Dissociation and reduction of covalent ß-lactoglobulin-quinone adducts by dithiothreitol, tris(2-carboxyethyl)phosphine, or sodium sulfite.

Covalent protein-phenol adducts, generated by reaction of protein nucleophiles with quinones, have recently attracted increased attention because the interactions change the functionality and physicochemical properties of proteins in biological and food systems. The formation of such covalent adducts between ß-lactoglobulin (ß-LG) and the quinone of 4-methylcatechol, 4-methylbenzoquinone (4MBQ), and subsequent reduction by dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or sodium sulfite was investigated by mass spectrometry. The results showed that 19.0 ± 8.8% of ß-LG reacted with 4MBQ when present in equimolar ratio at 20°C (pH 8.0) to yield the protein-phenol adduct (ß-LG-Q). Following treatment with sulfite, DTT, or TCEP, 75, 68, or 36%, respectively, of the formed ß-LG-Q adduct dissociated. Different reaction mechanisms were proposed for the reduction of ß-LG and ß-LG-Q by each of the reducing agents. These results show that on reductive sample preparation for analysis of protein samples, not only are protein polymers formed through oxidative disulfide bonds reduced into the individual protein constituents but also a large part of any protein-phenol adducts present will dissociate and, thus, give a false picture of the level of protein-protein interactions that have occurred in the sample.

Effect of white grape extract and modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties.

The oxidative stability of beef patties added 500ppm white grape extract (WGE), packed in four different modified atmospheres (MAP) with varying oxygen and carbon dioxide levels (70% or 0% O2, 30% or 0% CO2, balanced with N2 in all four combinations) and stored for up to 9days (4°C) was evaluated by a sensory panel, formation of TBARS, formation of protein carbonyl, appearance of myosin cross-links, and thiol loss. Formation of secondary lipid oxidation products, as detected by TBARS, and the rancidity, as perceived by sensory analysis, were inhibited in WGE beef patties independent of MAP compared to control beef patties. The protein carbonyl formation was also reduced in WGE beef patties, but no significant effects were observed in relation to different MAP. Loss of thiol groups in control beef patties was consistent with the formation of myosin cross-linkages. In the presence of WGE, thiol groups decreased faster but showed less myosin cross-link formation compared to control beef patties, indicating that WGE interacts with the thiol groups of the myofibrillar proteins, and thus reduces the cross-link formation in beef patties stored in high-oxygen MA.

Combination of light and oxygen accelerates formation of covalent protein-polyphenol bonding during chill storage of meat added 4-methyl catechol.

Plant polyphenols applied as natural antioxidant ingredients, are known to bind to cysteine residues on meat proteins. The aim of this study was to examine the effect of light exposure on the formation of cysteine-phenol adduct in meat added 4-methylcatechol (4MC), a model polyphenol, during storage through quantitative LC-MS/MS-based analysis. Cysteine-4-methylcatechol adduct (Cys-4MC) formation in meat added 1500 ppm 4-MC increased significantly (by 50%) when stored under light in oxygen at 4 °C for 7 days as compared to storage in the dark. This was reflected by a significant decrease in thiol concentrations in the same sample. Gel electrophoresis showed loss in myosin heavy chain (MHC), and a resulting increase in cross-linked MHC (CL-MHC) and larger protein polymers in samples added 4MC. Protein blots stained with nitroblue tetrazolium (NBT) showed intensive protein-polyphenol binding in the meat samples added 4MC, but no major differences between storage conditions.

Plant derived ingredients rich in nitrates or phenolics for protection of pork against protein oxidation.

A pork model system containg phenolic extracts (citrus, rosemary, and acerola), traditional Spanish food ingredients (paprika, garlic, and oregano), or natural nitrate sources (beet, lettuce, arugula, spinach, chard, celery, and watercress) were oxidized by an hydrophilic (OXHydro, 2,2'-azobis(2-amidinopropane)-dihydrochloride; AAPH) or lipophilic (OXLip, 2,2'-azobis(2,4-dimethylvaleronitrile; AMVN) radical initiator. Citrus as well as lettuce and spinach protected almost fully against protein thiol loss and showed efficient radical scavenging activity as determined by ESR spectroscopy in both oxidizing systems. Rosemary was an efficient radical scavenger in both systems, but behaved as a prooxidant on thiols in the OXHydro system. Acerola was also found to be prooxidative as determined by increased radical signal intensity especially in the OXLip system, assigned to high concentration of ascorbate in the extract. Natural nitrate sources, especially lettuce and spinach, are accordingly potential substitutes for synthetic phenolic antioxidants protecting against protein thiol oxidation and radical formation in pork.

Quantitation of Protein Cysteine-Phenol Adducts in Minced Beef Containing 4-Methyl Catechol.

Thiol groups of cysteine (Cys) residues in proteins react with quinones, oxidation products of polyphenols, to form protein-polyphenol adducts. The aim of the present work was to quantify the amount of adduct formed between Cys residues and 4-methylcatechol (4MC) in minced beef. A Cys-4MC adduct standard was electrochemically synthesized and characterized by liquid chromatography-mass spectrometry (LC-MS) as well as NMR spectroscopy. Cys-4MC adducts were quantified after acidic hydrolysis of myofibrillar protein isolates (MPIs) and LC-MS/MS analysis of meat containing either 500 or 1500 ppm 4MC and stored at 4 °C for 7 days under a nitrogen or oxygen atmosphere. The concentrations of Cys-4MC were found to be 2.2 ± 0.3 nmol/mg MPI and 8.1 ± 0.9 nmol/mg MPI in meat containing 500 and 1500 ppm 4MC, respectively, and stored for 7 days under oxygen. The formation of the Cys-4MC adduct resulted in protein thiol loss, and ca. 62% of the thiol loss was estimated to account for the formation of the Cys-4MC adduct for meat containing 1500 ppm 4MC. Furthermore, protein polymerization increased in samples containing 4MC as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the polymerization was found to originate from protein-polyphenol interactions as evaluated by a blotting assay with staining by nitroblue tetrazolium.

Mate extract is superior to green tea extract in the protection against chicken meat protein thiol oxidation.

Water soluble caffeic acid derivatives in aqueous extracts of mate (Ilex paraguariensis) showed a direct dose-dependent response, protecting protein thiols in a chicken breast meat model system when oxidation was initiated by a lipophilic radical initiator (2,2'-azobis(2,4-dimethylvaleronitrile; AMVN). In contrast, an inverse dose-dependent response was obtained when initiated by a hydrophilic radical initiator (2,2'-azobis(2-amidinopropane)dihydrochloride; AAPH), pointing toward a mechanism, where a specific concentration of water-soluble polyphenol yields optimum protection. Optimal concentrations of mate extract for the protection of thiols against radicals initiated in the lipid phase (AMVN) or in the aqueous phase (AAPH) were 0.1-0.5% and 0.01-0.1%, respectively. Green tea (Camellia sinensis) extract showed a pro-oxidative effect involving quinones from oxidation of pyrogallol-type catechins (epigallocatechin-3-gallate (EGCG), gallocatechin-3-gallate (GCG), and epicatechin-3-gallate (ECG)) and not the catechol-type catechins (catechin and epicatechin).

Light exposure accelerates oxidative protein polymerization in beef stored in high oxygen atmosphere.

Protein oxidation of beef patties stored in high oxygen modified atmosphere packaging for 9 days was investigated. Meat was either stored in the dark, under light, or in the dark with addition of FeCl2/H2O2/myoglobin (forced oxidation). SDS-PAGE analysis showed high degree of protein polymerization for meat exposed to light, compared to the other samples. Light exposure induced reducible (disulfide) and non-reducible cross-links, while mainly disulfides were formed in meat stored in the dark. Light exposure was responsible for 58% loss of free thiols (Cys residues). No significant loss of other amino acid residues was observed and none of the most common oxidation products of tryptophan, tyrosine, and phenylalanine were detected. Intrinsic fluorescence measurements of tryptophan showed 27% loss in samples exposed to light, which was ascribed to loss of protein solubility via protein polymerization rather than tryptophan oxidation. Protein carbonyls were mainly detected in forced oxidized samples at Day 0.

Antioxidant efficiency and mechanisms of green tea, rosemary or maté extracts in porcine Longissimus dorsi subjected to iron-induced oxidative stress.

Plant extracts from rosemary (RE), green tea (GTE), and maté (ME) were compared for the protection against iron-induced oxidation in porcine homogenates at total phenolic concentrations from 25 to 250 ppm. Lipid oxidation as indicated by TBARS was in all cases sufficiently suppressed, especially for RE. Hydrophobic RE retarded overall oxidation in the homogenates with an inverted dose-dependent response. Optimum delay of oxygen consumption was found at the lowest concentration applied, similar to protection against thiols and formation of protein radicals as measured by ESR, whereas the high concentration increased oxygen consumption and caused additionally thiol loss possibly due to thiol-quinone interactions, generating protein-phenol complexes. Hydrophilic ME or GTE increased the initial oxygen consumption rate as an indication of prooxidant activities at elevated concentrations. However, they were found to protect myoglobin and protein at those high concentrations with GTE being more efficient, possibly due to better chelation effect.

Protein Oxidation and Sensory Quality of Brine-Injected Pork Loins Added Ascorbate or Extracts of Green Tea or Maté during Chill-Storage in High-Oxygen Modified Atmosphere.

Background: Ascorbate is often applied to enhance stability and robustness of brine-injected pork chops sold for retail, but may affect protein oxidation, while plant extracts are potential substitutes. Methods: Brine-injected pork chops (weight-gain ~12%, NaCl ~0.9%) prepared with ascorbate (225 ppm), green tea extract (25 ppm gallic acid equivalents (GAE)), or maté extract (25 ppm GAE) stored (5 °C, seven days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, sensory quality, and protein oxidation compared to a control without antioxidant. Results: No significant differences were observed for green tea and maté extracts as compared to ascorbate when evaluated based on lipid oxidation derived off-flavors, except for stale flavor, which maté significantly reduced. All treatments increased the level of the protein oxidation product, α-aminoadipic semialdehyde as compared to the control, and ascorbate was further found to increase thiol loss and protein cross-linking, with a concomitant decrease in the sensory perceived tenderness. Conclusions: Green tea and maté were found to equally protect against lipid oxidation derived off-flavors, and maté showed less prooxidative activity towards proteins as compared to ascorbate, resulting in more tender meat. Maté is a valuable substitute for ascorbate in brine-injected pork chops.

Dose-Dependent Effects of Green Tea or Maté Extracts on Lipid and Protein Oxidation in Brine-Injected Retail-Packed Pork Chops.

Background: Phenolic plant extracts are added as antioxidants in meat to prevent lipid oxidation, but depending on the concentration applied, may affect proteins either through covalent interactions or by serving as a prooxidant. Methods: Brine-injected pork chops prepared with green tea extract (25-160 ppm gallic acid equivalents (GAE)), or maté extract (25-160 ppm GAE) and stored (5 °C, 7 days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, lipid oxidation by thiobarbituric acid reactive substances (TBARS), and protein oxidation evaluated by thiol loss and protein radical formation by electron spin resonance (ESR) spectroscopy, and compared to a control without antioxidant. Results: Extract of maté and green tea showed significant and comparable antioxidative effects against formation of TBARS in brine-injected pork chops for all concentrations applied compared to the control. Protein radical formation decreased significantly by addition of 25 ppm maté extract, but increased significantly by addition of 80-160 ppm green tea extract, when monitored as formation of protein radicals. Meanwhile, protein thiol groups disappeared when applying the extracts by reactions assigned to addition reactions of oxidized phenols from the extracts to protein thiols. Conclusion: Maté is accordingly a good source of antioxidants for protection of both lipids and proteins in brine-injected pork chops chill-stored in high-oxygen atmosphere, though the dose must be carefully selected.

Iron(II) Initiation of Lipid and Protein Oxidation in Pork: The Role of Oxymyoglobin.

Iron(II), added as FeSO4·7H2O, was found to increase the rate of oxygen depletion as detected electrochemically in a pork homogenate from Longissimus dorsi through an initial increase in metmyoglobin formation from oxymyoglobin and followed by formation of primary and secondary lipid oxidation products and protein oxidation as detected as thiol depletion in myofibrillar proteins. Without added iron(II), under the same conditions at 37 °C, oxygen consumption corresponded solely to the slow oxymyoglobin autoxidation. Long-lived myofibrillar protein radicals as detected by ESR spectroscopy in the presence of iron(II) were formed subsequently to oxymyoglobin oxidation, and their level was increased by lipid oxidation when oxygen was completely depleted. Similarly, the time profile for formation of lipid peroxide indicated that oxymyoglobin oxidation initiates both protein oxidation and lipid oxidation.

Quinone-induced protein modifications: Kinetic preference for reaction of 1,2-benzoquinones with thiol groups in proteins.

Oxidation of polyphenols to quinones serves as an antioxidative mechanism, but the resulting quinones may induce damage to proteins as they react through a Michael addition with nucleophilic groups, such as thiols and amines to give protein adducts. In this study, rate constants for the reaction of 4-methylbenzoquinone (4MBQ) with proteins, thiol and amine compounds were determined under pseudo first-order conditions by UV-vis stopped-flow spectrophotometry. The chemical structures of the adducts were identified by LC-ESI-MS/MS. Proteins with free thiols were rapidly modified by 4MBQ with apparent second order rate constants, k2 of (3.1±0.2)×10(4)M(-1)s(-1) for bovine serum albumin (BSA) and (4.8±0.2)×10(3)M(-1)s(-1) for human serum albumin at pH 7.0. These values are at least 12-fold greater than that for α-lactalbumin (4.0±0.2)×10(2)M(-1)s(-1), which does not contain any free thiols. Reaction of Cys-34 of BSA with N-ethylmaleimide reduced the thiol concentration by ~59%, which resulted in a decrease in k2 by a similar percentage, consistent with rapid adduction at Cys-34. Reaction of 4MBQ with amines (Gly, Nα-acetyl-l-Lys, Nε-acetyl-l-Lys and l-Lys) and the guanidine group of Nα-acetyl-l-Arg was at least 5×10(5) slower than with low-molecular-mass thiols (l-Cys, Nα-acetyl-l-Cys, glutathione). The thiol-quinone interactions formed colorless thiol-phenol products via an intermediate adduct, while the amine-quinone interactions generated colored amine-quinone products that require oxygen involvement. These data provide strong evidence for rapid modification of protein thiols by quinone species which may be of considerable significance for biological and food systems.

Competitive kinetics as a tool to determine rate constants for reduction of ferrylmyoglobin by food components.

Competitive kinetics were applied as a tool to determine apparent rate constants for the reduction of hypervalent haem pigment ferrylmyoglobin (MbFe(IV)O) by proteins and phenols in aqueous solution of pH 7.4 and I=1.0 at 25°C. Reduction of MbFe(IV)O by a myofibrillar protein isolate (MPI) from pork resulted in kMPI=2.2 ± 0.1 × 10(4)M(-1)s(-1). Blocking of the protein thiol groups on the MPI by N-ethylmaleimide (NEM) markedly reduced this rate constant to kMPI-NEM=1.3 ± 0.4 × 10(3)M(-1)s(-1) consistent with a key role for the Cys residues on MPI as targets for haem protein-mediated oxidation. This approach allows determination of apparent rate constants for the oxidation of proteins by haem proteins of relevance to food oxidation and should be applicable to other systems. A similar approach has provided approximate apparent rate constants for the reduction of MbFe(IV)O by catechin and green tea extracts, though possible confounding reactions need to be considered. These kinetic data suggest that small molar excesses of some plant extracts relative to the MPI thiol concentration should afford significant protection against MbFe(IV)O-mediated oxidation.

Green tea extract impairs meat emulsion properties by disturbing protein disulfide cross-linking.

The dose-dependent effects of green tea extract (100, 500, or 1500ppm) on the textural and oxidative stability of meat emulsions were investigated, and compared to a control meat emulsion without extract. All levels of green tea extract inhibited formation of TBARS as a measure for lipid oxidation. Overall protein thiol oxidation and myosin heavy chain (MHC) cross-linking were inhibited by 100ppm green tea extract without jeopardizing the textural stability, while increasing concentrations of extract resulted in reduced thiol concentration and elevated levels of non-reducible protein modifications. Addition of 1500ppm green tea extract was found to modify MHC as evaluated by SDS-PAGE combining both protein staining and specific thiol staining, indicating that protein modifications generated through reactions of green tea phenolic compounds with protein thiols, disrupted the meat emulsion properties leading to reduced water holding capacity and textural stability. Hence, a low dose of green tea extract preserves both the textural and the oxidative stability of the meat proteins.

Influence of the Oxidation States of 4-Methylcatechol and Catechin on the Oxidative Stability of ß-Lactoglobulin.

Chemical interactions between proteins and phenols affect the overall oxidative stability of a given biological system. To investigate the effect of protein-phenol adduct formation on the oxidative stability of ß-lactoglobulin (ß-LG), the protein was left to react with an equimolar concentration of 4-methylcatechol (4MC), catechin (Cat), or their respective quinone forms, 4-methylbenzoquinone (4MBQ) and catechin-quinone (CatQ), and subsequently subjected to metal-catalyzed oxidation by Fe(II)/H2O2 for 20 days at 37 °C. The reaction with 4MBQ resulted in 60% thiol loss and 22% loss of amino groups, whereas the addition of 4MC resulted in 12% thiol loss. The reaction with Cat or CatQ resulted in no apparent modification of ß-LG. The oxidative stability of ß-LG after reaction with each of 4MC, 4MBQ, Cat, or CatQ was impaired. Especially 4MC and 4MBQ were found to be pro-oxidative toward α-aminoadipic semialdehyde and γ-glutamic semialdehyde formation as well as the generation of fluorescent Schiff base products. The changes observed were ascribed to the redirection of oxidation as a result of the blocking of thiol groups but also to the oxidative deamination pathway, accelerating the production of semialdehydes and subsequently Schiff base structures.

Fatty acids and oxidative stability of meat from lambs fed carob-containing diets.

Male Comisana lambs were individually stalled and, for 56 days, were fed concentrates with 60% barley (n = 8 lambs), or concentrates in which barley was partially replaced by 24% or 35% carob pulp (n = 9 lambs in each group). The intramuscular fatty acids were analyzed and the color stability, lipid and protein oxidation were measured in fresh meat overwrapped with polyvinyl chloride film at 0, 3 or 6 days of storage at 4 °C in the dark. Carob pulp increased the concentration of polyunsaturated fatty acids (PUFA) in muscle, including the rumenic acid (P < 0.01), and reduced the saturated fatty acids (P < 0.01) and the n-6/n-3 PUFA ratio (P = 0.01). The meat did not undergo extensive oxidative deterioration and the diet did not affect the oxidative stability parameters. Therefore, carob in lamb diet could increase PUFA in muscle without compromising meat oxidative stability.

Competitive reduction of perferrylmyoglobin radicals by protein thiols and plant phenols.

Radical transfer from perferrylmyoglobin to other target species (myofibrillar proteins, MPI) and bovine serum albumin (BSA), extracts from green tea (GTE), maté (ME), and rosemary (RE), and three phenolic compounds, catechin, caffeic acid, and carnosic acid) was investigated by electron paramagnetic resonance (EPR) spectroscopy to determine the concentrations of plant extracts required to protect against protein oxidation. Blocking of MPI thiol groups by N-ethylmaleimide was found to reduce the rate of reaction of MPI with perferrylmyoglobin radicals, signifying the importance of protein thiols as radical scavengers. GTE had the highest phenolic content of the three extracts and was most effective as a radical scavenger. IC50 values indicated that the molar ratio between phenols in plant extract and MPI thiols needs to be >15 in order to obtain efficient protection against protein-to-protein radical transfer in meat. Caffeic acid was found most effective among the plant phenols.

Dietary citrus pulp improves protein stability in lamb meat stored under aerobic conditions.

The antioxidant effects of dried citrus pulp on proteins in lamb meat, when used as a replacement of concentrate in the feed, was studied using meat from 26 male Comisana lambs. The lambs of age 90 days had been grouped randomly to receive one of the three dietary treatments: (1) commercial concentrate with 60% barley (Control, n=8), (2) concentrate with 35% barley and 24% citrus pulp (Cp24, n=9), or (3) concentrate with 23% barley and 35% citrus pulp (Cp35, n=9). Slices from the longissimus thoracis et lomborum muscle were packed aerobically and stored for up to 6days at 4°C in the dark. The citrus pulp groups, Cp24 and Cp35, significantly decreased protein radicals and carbonyls, and preserved more thiols within six days of storage compared to the Control group. The citrus pulp groups significantly slowed down the rate of protein oxidation, indicating that dietary citrus pulp reduced oxidative changes in meat proteins.

Protein thiols undergo reversible and irreversible oxidation during chill storage of ground beef as detected by 4,4'-dithiodipyridine.

Quantification of protein thiols and disulfides in ground beef during storage under high-oxygen atmosphere at 4 °C was performed by thiol detection using 4,4'-dithiodipyridine (4-DPS) before and after disulfide reduction using sodium borohydride. Two independent storage trials were performed, and in trial 1, only reversible thiol oxidation was observed (thiol loss was 30%). In trial 2, irreversible thiol oxidation occurred during the first days of storage, while further loss of thiols was caused by reversible disulfide formation (thiol loss was 33%, of which ca. half was lost because of irreversible oxidation). The results were compared to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cross-linked myosin heavy chain formed by disulfide bonding. Both methods confirmed increasing disulfide formation because of thiol oxidation in meat during storage, but the 4-DPS method showed higher disulfide percentages than the SDS-PAGE method (22.2 ± 0.3% and 8.5 ± 1.2%, respectively). The 4-DPS assay provides an accurate method to evaluate the thiol-disulfide redox state in meat.

Thiol oxidation and protein cross-link formation during chill storage of pork patties added essential oil of oregano, rosemary, or garlic.

The effect of two levels (0.05% and 0.4%) of essential oil of rosemary, oregano, or garlic on protein oxidation in pork patties was studied during storage under modified atmosphere (MAP: 70% O2: 20% CO2: 10% N2) or under aerobic conditions (AE) at 4°C. The oxidative stability of the meat proteins was evaluated as loss of thiols for up to 9 days of storage, and as formation of myosin cross-links analyzed by SDS-PAGE after 12 days of storage. Protein thiols were lost during storage to yield myosin disulfide cross-links. Essential oils of rosemary and oregano were found to retard the loss of thiols otherwise resulting in myosin cross-links. Garlic essential oil, on the contrary, was found to promote protein oxidation, as seen by an extreme loss in thiol groups, and elevated myosin cross-link formation compared to control.