Sisse Jongberg

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.

Effect of green tea or rosemary extract on protein oxidation in Bologna type sausages prepared from oxidatively stressed pork.

Bologna type sausages were prepared from oxidatively stressed pork (UV-irradiation, 48 h, 5 °C) using a traditional recipe (control) or the same recipe but added green tea extract (500 ppm total phenolic compounds) or rosemary extract (400 ppm total phenolic compounds). Green tea and rosemary extracts protected against formation of TBARS and protein carbonyls. On the contrary, increased thiol loss and a distinct loss of myosin heavy chain and actin due to polymerization by reducible bonds as determined by SDS-page were found by addition of green tea extract. The enhanced protein polymerization was ascribed to the reaction between quinone compounds from the plant extracts and protein thiol groups to yield phenol-mediated protein polymerization. Analysis by ESR spectroscopy revealed increased radical intensities in sausages added plant extracts, which was ascribed to originate from protein-bound phenoxyl radicals, which may protect against other oxidatively induced protein modifications.

Thiol–Quinone Adduct Formation in Myofibrillar Proteins Detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.

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.

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.

4-methylcatechol inhibits protein oxidation in meat but not disulfide formation.

The interaction between phenolic compounds and protein thiols was investigated in minced beef with or without 500 ppm 4-methylcatechol (4-MC) that had been stored under oxygen or argon for 7 days (4 °C). Myofibrillar protein isolates were extracted, and the oxidative stability evaluated by the protein radical intensity measured by ESR spectroscopy was found to be improved by 4-MC as well as by storage without oxygen. Significant loss of thiols was found in samples stored under oxygen compared to argon, whereas an additional loss was found in samples with added 4-MC stored under oxygen. In beef with added 4-MC, LC-MS analysis showed formation of thiol-quinone adducts, which may explain the observed additional loss of thiols. Although storage without oxygen inhibited protein cross-link formation as evaluated by SDS-PAGE, both in presence and in the absence of 4-MC, no inhibitory effect of 4-MC was found on the formation of protein disulfide cross-links in beef stored under oxygen.

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.

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.

Phenolic Antioxidant Scavenging of Myosin Radicals Generated by Hypervalent Myoglobin

The scavenging activity of extracts of green tea (GTE), white grape (WGE), and rosemary (RE), all plant material with high phenolic content, and of the phenolic compounds 4-methylcatechol (4-MC), (+)-catechin, and carnosic acid toward long-lived myosin radicals generated by reaction with H2O2-activated myoglobin at room temperature (pH 7.5, I=1.0) was investigated by freeze-quench ESR spectroscopy. Myosin radicals were generated by incubating 16 μM myosin, 800 μM metmyoglobin, and 800 μM H2O2 for 10 min, and the phenolic extracts were subsequently added (1% (w/w) phenolic compounds relative to myosin). GTE was able to scavenge myosin radicals and reduce the radical intensity by 65%. Furthermore, a low concentration of 4-MC (33 μM) was found to increase the radical concentration when added to the myosin radicals, whereas a higher concentration of 4-MC and catechin (330 μM) was found to scavenge myosin radicals and reduce the overall radical concentration by ∼65%.

Peptides as antioxidants and carbonyl quenchers in biological model systems

Subjecting selected peptides to in vitro analyses covering their ability to interfere with the lipid oxidation chain reaction as well as to protect proteins from direct and indirect oxidation has provided the basis for a more detailed understanding of peptide-mediated protection in biological systems. The efficiency of peptides as radical scavengers and chain-breaking antioxidants in oxidizing lipid membranes was found to be low. Previous studies on antioxidative activity of peptides tend not to include comparisons with efficiencies of more well-documented antioxidants and/or use irrelevantly high dosages of peptides. The present study demonstrates that the effect of the investigated peptides towards oxidation in biological membrane systems is mainly a protection of vital proteins from being oxidatively modified. This protection is obtained through a prevention of lipid oxidation derived carbonylation (indirect protein oxidation) and through interference with aqueous radical species (direct protein oxidation), and it is only achieved if the peptides are present in high concentrations as sacrificial antioxidants.