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International audience; Freshness is a key parameter of fish quality assessment. This matrix is highly perishable thus, objective tools for freshness estimation are required. Fish spoilage results from both autolytic activities and exogenous actions of specific spoilage organisms (SSO). The flesh of marine fish is characterized by the presence of Trimethylamine N-oxide (TMAO) involved in the osmoregulation process. TMAO can be used by bacteria from the genus Vibrio, Photobacterium, Shewanella as final electron acceptor and its reduction, thanks to TMAO reductase encoded by torA, leads to the production of TMA responsible of the specific fishy odor of spoiled fish.An in silico approach using biofinformatics tools combining R software, NRDB, NRPS primer (Laboratoire ProBioGEM) and AmplifX was set up to design and select primers. Forty torA sequences of Vibrio, nine of Shewanella and nine of Photobacterium were used to select a set of twelve primers. After a first in vitro screening of primers (absence of important primer dimers, ability to amplify a unique PCR product), a pair of primers amplifying a 400 bp fragment was selected to further analysis. The pair was tested on bacterial DNA of S.putrefaciens, P.phosphoreum, P.damselae, V.vulnificus, V.alginolyticus and two strains isolated from a spoiled whiting, identified as Burkholderia cepacia and member of S.putrefaciens group. Then, primer concentrations and annealing temperature were optimized for the selected pair with DNA from Photobacterium and Vibrio strains.Results on bacterial DNA are encouraging for Vibrio and Photobacterium species with a good efficiency (> 90%) and an acceptable range of quantification (µg.mL-1 to pg.mL-1). Selected primers showed a poor specificity on S.putrefaciens and whiting isolated strains but a 400 bp fragment is nevertheless observed. Secondly, total DNA extract from a few fresh and spoiled fish samples have been analyzed thanks to the selected pair of primers.

International audience; In 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.