Ph. Gatellier

Antioxidant enzyme activities in beef in relation to oxidation of lipid and myoglobin

Lipid- and oxy-free radical generation has been implicated in oxidative processes which occur during meat maturation but the importance of the antioxidant enzyme (AOE) activity in these processes is not known. It was shown that metmyoglobin (MetMb) % and lipofuscin content were higher in colour-unstable muscles such as psoas major (PM) and diaphragma (D) compared to longissimus lumborum (LL) and tensor fasciae latae (TFL). Although Superoxide dismutase (SOD) activity is higher post mortem in PM and D muscles than in LL and TFL muscles, catalase and glutathione peroxidase (GPx) activities were higher only in D muscle. The higher AOE activity in colour-unstable muscles such as PM and D was not sufficient to prevent increased formation of MetMb and lipofuscin in these muscles compared to LL and TFL muscles.

Effect of cooking on protein oxidation in n-3 polyunsaturated fatty acids enriched beef. Implication on nutritional quality.

The effect of cooking on protein oxidation was investigated in M. Longissimus thoracis of eight Normand cows fed during a 100 days finishing period with two different diets: a conventional diet (concentrate/straw based diet) and a diet rich in n-3 polyunsaturated fatty acids (PUFAs), obtained by addition to the conventional diet of a mixture of extruded linseed and extruded rapeseed. After 11 days storage, at 4 degrees C under vacuum, meat was cooked by applying jets of steam. Three experimental heating treatments were tested: two with constant surface temperatures of 65 and 96 degrees C during 300s, and one with a continuous increasing surface temperature up to 207 degrees C. Protein oxidation was evaluated by the measurement of carbonyls, aromatic amino acids, and free thiols content. The formation of Schiff bases due to the reaction of proteins with aldehydic products of the lipid oxidation was also evaluated. Cooking resulted in a significant increase of carbonyl groups and Schiff bases as well as a significant degradation of tyrosine and tryptophan. Nevertheless, enrichment of the animal diet in n-3 PUFAs had minor effects on protein oxidation induced by cooking which are unlikely to be of nutritional significance.

Use of meat fluorescence emission as a marker of oxidation promoted by cooking.

Accumulation of fluorescent pigments in cooked bovine meat (M. Longissimus thoracis) was studied in relationship with the heating parameters (time and temperature). Muscles were aged at 4°C for 11days under vacuum before cooking. Meat cooking was performed by applying jets of steam. Three different heating treatments were tested: two with constant surface temperatures of 65 and 96°C for 300s, and one with a continuously increasing surface temperature up to 207°C. After extraction in water/dichloromethane/ethanol, fluorescence pigments were distributed between the apolar phase (emission 420-440nm after excitation at 360nm) and the polar phase, where two emission peaks were seen (emission 410-430 and 515nm after excitation at 360nm). Fluorescence in the two phases was little affected by heating at the two constant temperatures while it increased exponentially after 1min of treatment, as the varying temperature reached 141°C. The maximum fluorescence increases, measured in the extreme conditions of cooking (207°C/300s), were of 5000% in the apolar phase and 1700% in the polar phase. Thiobarbituric acid reactive substances (TBARS) and protein carbonyls were measured in parallel. The correlations between these two parameters and the fluorescence emission demonstrated that the interaction between proteins and aldehyde products of lipid peroxidation was mainly involved in the production of fluorescent pigments in cooked meat.

Autoxidation of purified myoglobin from two bovine muscles

To elucidate the behavioural differences between beef muscles from the viewpoint of colour stability, oxymyoglobin was extracted at 2 h post mortem, purified from two different muscles (longissimus lumborum (LL), stable and psoas major (PM), unstable) and the autoxidation rate was measured. Oxymyoglobin was isolated after separation from metmyoglobin by chromatography on DEAE-Sepharose and TSK SW 2000 columns, and its purity was controlled by electrophoresis and IEF. Over a wide range of pH values (5-9), temperatures (20-50°C) and ionic strength (0-500 mM), no difference was noted between autoxidation rates of LL and PM oxymyoglobin extracted at 2 h post mortem. Conversely, when myoglobin was extracted at 192 h post mortem, the autoxidation rate of PM oxymyoglobin was higher than LL myoglobin, particularly at elevated temperatures. These differences in autoxidation rates after extraction of myoglobin at 2h and 192 h post mortem were not associated with differences in Ea (approximately 23 kcal/mol).

Digestion study of proteins from cooked meat using an enzymatic microreactor.

A semi automatic flow procedure with photometric detection was developed for the study of meat protein digestion. This system comprised two independent flow pathways, gathered by two compartments. The gastric compartment was simulated by an ultrafiltration cell fitted with a 10 KDa cut off membrane and the intestinal compartment was simulated by a 1 KDa cut off dialysis membrane. The pathways were filled with solutions simulating digestive conditions. The proposed system was employed in digestion studies of whole protein extracts from raw and cooked (100°C) meat. A mathematical modelling for the determination of the digestive kinetic constants was established. The results show that meat cooking leads to an important decrease of protein digestibility by proteases of the digestive tract.

Early post-mortem sarcoplasmic proteome of porcine muscle related to protein oxidation

Oxidative deterioration or modifications of proteins which appear during meat storage and processes can result in the impairment of technological, sensorial and nutritional qualities. Improving the quality involves a better understanding of the biochemical mechanisms responsible for protein oxidation in meat. For that purpose, an analysis was conducted to investigate the relationships between the early post-mortem sarcoplasmic proteome, which contains the majority of enzymes involved in the oxidative process, and protein oxidation generated during meat storage and cooking. This study was performed in Longissimus lumborum pig muscle. In order to have sufficient variability in the proteome and in the meat oxidation level, five groups of 10 animals issued from two different breeds and raised in three different rearing systems were analysed. Protein oxidation was estimated by the measurement of carbonyl groups after 1 and 4days of refrigerated storage, and after 100°C experimental cooking of the 4days aged meat. Significant correlations (p<0.05) were observed between the level of carbonyl groups and the intensities of 104 spots of the 2D electrophoresis, out of which 52 were clearly identified. The possible involvement of some proteins in the muscle oxidative stress leading to protein oxidation is discussed.

NMR relaxation of water protons in normal and malignant hyperthermia-susceptible pig muscle.

(1)H-NMR has been used to study the evolution of water proton transversal relaxation times in ageing skeletal muscles of normal and halothane-positive Pietrain pigs. Malignant hyperthermia was confirmed by the caffeine contracture test. Lactic acid, creatine phosphate, and ATP levels in muscle biopsies were measured by biochemical analysis. The NMR dynamic results revealed malignant hyperthermia, but knowledge of animal age and muscle type improved significantly the detection ability. The NMR results revealed large differences between muscles. Halothane sensitivity detection seems to be less affected by animal age, than by muscle effect but discrimination was more efficient in the older animal group. It is concluded that (1) H-NMR is a suitable method for diagnosing halothane sensitivity on a well identified muscle biopsy and that water dynamics might be related to acidosis in muscle fibres.

A rapid method of oxymyoglobin purification

Oxymyoglobin was isolated from bovine Longissimus lumborum muscle by precipitation with ammonium sulfate and purification in rapid conditions with only one chromatographic step on Mono-Q HR column with a HPLC system. Purity of oxymyoglobin was controlled by SDS-polyacrylamide gel electrophoresis and isoelectrofocusing.