Marianne N. Lund

Protein oxidation in muscle foods: a review.

Protein oxidation in living tissues is known to play an essential role in the pathogenesis of relevant degenerative diseases, whereas the occurrence and impact of protein oxidation (Pox) in food systems have been ignored for decades. Currently, the increasing interest among food scientists in this topic has led to highlight the influence that Pox may have on meat quality and human nutrition. Recent studies have contributed to solid scientific knowledge regarding basic oxidation mechanisms, and in advanced methodologies to accurately assess Pox in food systems. Some of these studies have provided insight into the reactions involved in the oxidative modifications undergone by muscle proteins. Moreover, a variety of products derived from oxidized muscle proteins, including cross-links and carbonyls, have been identified. The impact of oxidation on protein functionality and on specific meat quality traits has also been addressed. Some other recent studies have shed light on the complex interaction mechanisms between myofibrillar proteins and certain redox-active compounds such as tocopherols and phenolic compounds. This paper is devoted to review the most relevant findings on the occurrence and consequences of Pox in muscle foods. The efficiency of different anti-oxidant strategies against the oxidation of muscle proteins is also reported.

High-oxygen packaging atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during chill storage

The effect of modified atmosphere packaging (70% O(2)/30% CO(2)) and skin packaging (no oxygen) on protein oxidation and texture of longissimus dorsi was investigated during storage for 14 days at 4°C. High oxygen atmosphere resulted in reduced tenderness and juiciness and SDS-PAGE revealed cross-linking of myosin heavy chain through disulfide bonding, and the content of protein thiols was reduced indicating protein oxidation. Myofibril fragmentation was reduced in meat stored in high oxygen atmosphere indicating less proteolysis and/or cross-linking of proteins. Protein carbonyl content was not affected by the packaging atmospheres. This study shows that packaging in modified atmosphere containing a high level of oxygen can result in protein cross-linking and reduced tenderness and juiciness of the meat.

The combined effect of antioxidants and modified atmosphere packaging on protein and lipid oxidation in beef patties during chill storage.

Effect of rosemary extract and ascorbate/citrate (1:1) in combination with modified atmosphere packaging (100% N(2), 80% O(2)/20% N(2)) on protein and lipid oxidation in minced beef patties during storage in the dark for up to 6 days at 4°C was investigated. A high level of oxygen in the packaging atmosphere was found to increase both lipid and protein oxidation during storage as evaluated by TBARS analysis of secondary lipid oxidation products and by 2,4-dinitrophenylhydrazine derivatization of protein carbonyls. Both antioxidant systems tested were found to inhibit lipid oxidation but not protein oxidation. In contrast, ascorbate/citrate was found to promote protein oxidation. Rosemary extract was found to regenerate or protect α-tocopherol whereas the packaging atmospheres had no effect on α-tocopherol stability. In high oxygen atmospheres both antioxidants protected the fresh red meat colour with ascorbate/citrate being more efficient than the rosemary extract, whereas no effect of antioxidant on meat colour was found in beef patties stored in 100% nitrogen.

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.

Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.

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.

Effects of dietary soybean oil on lipid and protein oxidation in pork patties during chill storage.

The effect of dietary soybean oil on lipid and protein oxidation in low and high fat pork patties made from quadriceps femoris during chill storage in a high oxygen atmosphere packaging (80% O(2)/20% CO(2)) in the dark for 7 days was investigated. Pigs were fed either a standard diet or a diet added 2% soybean oil. After slaughter high fat pork patties were prepared for both feeding regimes by addition of back fat from pigs fed the same diet whereas low fat pork patties were prepared without addition of back fat. The 2% soybean diet increased the amount of unsaturated fat in the pork. Secondary lipid oxidation products determined as thiobarbituric acid reactive substances (TBARS) were found to increase in the pork patties with increased unsaturated fat. Increased unsaturated fat in the pork patties had no effect on protein oxidation determined as free protein thiol content and protein carbonyl content. A small, but significant increase in protein oxidation was found in the high fat pork patties independent on dietary fat. In conclusion, protein oxidation is unaffected by dietary fat in pork patties during chill storage for periods normally used in retail trade, and lipid and protein oxidation are not coupled under these conditions.

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.

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.