin reducing activity and higher TBARS values (P < .05), but deeper oxygen penetration depth (P < .05) that may have masked discoloration resulting from metmyoglobin formation under the surface. While both 60%O2-MAP and 80%O2-MAP improved the appearance of dark-cutting beef, 60%O2-MAP may be preferred owing to the lower level of lipid oxidation.

Structure and biological activities of polysaccharide purified from Senegrain seed.

A polysaccharide named Senegrain Water-Soluble Polysaccharide (SWSP) was extracted and purified from Senegrain seeds. The physicochemical properties of SWSP were evaluated by Thin-layer chromatography (TLC), High Performance Liquid Chromatography (HPLC), Scanning Electron Microscopy (SEM), and Differential Scanning Colorimeter (DSC). TLC results showed that SWSP is a gluconic acid polymer. It had a network with a large number of cavities. This polysaccharide with a semi-crystalline structure has an average molecular weight of 47.42 kDa. On other trend, obtained data exhibited potent antidiabetic and antibacterial activities of SWSP. Additionally, the antioxidant activities of SWSP were studied in vitro, and demonstrated that this polymer displayed an interesting ABTS radical scavenging activity as well as a protective effect on the hydroxyl radical-induced DNA damage. The effects of SWSP on oxidative processes in minced raw beef during refrigerated (4 °C) storage were investigated. The results proved that SWSP decreased the TBARS value and inhibited the MetMb accumulation during the period of refrigeration storage. Overall, the finding demonstrated the potential applications of SWSP in food industries.

Carbon Chain Length of Lipid Oxidation Products Influence Lactate Dehydrogenase and NADH-Dependent Metmyoglobin Reductase Activity.

The biochemical basis of lower metmyoglobin reducing activity (MRA) in high-oxygen modified atmospheric packaged (HiOx-MAP) beef than those in vacuum and polyvinyl chloride (PVC) packaging is not clear. To explore this, the effects of lipid oxidation products with varying carbon chain length on lactate dehydrogenase (LDH) and NADH-dependent metmyoglobin reductase activity were evaluated. Surface color, MRA, and lipid oxidation of beef longissimus lumborum steaks (n = 10) were measured during 6-day display. Further, two enzymes, LDH and NADH-dependent metmyoglobin reductase (n = 5), critical for MRA were incubated with or without (control) lipid oxidation products of varying carbon chain length: malondialdehyde (3-carbon), hexenal (6-carbon), and 4-hydroxynonenal (9-carbon). Steaks in HiOx-MAP had greater (P < 0.05) redness than vacuum and PVC, but had lower (P < 0.05) MRA and greater (P < 0.05) lipid oxidation on day 6. LDH and NADH-dependent metmyoglobin reductase activities were differentially influenced by lipid oxidation products (P < 0.05). The results indicate that the difference in reactivity of various lipid oxidation products on LDH (HNE > MDA = hexenal) and NADH-dependent metmyoglobin reductase (HNE = MDA > hexenal) activity could be responsible for lower MRA in HiOx-MAP.

Lipid protection by polyphenol-rich apple matrices is modulated by pH and pepsin in in vitro gastric digestion.

Lipid oxidation takes place in the gastric tract after the ingestion of a Western diet rich in ω-6 polyunsaturated fatty acids (PUFA) and red meat (heme iron). The incorporation of oxidation products such as 4-hydroxy-2-nonenal (4-HNE) into low-density lipoproteins is further correlated to endothelial dysfunction. Gastric postprandial stress could thus be reduced by antioxidant phytomicronutrients. The aim of this study was to investigate dietary lipid oxidation and its inhibition by apple polyphenols under different matrix forms (fresh fruit, puree, extract) under in vitro gastric digestion conditions. A deep insight was given into the two factors pH and pepsin governing the metmyoglobin-initiated lipid oxidation of sunflower oil-in-water emulsions simulating the physical state of dietary lipids. Our results first showed that pepsin accelerated lipid oxidation at pH 5 through the formation of a micro-metmyoglobin form likely displaying a higher accessibility to lipids. Spectroscopic studies further highlighted the formation of a reversible unfolded metmyoglobin form at pH 3 which was shown to be more pro-oxidant in the absence of pepsin. At nutritional levels, the three apple matrices inhibited less efficiently the accumulation of lipid-derived conjugated dienes and 4-HNE at pH 5 when pepsin was present whereas at pH 3 the opposite was true. High initial bioaccessibilities of monomeric phenolic compounds were evidenced for both puree (57-74%) and the phenolic extract (79-96%) compared to fresh apple (1-14%) supporting their greater antioxidant capacity. By contrast, the bioaccessibility of dimer B2 was low for all matrices suggesting non-covalent binding to apple pectins.

Effects of malondialdehyde as a byproduct of lipid oxidation on protein oxidation in rabbit meat.

The effects of malondialdehyde (MDA) as a byproduct of lipid oxidation on myoglobin and myofibrillar proteins (MP) oxidations in muscle homogenates containing components native to rabbit muscle were investigated. For myoglobin, MDA could lead to increase in metmyoglobin percentage. For MP, MDA could promote protein carbonylation and loss of tryptophan fluorescence. In addition, MDA could affect reactive oxygen species (ROS)-generating system by promoting the formation of hypervalent myoglobin species and release of non-heme iron. The result of MDA-MP adducts fluorescence intensity indicated that ROS-generating systems may be the main reason for protein carbonylation at the later of incubation treatment. SDS-PAGE analysis revealed that the ability of ROS-generating systems to facilitate protein oxidation was enhanced with MDA, which was responsible for the formation of protein cross-linking throughout incubation treatment. Taken together, the ability of MDA on promoting the oxidation of MP in rabbit muscle homogenates may be relied on both adduct reactions and the influence on ROS-generating system.

Mitochondrial Degeneration, Depletion of NADH, and Oxidative Stress Decrease Color Stability of Wet-Aged Beef Longissimus Steaks.

Interrelationship between mitochondria and myoglobin function influence beef color. NADH level in postmortem muscle is an important determinant of mitochondrial activity and metmyoglobin reduction. Increased aging time promotes discoloration of steaks; however, the mechanism of this effect is not clear. The objective was to characterize the role of wet-aging in beef longissimus lumborum muscle mitochondrial function and to characterize the global metabolome to determine the mechanism of that can regenerate NADH. Beef longissimus lumborum muscles were randomly assigned to 3, 7, 14, 21, and 28 days aging periods. Surface color, biochemical, mitochondrial, and metabolite profiles were determined at each aging period and at the end of 6-day display. During 6-day display, sections aged for 28 days had 30.4% decrease in redness than sections aged for 3 days. Aging time decreased (P <0.05) muscle oxygen consumption, mitochondrial protein content, and antioxidant capacity. Metabolites such as fumaric acid, creatinine, and fructose, that can take part in glycolytic/TCA cycle and regenerate NADH decreased (P <0.05) with aging and display time. In support, NADH levels also decreased (P <0.05) with aging time, but aging time had no effect (P = 0.44) on NADH-dependent reductase activity. The results suggest that decreased color stability in aged beef can be attributed to increased mitochondrial damage, depletion of metabolites that can regenerate NADH, and increased oxidative stress. PRACTICAL APPLICATION: Beef aging time results in increased discoloration of steaks under retail display. The current research determines the fundamental basis of lower color stability in aged beef. The results indicate that mitochondrial degeneration, depletion of metabolites that produce NADH, and increased oxidative stress can limit shelf-life of aged steaks. Hence, application of post-harvest strategies to minimize mitochondrial damage and oxidative changes may have the potential to increase shelf-life of aged beef.

Mechanisms of HNO Reactions with Ferric Heme Proteins.

Many HNO-scavenging pathways exist to regulate its biological and pharmacological activities. Such reactions often involve ferric heme proteins and form an important basis for HNO probe development. However, mechanisms of HNO reactions with ferric heme proteins are largely unknown. We performed a computational investigation using metmyoglobin and catalase as representative ferric heme proteins with neutral and negatively charged axial ligands to provide the first detailed pathways. The results reproduced experimental barriers well with an average error of 0.11 kcal mol-1 . The rate-limiting step was found to be dissociation of the resting ligand or HNO coordination when there is no resting ligand. For both heme proteins, in contrast to the non-heme case, the reductive nitrosylation step was found to be barrierless proton-coupled electron transfer, which provides the major thermodynamic driving force for the overall reaction. The origin of the difference in reactivity between metmyoglobin and catalase was also revealed.

Technological characteristics of pre- and post-rigor deboned beef mixtures from Holstein steers and quality attributes of cooked beef sausage.

The objective of this study was to determine the effects of deboning time (pre- and post-rigor), processing steps (grinding - GB; salting - SB; batter formulation - BB), and storage time on the quality of raw beef mixtures and vacuum-packaged cooked sausage, produced using a commercial formulation with 0.25% phosphate. The pH was greater in pre-rigor GB and SB than in post-rigor GB and SB (P < .001). However, deboning time had no effect on metmyoglobin reducing activity, cooking loss, and color of raw beef mixtures. Protein solubility of pre-rigor beef mixtures (124.26 mg/kg) was greater than that of post-rigor beef (113.93 mg/kg; P = .071). TBARS were increased in BB but decreased during vacuum storage of cooked sausage (P ≤ .018). Except for chewiness and saltiness being 52.9 N-mm and 0.3 points greater in post-rigor sausage (P = .040 and 0.054, respectively), texture profile analysis and trained panelists detected no difference in texture between pre- and post-rigor sausage.

Oxidative stability of beef from steers finished exclusively with concentrate, supplemented, or on legume-grass pasture.

The objective of this study was to evaluate the effects of three finishing systems based on concentrate and legume-grass pasture on beef. Steers were finished for 91 days with an exclusively whole corn grain-based (GRAIN) diet, grazed on legume-grass pasture plus 1.4% of body weight of whole corn grain supplementation (SUPP), or grazed on legume-grass pasture (PAST) only. Lipid and myoglobin oxidation, pH, objective color, and α-tocopherol concentrations were evaluated on M. longissimus thoracis steaks. Dietary treatments did not affect pH and minimally affected protein carbonylation. Steaks from steers fed GRAIN were less red, showed higher lipid oxidation during retail display, and higher metmyoglobin formation from day 7 to 13 when compared to PAST. Levels of α-tocopherol were higher in steaks from steers fed diets containing legume and grass. Inclusion of roughage in finishing diets is essential to maintain retail color and prevent lipid and myoglobin oxidation.