Differentiating between fresh and frozen-thawed fish fillets by muscle fibre permeability measurement

International audienceThere is no comprehensive method for differentiating between fresh and frozen-thawed fish fillets. This is an ongoing problem, particularly in relation to regulations. In this study, we showed the relevance of using the properties of mitochondria to discriminate fresh fish fillets from frozen-thawed fish fillets. The use of red muscle fibres of Gilthead sea bream allowed us to leave mitochondria in their physiological environment and to avoid possible alterations of mitochondrial membranes during isolation steps. The impact of freezing on fillets was evaluated by measuring the permeability of fibres and mitochondrial membranes to nicotinamide adenine dinucleotide + hydrogen (NADH). NADH permeability of fresh fillet fibres stored at 4°C was compared to the permeability of fibres extracted from frozen-thawed fillets. Two approaches were used to measure permeability changes: a spectrophotometric method that measured consumption of NADH by complex I, and an oxygraphic approach that measured stimulation of O 2 consumption by NADH. Fibres from frozen-thawed fillets were more permeable to NADH and were less sensitive to the permeabilizer alamethicin. The 2 sensitivity of this method allowed us to clearly detect red muscle fibres from frozen-thawed fish versus fresh fish fillets

Differentiating between fresh and frozen-thawed fish fillets by muscle fibre permeability measurement

International audienceThere is no comprehensive method for differentiating between fresh and frozen-thawed fish fillets. This is an ongoing problem, particularly in relation to regulations. In this study, we showed the relevance of using the properties of mitochondria to discriminate fresh fish fillets from frozen-thawed fish fillets. The use of red muscle fibres of Gilthead sea bream allowed us to leave mitochondria in their physiological environment and to avoid possible alterations of mitochondrial membranes during isolation steps. The impact of freezing on fillets was evaluated by measuring the permeability of fibres and mitochondrial membranes to nicotinamide adenine dinucleotide + hydrogen (NADH). NADH permeability of fresh fillet fibres stored at 4°C was compared to the permeability of fibres extracted from frozen-thawed fillets. Two approaches were used to measure permeability changes: a spectrophotometric method that measured consumption of NADH by complex I, and an oxygraphic approach that measured stimulation of O 2 consumption by NADH. Fibres from frozen-thawed fillets were more permeable to NADH and were less sensitive to the permeabilizer alamethicin. The 2 sensitivity of this method allowed us to clearly detect red muscle fibres from frozen-thawed fish versus fresh fish fillets

Differentiating between fresh and frozen-thawed fish fillets by mitochondrial permeability measurement

International audienceIn this study, we investigated the properties of mitochondria to discriminate fresh from frozen-thawed fish fillets. Mitochondria were isolated from gilthead seabream fillets and the impact of freezing was evaluated by measuring the permeability of mitochondrial membranes. Freezing led to permeabilization of mitochondrial inner membranes to reduced nicotinamide adenine dinucleotide (NADH). The increase in permeability related to freezing shock was compared to the physiological permeabilization of mitochondria isolated from gilthead seabream fillets stored at 4°C. Two approaches were chosen to measure the increase in permeability: a spectrophotometric method to measure the consumption of NADH by complex I, and an oxygraphic method to measure O2 consumption by respiratory chains after exposure of mitochondria to NADH. Mitochondria isolated from frozen-thawed fillets were highly permeable to NADH and were no longer sensitive to a membrane permeabilizing agent: alamethicin. Altogether, our scientific approach allowed us to discriminate mitochondria isolated from fillets that have been exposed or not to a freezing shock (-80°C) and thus to discriminate between fresh and frozen-thawed fish fillets

Assessment of freshness and freeze-thawing of sea bream fillets (<em>Sparus aurata</em>) by a cytosolic enzyme: Lactate dehydrogenase

International audienceThe evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8 day storage period at +4 degrees C. A significant increase of lactate dehydrogenase release was observed, especially after 5 days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal a-D-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of a-D-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets

Mitochondrial activity as an indicator of fish freshness

International audienceThe current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (Delta Psi(m)) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at + 4 degrees C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules Delta Psi(m) and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at + 4 degrees C. Mitochondrial activity constituted a reliable and early indicator of fish freshness

Use of ratiometric probes with a spectrofluorometer for bacterial viability measurement

International audienceAssessment of microorganism viability is useful in many industrial fields. A large number of methods associated with the use of fluorescent probes have been developed, including fluorimetry, fluorescence microscopy, and cytometry. In this study, a microvolume spectrofluorometer was used to measure the membrane potential variations of Escherichia coli. In order to estimate the sensitivity of the device, the membrane potential of E. coli was artificially disrupted using an ionophore agent: carbonyl cyanide 3-chlorophenylhydrazone. The membrane potential was evaluated using two ratiometric methods: a Rhodamine 123/4',6-diamidino-2-phenylindole combination and a JC-10 ratiometric probe. These methods were used to study the impact of freezing on E. coli, and were compared with the conventional enumeration method. The results showed that it was beneficial to use this compact, easy-to-use, and inexpensive spectrofluorometer to assess the viability of bacterial cells via their membrane potential

Measurement of fish freshness: Flow cytometry analysis of isolated muscle mitochondria

International audienceMitochondria are real sensors of the physiological status of tissues. After the death of an animal, they maintain physiological activity for several days. This activity is highly dependent on the availability of nutrients in the tissue. In this study, flow cytometry was used to measure the membrane potential of mitochondria isolated from European seabass (Dicentrarchus labrax) red muscle stored in ice for seven days in order to characterize fish freshness. Two probes, TMRM and Rhodamine 123, were used to measure mitochondrial potential. During the first few days (D0 to D3), isolated mitochondria maintained high potential, and then lost their potential (from D3 to D5), but were always re-polarizable after addition of substrates (glutamate, malate and succinate). From D7, the mitochondria were more strongly depolarized and were difficult to repolarize by the substrates. Using flow cytometry, we demonstrated that mitochondria were an excellent marker to confirm seabass freshness

Chemical structure and biological activity of a highly branched (1 → 3,1 → 6)- _-d-glucan from Isochrysis galbanaIrina.

International audiencetA highly branched (1 → 3,1 → 6)- -d-glucan was isolated from the microalga Isochrysis galbana Parke(Isochrysidales, Haptophyta). The polysaccharide structure was analyzed by methylation and Smithdegradation, as well as by ESI and MALDI TOF mass spectrometry and NMR spectroscopy. The glucanwas shown to contain a (1 → 6)-linked backbone, where every residue is substituted at position 3 byGlc, which in turn may be substituted at C-6 by a single Glc or by rather short (up to tetrasaccharide)oligosaccharide chains. All the 3-linked Glc residues are present in these side chains. In the biologicalactivity experiments it was demonstrated that the polysaccharide directly inhibits the proliferation ofU937 human leukemic monocyte lymphoma cells and therefore has potential anti-tumor activity