Search

International audience; Lactococcus cremoris and Lactococcus lactis are among the most extensively exploited species of lactic acid bacteria in dairy fermentations. The cell wall of lactococci, like other Gram-positive bacteria, possesses a thick peptidoglycan layer, which may incorporate cell wall polysaccharides (CWPS), wall teichoic acids (WTA), and/or lipoteichoic acids (LTA).In this study, we report the isolation, purification and structural analysis of the carbohydrate moieties of glycolipids (GL) and LTA of the L. cremoris model strain 3107. Chemical structures of these compounds were studied by chemical methods, NMR spectroscopy and positive and negative mode ESI MS. We found that the LTA of strain 3107 is composed of short chains of 1,3-polyglycerol phosphate (PGP), attached to O-6 of the non-reducing glucose of the kojibiose-Gro backbone of the glycolipid anchor. Extraction of cells with cold TCA afforded the detection of 1,3-glycerol phosphate chains randomly substituted at O-2 of glycerol by D-Ala. Unlike the LTA of L. lactis strains studied to date, the PGP backbone of the LTA of L. cremoris 3107 did not carry any glycosyl substitution. The deacylated glycolipid fraction contained the free kojibiose-Gro oligosaccharide, identical to the backbone of the GL anchor of LTA, and its shorter fragment α-Glc-1-Gro. These OS may have originated from the GL precursors of LTA biosynthesis.

International audience; Nanoplastics (NPLs), the presence in the environment of which was considered only “highly plausible” until recently, have become the focus of environmental and ecotoxicological studies. However, up to know, little is known about the potential risks of NPLs to human health. In this review, we provide an overview of the evidence of a specific impact of NPLs on human digestive health reported to date. We focus on the different sources of oral exposure to NPLs, including food packaging, food and beverages. We then summarize the toxicological effects of such exposure on the digestive ecosystem in vitro and in vivo in rodents. Importantly, gut toxicity results should be assessed with the type of NPL model materials employed, due to the critical influence of their chemical and physical properties. As a result, we thoroughly describe NPLs with their source, chemical composition and physicochemical behavior to emphasize the lack of NPL characterization and/or model materials. Finally, we propose avenues for interdisciplinary studies at the interface of toxicology and chemistry, with a view to achieving appropriate scientific assessments of the risks to gut health posed by NPLs, and improvements in their management.

International audience; This paper presents a new Remote Hyperspectral Imaging System (RHIS) embedded on an Unmanned Aquatic Drone (UAD) for plastic detection and identification in coastal and freshwater environments. This original system, namely the Remotely Operated Vehicle of the University of Littoral Côte d’Opale (ROV-ULCO), works in a near-field of view, where the distance between the hyperspectral camera and the water surface is about 45 cm. In this paper, the new ROV-ULCO system with all its components is firstly presented. Then, a hyperspectral image database of plastic litter acquired with this system is described. This database contains hyperspectral data cubes of different plastic types and polymers corresponding to the most-common plastic litter items found in aquatic environments. An in situ spectral analysis was conducted from this benchmark database to characterize the hyperspectral reflectance of these items in order to identify the absorption feature wavelengths for each type of plastic. Finally, the ability of our original system RHIS to automatically recognize different types of plastic litter was assessed by applying different supervised machine learning methods on a set of representative image patches of marine litter. The obtained results highlighted the plastic litter classification capability with an overall accuracy close to 90%. This paper showed that the newly presented RHIS coupled with the UAD is a promising approach to identify plastic waste in aquatic environments.

International audience; Dans le cadre de cette étude, la contamination en microplastiques a été évaluée dans une rivière à la fois en surface (0-25cm) et en subsurface (25-50cm). La partie la plus profonde de la colonne d’eau (10 derniers cm) a également été échantillonnée ainsi les sédiments. Trois campagnes d’échantillonnage ont été menées lors de différentes conditions météorologiques : durant une forte pluie au printemps, après une forte pluie en automne et lors d’une période sèche en hiver. Les résultats montrent que les eaux de subsurface contiennent autant de particules que les eaux de surface, quelles que soient les conditions environnementales.Des concentrations en particules 3 à 11 fois supérieures ont été retrouvées lors des fortes pluies au printemps comparées aux autres campagnes d’échantillonnage dans tous les compartiments étudiés dans la colonne d’eau. Tous compartiments confondus, le type de particules observé est impacté par les conditions météorologiques avec 86% à 96% de fibres parmi les particules échantillonnées en hiver, comparé aux autres campagnes au printemps et en automne où les fibres représentent respectivement 31% et 75%.Lors de cette étude 73,5% des particules analysées sont des polymères qui ont une densité théorique inférieure à 1 dans les échantillons de surface et subsurface, alors que cette proportion tombe à 40,8% pour les échantillons de fond et de sédiments ce qui indique une répartition des particules selon leur densité dans la colonne d’eau.Cette étude souligne l’importance de prendre en compte la subsurface ainsi que les variations saisonnières pour évaluer les types et niveaux de contamination microplastiques dans les environnements fluviaux.

Fish consumption in France has increased by 20% in 20 years, from 19.1 kg/inhabitant/year in 2000 to 23.9 kg/inhabitant/year in 2020. This increase leads to an overexploitation of traditional fishing areas that are no longer sufficient to supply the fresh market. To overcome this supply problem, the share of aquaculture has grown considerably over the last 20 years, from 25.7% to 46%. The exploitation of new fishing areas that are increasingly far from consumption sites is also another alternative to these supply shortages, which increases travel from fishing to consumer. As a consequence, fraudulent practices of selling frozen/ thawed aquatic products in the fresh market often occur. The development of a reliable tool for distinguishing fresh and frozen/thawed products remains a major challenge for the seafood sector. The thesis aimed to explore the use of mitochondria as a marker of freshness and freezing of fish. Mitochondria are mostly present in red muscle tissue. Therefore, our study focused on this tissue for the three fish species studied: sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss). We have found that freezing leads to profound structural changes in mitochondria, with an alteration of the outer and inner membranes (visible in electron microscopy). The alteration of mitochondrial membranes contributes to the total loss of mitochondrial potential of isolated mitochondria. This loss of potential was analyzed in flow cytometry using two potential probes: TMRM and Rhodamine 123. The loss of potential was observed on fresh fish fillet on Day 9, which limits the use of the loss of potential as a marker of freezing. Mitochondrial permeabilization by freezing/thawing shock was first studied by measuring the NADH permeability of isolated mitochondria. The level of permeabilization was normalized by comparison to permeabilization by a known channel-forming antibiotic alamethicin. This permeabilization was followed by the spectrophotometric measurement of the rate of consumption of NADH by mitochondria and the activation of oxygen consumption by oxygraphy. Freezing/thawing was shown to lead to NADH permeabilization of mitochondria and activation of enzyme complexes which results in increased oxygen consumption. The results obtained on isolated mitochondria were confirmed on entire muscle fibres, thus allowing to skip the mitochondrial extraction steps. Freezing leads to rupture of mitochondrial membranes and plasma membranes. Muscle fibres from frozen muscle are more permeable to NADH and therefore consume it faster than fresh fibres. A greater stimulation of oxygen consumption by thawed fibers was also observed. Maintaining a certain integrity of mitochondria in red muscle makes it possible to explore mitochondrial functions and use them as a marker for freezing/thawing fish fillets. This work showed that mitochondria isolated or present in the fibers of fresh fillets were sensitive to the action of a permeabilizer. Conversely, mitochondria and fibres isolated from frozen fillets were insensitive to alamethicin. This reflects a high level of alteration of mitochondrial membranes in these products. This technique, which uses red muscle, appears to be effective in discriminating between frozen aquatic products and fresh products. It is necessary to define whether it can be adapted to the white muscle of fish fillets.; La consommation de poisson en France a augmenté de 20 % en 20 ans, passant de 19,1 kg/hab/an en 2000 à 23,9 kg/hab/an en 2020. Cette hausse se traduit par une surexploitation de zones traditionnelles de pêche qui ne sont plus suffisantes pour alimenter le marché du frais. Pour pallier ce problème d’approvisionnement, la part de l’aquaculture s’est considérablement développée ces 20 dernières années en passant de 25,7 % à 46 %. L’exploitation de nouvelles zones de pêche de plus en plus éloignées des sites de consommation est aussi une autre alternative à ces problèmes d’approvisionnement. Si de nombreux produits aquatiques sont connus pour être commercialisés à l’état décongelé, l’alimentation du marché du frais par des produits décongelés constitue une fraude. La mise au point d’un outil fiable de détection des produits congelés/décongelés reste un enjeu majeur pour la filière des produits de la mer. Elle permettrait de détecter les fraudes, la congélation/décongélation pouvant entraîner des modifications au niveau du produit. La thèse avait pour but d’explorer l’utilisation des mitochondries comme marqueurs de fraîcheur et de décongélation. Les mitochondries sont particulièrement présentes dans le tissu musculaire rouge. C’est pourquoi notre étude s’est concentrée sur ce tissu pour les trois espèces de poissons étudiées : le bar (Dicentrarchus labrax), la daurade royale (Sparus aurata) et la truite arc en ciel (Oncorhynchus mykiss). La congélation va entraîner de profondes modifications structurales des mitochondries, avec une altération des membranes externes et internes (visible en microscopie électronique). L’altération des membranes mitochondriales va participer à la perte totale du potentiel mitochondrial des mitochondries isolées. Cette perte du potentiel a été analysée en cytométrie de flux grâce à deux sondes de potentiel : le TMRM et la Rhodamine 123. La perte de potentiel peut cependant être observée sur du filet de poisson frais à J9, ce qui limite l’utilisation de la perte du potentiel comme marqueur de décongélation. La perméabilisation des mitochondries par le choc congélation/décongélation a été étudiée en mesurant la perméabilité au NADH de mitochondries isolées. Le niveau de perméabilisation a été normalisé en utilisant un perméabilisant : l’alaméthicine. Cette perméabilisation a été suivie par la mesure de la vitesse de consommation du NADH par les mitochondries en spectrophotométrie et l’activation de la consommation de d’oxygène en oxygraphie. La congélation/décongélation conduit à une perméabilisation au NADH des mitochondries et à une activation des complexes enzymatiques qui se traduit par un regain de consommation d’oxygène. Pour s’affranchir des étapes d’extraction des mitochondries, ces résultats ont été confirmés sur fibres musculaires. La congélation entraîne une rupture des membranes mitochondriales et des membranes plasmiques. Les fibres musculaires issues du muscle décongelé sont plus perméables au NADH et le consomment donc plus rapidement que les fibres fraîches. Une stimulation plus importante de la consommation d’oxygène par les fibres décongelées est aussi observée. Le maintien d’une certaine intégrité des mitochondries dans le muscle rouge permet d’explorer les fonctions mitochondriales et de les utiliser comme marqueur de congélation /décongélation des filets de poisson. Ces travaux ont montré que les mitochondries isolées ou présentes dans les fibres de filets frais étaient sensibles à l’action d’un perméabilisant. A l‘inverse, les mitochondries et fibres isolées de filets décongelés étaient insensibles à l’alaméthicine. Ceci traduit un niveau élevé d’altération des membranes mitochondriales dans ces produits. Cette technique, qui utilise le muscle rouge, semble efficace pour discriminer les produits aquatiques décongelés des produits frais. Il reste à définir si celle-ci peut s’adapter au muscle blanc des filets de poisson.

Fish consumption in France has increased by 20% in 20 years, from 19.1 kg/inhabitant/year in 2000 to 23.9 kg/inhabitant/year in 2020. This increase leads to an overexploitation of traditional fishing areas that are no longer sufficient to supply the fresh market. To overcome this supply problem, the share of aquaculture has grown considerably over the last 20 years, from 25.7% to 46%. The exploitation of new fishing areas that are increasingly far from consumption sites is also another alternative to these supply shortages, which increases travel from fishing to consumer. As a consequence, fraudulent practices of selling frozen/thawed aquatic products in the fresh market often occur. The development of a reliable tool for distinguishing fresh and frozen/thawed products remains a major challenge for the seafood sector.The thesis aimed to explore the use of mitochondria as a marker of freshness and freezing of fish.Mitochondria are mostly present in red muscle tissue. Therefore, our study focused on this tissue for the three fish species studied: sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss). We have found that freezing leads to profound structural changes in mitochondria, with an alteration of the outer and inner membranes (visible in electron microscopy). The alteration of mitochondrial membranes contributes to the total loss of mitochondrial potential of isolated mitochondria. This loss of potential was analyzed in flow cytometry using two potential probes: TMRM and Rhodamine 123. The loss of potential was observed on fresh fish fillet on Day 9, which limits the use of the loss of potential as a marker of freezing.Mitochondrial permeabilization by freezing/thawing shock was first studied by measuring the NADH permeability of isolated mitochondria. The level of permeabilization was normalized by comparison to permeabilization by a known channel-forming antibiotic alamethicin. This permeabilization was followed by the spectrophotometric measurement of the rate of consumption of NADH by mitochondria and the activation of oxygen consumption by oxygraphy. Freezing/thawing was shown to lead to NADH permeabilization of mitochondria and activation of enzyme complexes which results in increased oxygen consumption.The results obtained on isolated mitochondria were confirmed on entire muscle fibres, thus allowing to skip the mitochondrial extraction steps. Freezing leads to rupture of mitochondrial membranes and plasma membranes. Muscle fibres from frozen muscle are more permeable to NADH and therefore consume it faster than fresh fibres. A greater stimulation of oxygen consumption by thawed fibers was also observed.Maintaining a certain integrity of mitochondria in red muscle makes it possible to explore mitochondrial functions and use them as a marker for freezing/thawing fish fillets. This work showed that mitochondria isolated or present in the fibers of fresh fillets were sensitive to the action of a permeabilizer. Conversely, mitochondria and fibres isolated from frozen fillets were insensitive to alamethicin. This reflects a high level of alteration of mitochondrial membranes in these products. This technique, which uses red muscle, appears to be effective in discriminating between frozen aquatic products and fresh products. It is necessary to define whether it can be adapted to the white muscle of fish fillets.; La consommation de poisson en France a augmenté de 20 % en 20 ans, passant de 19,1 kg/hab/an en 2000 à 23,9 kg/hab/an en 2020. Cette hausse se traduit par une surexploitation de zones traditionnelles de pêche qui ne sont plus suffisantes pour alimenter le marché du frais. Pour pallier ce problème d’approvisionnement, la part de l’aquaculture s’est considérablement développée ces 20 dernières années en passant de 25,7 % à 46 %. L’exploitation de nouvelles zones de pêche de plus en plus éloignées des sites de consommation est aussi une autre alternative à ces problèmes d’approvisionnement.Si de nombreux produits aquatiques sont connus pour être commercialisés à l’état décongelé, l’alimentation du marché du frais par des produits décongelés constitue une fraude. La mise au point d’un outil fiable de détection des produits congelés/décongelés reste un enjeu majeur pour la filière des produits de la mer. Elle permettrait de détecter les fraudes, la congélation/décongélation pouvant entraîner des modifications au niveau du produit.La thèse avait pour but d’explorer l’utilisation des mitochondries comme marqueurs de fraîcheur et de décongélation. Les mitochondries sont particulièrement présentes dans le tissu musculaire rouge. C’est pourquoi notre étude s’est concentrée sur ce tissu pour les trois espèces de poissons étudiées : le bar (Dicentrarchus labrax), la daurade royale (Sparus aurata) et la truite arc en ciel (Oncorhynchus mykiss). La congélation va entraîner de profondes modifications structurales des mitochondries, avec une altération des membranes externes et internes (visible en microscopie électronique). L’altération des membranes mitochondriales va participer à la perte totale du potentiel mitochondrial des mitochondries isolées. Cette perte du potentiel a été analysée en cytométrie de flux grâce à deux sondes de potentiel : le TMRM et la Rhodamine 123. La perte de potentiel peut cependant être observée sur du filet de poisson frais à J9, ce qui limite l’utilisation de la perte du potentiel comme marqueur de décongélation.La perméabilisation des mitochondries par le choc congélation/décongélation a été étudiée en mesurant la perméabilité au NADH de mitochondries isolées. Le niveau de perméabilisation a été normalisé en utilisant un perméabilisant : l’alaméthicine. Cette perméabilisation a été suivie par la mesure de la vitesse de consommation du NADH par les mitochondries en spectrophotométrie et l’activation de la consommation de d’oxygène en oxygraphie. La congélation/décongélation conduit à une perméabilisation au NADH des mitochondries et à une activation des complexes enzymatiques qui se traduit par un regain de consommation d’oxygène.Pour s’affranchir des étapes d’extraction des mitochondries, ces résultats ont été confirmés sur fibres musculaires. La congélation entraîne une rupture des membranes mitochondriales et des membranes plasmiques. Les fibres musculaires issues du muscle décongelé sont plus perméables au NADH et le consomment donc plus rapidement que les fibres fraîches. Une stimulation plus importante de la consommation d’oxygène par les fibres décongelées est aussi observée.Le maintien d’une certaine intégrité des mitochondries dans le muscle rouge permet d’explorer les fonctions mitochondriales et de les utiliser comme marqueur de congélation/décongélation des filets de poisson. Ces travaux ont montré que les mitochondries isolées ou présentes dans les fibres de filets frais étaient sensibles à l’action d’un perméabilisant. A l‘inverse, les mitochondries et fibres isolées de filets décongelés étaient insensibles à l’alaméthicine. Ceci traduit un niveau élevé d’altération des membranes mitochondriales dans ces produits. Cette technique, qui utilise le muscle rouge, semble efficace pour discriminer les produits aquatiques décongelés des produits frais. Il reste à définir si celle-ci peut s’adapter au muscle blanc des filets de poisson.

Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species.

International audience; The lack of one standardized method to evaluate microplastic pollution in different aquatic environments worldwide represent a gap to fill for the scientist's community. To help overcome this challenge, we adapted an aquatic drone, named Jellyfishbot®, to sample microplastics. The aquatic drone has been compared with the actual most used method for sampling MPs in surface waters: the Manta net. In order to test the reliability of the aquatic drone in different environments, samples were collected in a river and coastal waters sites. The results obtained with the two methods were similar in term of MPs abundances, shapes and colors. It provides also a better reproducibility and more accurate sampling of MPs located in the surface waters mainly the lighter and smaller ones. This sampling method has the advantage of combining the benefits of Manta net sampling (i.e. a representative surface water sampling method that covers a large sampling area and volume (several tens m3) with those of pump filtration and grab sampling (easy access to confined and hard-to-reach areas). This new sampling method could be applied in different aquatic environments making it possible to compare the data and hence become a new standardized approach to evaluate microplastic pollution levels.

International audience

International audience; To investigate the microbial community in the marine environment by molecular approaches, it is important to extract DNA in sufficient quantity and purity. The presence of inhibitors in the samples can lead to false negative results or a loss of information, but can be highlighted by a process control in the experiments. We compared seven bacterial DNA extraction methods on marine samples: fish skin, gills and guts, mollusk meat, phytoplankton and zooplankton. A process control (Listeria monocytogenes) was added in half of the samples. The performance of the DNA extraction methods were compared to produce the more pure and concentrated DNA for qPCR amplification targeting the bacterial tuf gene and the process control hlyA gene. The purity and concentration of DNA were determined by spectrophotometry assays. The results showed that the highest purity and concentration of DNA were obtained using the PowerBiofilm and PureLink Microbiome kits. The qPCR data confirmed these kits produced better bacterial DNA purity and concentration with higher amplification efficiency. In some samples, the presence of inhibitors was detected by qPCR targeting the hlyA gene, showing that the samples were heterogeneous contaminated with inhibitors. The DNA extracts are suitable for genetic downstream applications in the marine environment.

International audience; There 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.

International audience; Les additifs plastiques, ajoutés aux polymères au cours de leur formulation, participent aux dangers que posent l’utilisation et le rejet de plastique dans l’environnement. Cet impact dit « chimique » a été démontré à de multiples reprises. En effet, une fois dans l’environnement, les additifs plastiques peuvent être libérés de la matrice polymérique et diffuser dans l’eau environnante ou au sein des organismes après ingestion de particules de plastiques. L’utilisation de certains additifs est controversée car ils se sont révélés être toxiques pour l’environnement, les organismes marins et l’Homme. Afin d’avoir une meilleure compréhension de l’impact chimique de ces additifs, il est important de les identifier, et idéalement de les quantifier. Après avoir sélectionné une soixantaine de ces additifs (plastifiants, retardateurs de flammes, antioxydants, …), une méthode unique pour l’analyse de l'ensemble ces molécules a été développée en pyrolyse couplée à la chromatographie gazeuse et la spectrométrie de masse haute résolution (Py-GC-HRMS). La méthode développée à ensuite permis d’identifier la présence de divers additifs plastiques dans des échantillons de barquettes plastiques alimentaires en polypropylène (PP) et en acide polylactique (PLA). Au vu des additifs mis en évidence dans ces échantillons, et du fait de la forte présence de barquettes alimentaires dans l’environnement, une étude complémentaire s’est portée sur l’impact potentiel de ces additifs sur les stades de reproduction et les jeunes stades de vies de l’huitre creuse (Crassostrea gigas) (une espèce sentinelle, ingénieure et mondialement commercialisée), après exposition à des lixiviats de ces échantillons.

International audience; The marine environment is a potential natural reservoir of antimicrobial resistance genes (ARGs), subject to anthropogenic effluents (wastewater, industrial, and domestic), and known as a final receiving system. The aim of this study was to investigate the abundance and geographical distribution of the three bla TEM , sul1 , and intI1 genes, proposed as indicators of contamination to assess the state of antimicrobial resistance in environmental settings, added to the tetA gene and the microbial population ( tuf gene) in the English Channel and North Sea areas. Bacterial DNA was extracted from 36 seawater samples. The abundance of these genes was determined by quantitative PCR (qPCR) and was analyzed in association with environmental variables and geographical locations to determine potential correlations. The bla TEM and tetA genes were quantified in 0% and 2.8% of samples, respectively. The sul1 and intI1 genes were detected in 42% and 31% of samples, respectively, with an apparent co-occurrence in 19% of the samples confirmed by a correlation analysis. The absolute abundance of these genes was correlated with the microbial population, with results similar to the relative abundance. We showed that the sul1 and intI1 genes were positively correlated with dissolved oxygen and turbidity, while the microbial population was correlated with pH, temperature and salinity in addition to dissolved oxygen and turbidity. The three tetA , sul1 , and intI1 genes were quantified in the same sample with high abundances, and this sample was collected in the West Netherlands coast (WN) area. For the first time, we have shown the impact of anthropogenic inputs (rivers, man-made offshore structures, and maritime activities) and environmental variables on the occurrence of three indicators of environmental contamination by antimicrobial resistance in the North Sea and English Channel seawaters.

National audience; Environmental pollution from plastics and microplastics (MPs) is a threat to the environment and wildlife on multiple aspects regarding exposition of different hazards. One of them is due to the chemical impact of the organic plastic additives (OPAs). Various additives can be added to polymers during the manufacturing process to modify and improve their physical properties. The analysis of OPAs has shown a growing importance since their use has become controversial as some of the additives were found to be toxic to marine organism and humans. In order to have a better understanding of the chemical impact of those additives, it is important to identify and, ideally quantify the additives included in the polymeric matrices. The analysis of OPAs and polymers is an analytical challenge due to the diversity of their chemical composition, the fact that they are most of the time mixed, and the numerous steps needed for the sample preparation with “conventional” approaches such as solvent extraction (SE). Thus, implementing easy and quick screening analysis methods, able to analyse plastic additives, have become a focus of interest. Pyrolysis coupled to a gas chromatography and a high-resolution mass spectrometer (Py-GC-HRMS) is an analytical tool that enable a relatively quick and easy analysis. This technique minimize or even remove the steps for sample preparation, limiting their potential contamination prior the analysis.The goal of this study is to develop a method using Py-GC-HRMS to identify and, if possible, quantify OPAs included in a polymeric matrix. A single method for the analysis of various selected plastic additives has been implemented and benefits the constitution of a database in GC/MS-Orbitrap with a wide variety of plastic additives. This method has shown its ability to characterise several additives is plastic packaging samples. The development of the quantitative method is still on going.

Four b acteriophage i nsensitive m utants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following a challenge with the Brussowvirus SW13. The BIMs displayed an altered, sedimentation phenotype. Whole genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family-2-glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated r hamnose- g lucose p olymer (Rgp) biosynthesis locus (designated here as the rgp gene cluster). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis p oly s accharide p ellicle (PSP) initiator, WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescent labelling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide which is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. Importance: Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements which specify the surface receptor(s) is apparent. Our research identifies a single gene which is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

International audience; To address the issues related to high perishability and limited shelf life of food proteins from muscle origin, different innovative processing, and preservation techniques, as well as analytical methodological approaches have been developed to meet environmental challenges and consumer demand for food of high quality and sustainable production supported by circular economy principles. This development has been enhanced and increased during the ongoing age of the fourth industrial revolution (Industry 4.0), which has been gaining momentum since 2015, coming up with a range of automated and digitized technologies. This review provides an updated overview of the recently developed thermal and nonthermal processing and preservation technologies, along with selected advanced analytical techniques used in the industry of muscle foods. Particular attention has been given to Industry 4.0 technologies and their role in achieving smart production with high automation and digitalization rates. As in other industry sectors, technology breakthroughs are reshaping the food industry, including the muscle food sector. Emerging technologies, such as pulsed electric field, high-pressure processing, ohmic heating, nanotechnology, advanced mass spectrometry and hyperspectral imaging sensors are among the key elements in the current food revolution 4.0. Although Industry 4.0 offers countless possibilities, more studies are still needed to capture its full potential and further harness its technologies to solve current challenges and move forward toward Industry 5.0.

International audience; The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

International audience; Plastic food packaging represents 40 % of the plastic production worldwide and belongs to the 10 most commonly found items in aquatic environments. They are characterized by high additives contents with >4000 formulations available on the market. Thus they can release their constitutive chemicals (i.e. additives) into the surrounding environment, contributing to chemical pollution in aquatic systems and to contamination of marine organism up to the point of questioning the health of the consumer. In this context, the chemical and toxicological profiles of two types of polypropylene (PP) and polylactic acid (PLA) food packaging were investigated, using in vitro bioassays and target gas chromatography mass spectrometry analyses. Plastic additives quantification was performed both on the raw materials, and on the material leachates after 5 days of lixiviation in filtered natural seawater. The results showed that all samples (raw materials and leachates) contained additive compounds (e.g. phthalates plasticizers, phosphorous flame retardants, antioxidants and UV-stabilizers). Differences in the number and concentration of additives between polymers and suppliers were also pointed out, indicating that the chemical signature cannot be generalized to a polymer and is rather product dependent. Nevertheless, no significant toxic effects was observed upon exposure to the leachates in two short-term bioassays targeting baseline toxicity (Microtox® test) and Pacific oyster Crassostrea gigas fertilization success and embryo-larval development. Overall, this study demonstrates that both petrochemical and bio-based food containers contain harmful additives and that it is not possible to predict material toxicity solely based on chemical analysis. Additionally, it highlights the complexity to assess and comprehend the additive content of plastic packaging due to the variability of their composition, suggesting that more transparency in polymer formulations is required to properly address the risk associated with such materials during their use and end of life.

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.

International audience; The interest of studying organic plastic additives (OPAs) is growing since many of them are considered as toxic for human beings, marine organisms and have been found in the ecosystem. The impact and the extent of this pollution is not fully understood because of the lack of methods able to comprehensively analyze the wide diversity of OPAs. This study aims at offering a quick and efficient way to identify specific harmful plastic additives in polymer materials. For this purpose, a general method, for identifying 49 OPAs, has been successfully developed using pyrolyzer/thermal desorption-gas chromatography coupled to a high-resolution mass spectrometer (Py/TD-GC-HRMS). The retention indices were calculated for each compound and a high-resolution mass spectra database for plastic additives was implemented. This method was then applied to characterize the OPAs included in food containers that are frequently found in the environment. Four samples made of polypropylene (PP) and polylactic acid (PLA) coming from two distinct suppliers were then selected. A total of 10 additives, which were plasticizers (phthalates and adipates), antioxidants (bisphenol B) and phosphorous flames retardants, were found in these food packaging. Generally, higher peak signals were detected in the PP samples in comparison to PLA samples, except for tributyl phosphate and bisphenol B that were exclusively found in PLA samples. Moreover, the experimentations pointed out additives differences between suppliers, for products intended for the same use and made of the same polymer, for both PP and PLA samples highlighting the complexity of identifying and generalizing the additive content of plastic materials.

National audience; La pollution plastique est une menace pour l’environnement et les organismes. Elle les expose à différents dangers. Outre l’impact « physique » lié à l’ingestion de déchets plastique, l’impact « chimique », dû aux additifs plastiques ajoutés aux polymères au cours de leur formulation, est lui aussi problématique. L’utilisation de certains additifs est controversée du fait que certaines molécules se sont révélées être toxiques pour l’environnement, les organismes marins et l’Homme. De ce fait, l’analyse des additifs plastiques montre un intérêt grandissant.Afin d’avoir une meilleure compréhension de l’impact chimique de ces additifs, il est important de les identifier, et idéalement de les quantifier. Leur analyse dans les matrices polymériques est un challenge analytique pour différentes raisons : la diversité de nature chimique des additifs, leur présence généralement en mélange au sein des polymères, ainsi que les nombreuses étapes nécessaires pour la préparation des échantillons avec les approches plus « conventionnelles » telles que l’extraction au solvant. De ce fait, développer des méthodes analytiques relativement faciles et rapides présente un réel intérêt. Ainsi la pyrolyse couplée à la chromatographie gazeuse et la spectrométrie de masse haute résolution (Py-GC-HRMS) est un outil analytique qui permet une analyse relativement facile et rapide d’un échantillon. Cette technique permet de limiter voire de s’affranchir des étapes de préparation des échantillons, limitant alors leur potentielles contaminations avant l’analyse.Le but de cette étude est de développer une méthode en Py-GC-HRMS haute résolution pour l’identification et la quantification d’additifs plastiques organiques. Une méthode unique pour l’analyse de l'ensemble des additifs sélectionnés a été développée et a permis la constitution d’une base de donnée haute résolution contenant une large variété d’additifs plastiques. Cette méthode a ensuite été appliquée sur certains plastifiants dans le but de les quantifier. Elle montre son utilité pour la caractérisation et le dosage d’additifs dans différents échantillons de matrices polymériques.

National audience; En dix ans, les recherches sur les particules plastiques, nano- (NP) et microplastiques (MP), et leurs contaminants associés se sont largement développées. L’amélioration des connaissances de ces familles de contaminant s’étant accrue, les pratiques analytiques ont dû évoluer et être adaptées. La garantie de la qualité des résultats générés ne peut être obtenue que par la mise en commun des expertises, ainsi que la mise en place d’infrastructures et l’utilisation d’appareillages complémentaires.Dans le cadre du CPER Marco, différents laboratoires (Anses, LOG et ULCO) ont mis en place la plateforme 3PAC de façon à partager leurs équipements analytiques, méthodes, expertises et connaissances.En terme d’infrastructure, de nombreuses possibilités existent. Tout d’abord des systèmes d’aquarium avec un environnement contrôlé sont disponibles pour réaliser des études d’exposition. Une pièce « microplastique » permet de réaliser des manipulations (digestion, filtration) sous atmosphère maîtrisée et ainsi limiter les risques de contamination. La caractérisation des particules (taille, forme) peut être réalisée à l’aide de divers équipements : loupes binoculaires ou directement par microscopie via l’utilisation des capteurs optiques du µ-Raman ou du µ-FT-IR. Enfin, l’identification des particules ou des contaminants associés est rendue possible par l’utilisation de techniques complémentaires soit spectroscopiques (µ-Raman, µ-FT-IR), soit spectrométriques (Py-GC/MS et Py-GC/Orbitrap). L’acquisition de nouveaux équipements, complémentaires, est programmée de façon à venir enrichir les possibilités analytiques (microscopie confocale, technologie light scattering).Dès aujourd’hui, l’expertise des différents partenaires permet de couvrir un large champ de recherches allant des études en laboratoire, aux prélèvements sur le terrain. A titre d’exemple, des travaux récents ont porté sur l’étude de sédiments et organismes prélevés in situ, la réalisation d’études d’encagement d’organismes marins, ou encore l’exposition de bivalves en conditions contrôlées.Désormais, l’ambition de la plateforme 3PAC est de répondre aux appels à projets nationaux et internationaux portant sur l’étude des particules plastiques et de leurs conséquences. Ces projets seront menés au sein d’un écosystème analytique idoine, regroupant expertises, technologies et infrastructures.

International audience; The objective of this study is to determine the lighting conditions for an optimal production of Arthrospira (Spirulina) platensis quantitatively and qualitatively for the process most commonly used by producers. A design of experiments made it possible to obtain surface modelling of responses which were validated under pilot conditions. Spirulina growth, protein and pigment content were modelled as a function of light intensity and duration of illumination. For each of them, the optima have been determined: 14 h (hours light exposure)/10 Klux (light intensity) for the growth rate; 11.8 h/7.2 Klux for phycocyanin rate; 6.2Klux/12.6 h for chlorophyll rate; 6.6Klux/12.6 h for carotene rate. The protein rate is the lowest when lighting conditions are of 10.6 Klux and 14.4 h. It is then possible for the producer to partially obscure the ponds and/or increase the light intensity using additional lighting.

Copepods represent an interesting alternative or a complement live food to brine shrimps and rotifers commonly used in aquaculture. They constitute the natural prey of many fish species and therefore do not require a potential nutritional enrichment. But an optimization of the microalgal diets used to feed copepods is essential to improve their mass culture. This study examined the effects of seven microalgal diets, namely single-species diets of Rhodomonas salina (R), Tisochrysis lutea (T), and Pavlova lutheri (=Diacronema lutheri) (P), two-species diets (R + T, T + P, and R + P), and a three-species diet (R + T + P), on the fatty acid and monosaccharide composition of the cyclopoid copepod Paracyclopina nana as well as its reproductive investment. Experiments were run during 15 days in 10-L beakers; starting with nauplii collected from a large 300-L batch culture. Copepods fatty acid contents were studied, particularly the relative amounts of docosahexaenoic acid (DHA) and eicosa-pentaenoic acid (EPA). The R + T, R, and T diets induced the highest total fatty acid amount in copepods. R + T and R also generated the lowest DHA/EPA ratios in copepods due to high EPA contents. The highest value of total monosaccharides was found in copepods fed with R + T + P. Diets R + T and R induced the greatest prosome volumes and clutch volumes in ovigerous females. Both prosome volume and clutch volume in P. nana ovigerous females were correlated to the individual EPA amount. The results demonstrated that all diets including R. salina enhanced the productivity of P. nana in mass culture, particularly when combined with T. lutea. R. salina, and T. lutea induced complementary fatty acid and monosaccharide profiles, confirming that R + T represents the best microalgae combination for productive culture of P. nana. Conversely, P. lutheri did not enhance the nutritional profile nor the fecundity of P. nana in the culture. This study is the first to demonstrate that R. salina is a suitable microalga for productive mass culture of P. nana for use as live food in aquaculture.

National audience; La pollution plastique est une menace pour l’environnement et les organismes. Elle les expose à différents dangers. Outre l’impact « physique » lié à l’ingestion de déchets plastique, l’impact chimique, dû aux additifs plastiques ajoutés aux polymères au cours de leur formulation, est lui aussi problématique. En effet, une fois dans l’environnement, les additifs plastiques peuvent être libérés de la matrice polymérique et diffuser dans l’eau environnante ou au sein des organismes après ingestion de particules de plastiques. L’utilisation de certains additifs est controversée car ils se sont révélés être toxiques pour l’environnement, les organismes marins ou l’Homme.Afin d’avoir une meilleure compréhension de l’impact de ces additifs sur les organismes, il est important de les identifier et idéalement de les quantifier. L’analyse des additifs plastiques est un challenge analytique pour différentes raisons. En comparaison avec les approches plus "traditionnelles", la pyrolyse couplée à une chromatographie gazeuse et spectrométrie de masse (Py-GC-MS) est un outil analytique qui permet une analyse relativement facile et rapide d’un échantillon. Cette technique permet une analyse quasi-directe des échantillons limitant alors les potentielles contaminations des échantillons lors des étapes de préparation précédant l’analyse.Le but de cette étude est de développer une méthode en Py-GC-MS pour l’identification et, si possible, la quantification d’additifs plastiques organiques. Après avoir sélectionné une soixantaine de ces additifs selon leur nocivité et utilisation (plastifiants, retardateurs de flammes, antioxydants, …), une méthode unique pour l’analyse de l'ensemble ces molécules a été développée et a permis la constitution d’une base de donnée haute résolution. Cette méthode sera appliquée sur divers échantillons pour identifier, voire quantifier, les additifs plastiques contenus dans plusieurs matrices polymériques. De plus, la mise en place d'une méthode de pyrolyse avec un gradient de température permet également de caractériser le type de polymère sur le même échantillon.

International audience; Assessment 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.

International audience; Vibriosis is a recurrent disease in marine aquaculture environments. Monitoring the dynamics of bacterial populations in several compartments of the aquaculture infrastructure is of paramount importance to understand the occurrence of the disease. In the present study, the fish farm, located in the north of France, is a flow-through system where the tanks are supplied by two water inlets. Water from breeding tanks, and inlets and the biofilms in the tanks were sampled weekly in a seabass farm for 7 months. DNA was extracted from 477 samples to sequence the variable V4-V5 region of the 16S rRNA gene and analyze the sequences using the QIIME2 platform. Proteobacteria (54.52%) and Bacteroidetes (26.85%) composed more than 80% of the phylum diversity. Alpha and beta-diversity indicators showed that bacterial communities were more diverse in samples from biofilms and tanks than in samples from water inlets. Some families were specific to one compartment, e.g. ammoniumoxidizing bacteria in biofilms, whereas Vibrionaceae family isolates were mostly found in tank water samples. Taxa were determined up to the species level for the Vibrionaceae. Besides, all six determined genera of Vibrionaceae were identified, with mostly Vibrio and Photobacterium isolates detected in almost all samples. Vibrionaceae persisted over time in the biofilms. Our results suggest that biofilms may be a potential reservoir in case of dysbiosis. This study helps better understand microbial interactions, especially with Vibrionaceae in aquaculture environments.

International audience; Analysis of organic plastic additives (OPAs) associated to plastic polymers is growing. The current review outlines the characteristics and the development of (multi-step) pyrolysis coupled with a gas chromatography mass spectrometer (Py-GC/MS) for the identification and semi-quantification of OPAs. Compared to traditional methods, Py-GC/MS offers advantages like suppressing extensive steps of preparation, limiting contamination due to solvents and the possibility to analyse minute particles. Its key advantage is the successive analysis of OPAs and the polymeric matrix of the same sample. Based on the studied articles, numerous methods have been described allowing identification and, in some case, semi-quantification of OPAs. There is nevertheless no gold standard method, especially given the huge diversity of OPAs and the risks of interferences with polymers or other additives, but, among other parameters, a consensus temperature seems to arise from studies. More broadly, this review also explores many aspects on the sample preparation like weight and size of particles and calibration strategies. After studying the various works, some development prospects emerge and it appears that methodological developments should focus on better characterizing the limits of the methods in order to consider which OPAs can be quantified and in which polymers this is feasible.

National audience; La pollution plastique est une menace pour l’environnement et les organismes. Elle les expose à différents dangers. Un de ces dangers est l’impact chimique due aux additifs plastiques organiques. Différents types d’additifs peuvent être ajoutés au polymères plastiques au cours de leur formulation afin de leur conférer certaines propriétés. L’utilisation de certains additifs est controversée du fait que certaines molécules se sont révélées être toxique pour l’environnement, les organismes marins et l’homme. De ce fait, l’analyse des additifs plastiques montre un intérêt grandissant.Afin d’avoir une meilleure compréhension de l’impact chimique de ces additifs, il est important d’identifier, et idéalement quantifier les additifs inclus dans les matrices polymériques. Leur analyse dans les polymères est un challenge analytique du fait de la diversité de la composition chimiques des additifs et du fait qu’il soit, en général, en mélange au sein des polymères. De ce fait, développer des méthodes analytiques de screening relativement facile et rapide présente un réel intérêt. Le but de cette étude est de développer une méthode en pyrolyse couplée à une chromatographie gazeuse et spectrométrie de masse (Py-GC/MS) pour l’identification et, si possible, la quantification d’additifs plastiques organiques. Cette méthode permettra également de caractériser le type de polymère sur le même échantillon. La première étape a consisté à développer une méthode commune en GC/MS-Orbitrap pour l’analyse de différents additifs plastiques ainsi que de constituer une base de donnée haute résolution contenant une large variété d’additifs plastiques.

National audience; La recherche concernant les nano- (NP) et microplastiques (MP) s’est largement développée ces dernières années avec une meilleure connaissance de la façon dont les expérimentations doivent être conduites. Ainsi, il est désormais clair que ce sujet nécessite un investissement important en terme d’expertise, d’infrastructure, ou d’appareillage, de manière à garantir la production de résultats de qualité.Dans le cadre d’un contrat plan Etat/Région (CPER Marco), différents laboratoires du Boulonnais (Anses, LOG et ULCO) ont mis en place une plateforme (3PAC) de façon à partager leurs équipements analytiques, méthodes, expertises et connaissances.Parmi les infrastructures à disposition à l’heure actuelle sont retrouvés : des systèmes d’aquarium avec un environnement contrôlé pour réaliser des études d’exposition, une pièce « microplastique » permettant de réaliser des manipulations (digestion, filtration) sous atmosphère maîtrisée et ainsi limiter les risques de contamination. La caractérisation des particules (taille, forme) est ensuite réalisée à l’aide de divers équipements : une loupe binoculaire ou directement via l’utilisation du µ-Raman. Enfin l’identification des particules est rendue possible par l’utilisation de techniques spectroscopiques (µ-Raman) ou spectrométriques (Py-GC/MS et Py-GC/Orbitrap). L’acquisition de nouveaux équipements, complémentaires, est d’ores et déjà programmée de façon à venir enrichir les possibilités analytiques (microscopie confocale, µ-FT-IR, light scattering).Dès aujourd’hui, l’expertise des différents partenaires permet de couvrir un large champ de recherches allant des études en laboratoire, aux prélèvements sur le terrain. A titre d’exemple, des travaux récents ont porté sur l’étude de sédiments et organismes prélevés in situ, la réalisation d’études d’encagement d’organismes marins, ou encore l’exposition de bivalves en conditions contrôlées.L’ambition de la plateforme 3PAC est de répondre aux appels à projet nationaux et internationaux portant sur l’étude des particules plastiques et de leurs conséquences, en proposant un écosystème analytique idoine, regroupant expertises, technologies et infrastructures.

International audience; Environmental pollution from plastics and microplastics (MPs) is a threat to the environment and wildlife on multiple aspects regarding exposition of different hazards. One of them is the impact of the organic plastic additives (OPAs). Various chemical additives can be added to polymers during the manufacturing process to modify and improve their physical properties for example. The analysis of OPAs has shown a growing importance since their use has become controversial as some of the additives were found to be toxic to marine organism and human being. In order to have a better understanding of the chemical impact of these additives, it is important to identify and, ideally quantify the ones included in the polymeric matrices. The analysis of OPAs and polymers is an analytical challenge due to the diversity of their chemical composition and the fact that they are most of the time several into a single plastic. Implementing easy and quick screening analysis methods, able to analyze plastic additives, have become a focus of interest. The goal of this study is to develop such a method, with pyrolysis coupled to a gas chromatography and mass spectrometery (Py-GC/MS) for the identification and, if possible, the quantification of OPAs and, consecutively, of the polymeric matrix they are included in. A first step consisted in constituting a database in GC/MS-Orbitrap with a wide variety of plastic additives. So far, a common method in GC/MS-Orbitrap has been developed for the analysis of various plastic additives with standards solution. The selection of the main additives studied was based on their use and toxicity.

National audience; S’appuyant sur l’expertise conjointe de trois entités de l’Anses, le laboratoire d’hydrologie de Nancy (LHN), le laboratoire de sécurité des aliments de Boulogne-sur-Mer (LSAl) et l’Unité d’évaluation des risques liés à l’eau (UERE), le projet interne MicrEAU avait pour objectif de développer une méthode robuste pour la recherche de microplastiques (MP) dans les eaux de consommation et de réaliser, en parallèle, une synthèse et une veille bibliographique sur cette même thématique.L’étude bibliographique a porté sur la recherche de MP dans l’eau du robinet et les eaux conditionnées. Les principes de la revue systématique ont été appliqués : élaboration d’une requête pour la recherche de publications, élaboration de critères de sélection, extraction des données via une grille de lecture, évaluation de la qualité des études, rédaction d’une synthèse.À l’échelle du laboratoire, un développement analytique a été engagé en se basant sur les travaux préalables du LSAl et sur les données de la littérature (choix des types de verrerie, solutions de travail, etc.). Un focus spécifique a été réalisé sur la maîtrise de l’extraction de MP de la matrice eau avant caractérisation. Les données concernant le devenir des MP dans les filières de traitement de l’eau potable et l’état de la contamination des eaux du robinet et eaux conditionnées par des MP restent limitées. Différentes méthodes existent pour quantifier et identifier les MP, avec des performances variables, rendant la comparaison des résultats difficile. Au-delà de la présentation de données de contamination des eaux de consommation, la communication portera sur les points de vigilances et axes de progrès pour le dénombrement et l’identification de MP dans l’eau. Le développement de méthodes fiables est en effet nécessaire avant d’évaluer l’exposition de la population aux MP.

International audience; Vibrio bacteria, and particularly members of the Harveyi clade, are the causative agents of vibriosis. This disease is responsible for mass mortality events and important economic losses on aquaculture farms. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. 16S rRNA gene sequencing is generally considered to be the gold standard for bacterial identification but the cost and long processing time make it difficult to apply for routine identification. In contrast, MALDI-TOF MS offers rapid diagnosis and is commonly used in veterinary laboratories today. The major limiting factor for using this technique is the low environmental bacterial diversity in the commonly available databases. Here, we demonstrate that the sole use of the commercially available Bruker BioTyper database is not fully adequate for identifying Vibrio bacteria isolated from aquaculture farms. We therefore developed a new in-house database named Luvibase, composed of 23 reference MALDI-TOF mass spectra profiles obtained from Vibrio collection strains, mostly belonging to the Harveyi clade. The comparison of the accuracy of MALDI-TOF MS profiling and 16S rRNA gene sequencing revealed a lack of resolution for 16S rRNA gene sequencing. In contrast, MALDI-TOF MS profiling proved to be a more reliable tool for resolving species-level variations within the Harveyi clade. Finally, combining the Luvibase with the Bruker ver.9.0.0.0 database, led to successful identification of 47 Vibrio isolates obtained from moribund abalone, seabass and oysters. Thus, the use of Luvibase allow for increased confidence in identifying Vibrio species belonging to the Harveyi clade.

International audience; Research and development of methods to assess fish freshness continues to be a major challenge for the fishing industry. At the same time, consumers are now increasingly attentive to food quality, including fish freshness and product history. Here, we propose a reliable, rapid and easy-to-apply fluorimetric approach to assess fish freshness using a micro-volume fluorimeter. Mitochondrial functions were assessed in European seabass (Dicentrarchus labrax) fillets at different durations of storage at +4 °C: from Day 0 to Day 8. We found that mitochondrial respiration and mitochondrial membrane potential (ΔΨm) were significantly disrupted after 4 days of storage at +4 °C. The spectral properties (emission peak and fluorescence intensity) of mitochondrial membrane potential probes rhodamine 123 (Rh123) and tetramethylrhodamine methyl ester (TMRM) were strongly affected by the ΔΨm integrity of the fish fillets. We highlighted two categories of fish quality as a function of ΔΨm: Day 0 to Day 3, ΔΨm was preserved; and D4 to D7, ΔΨm was disrupted. Thus, evaluation of ΔΨm constitutes an early and reliable predictive indicator of fish freshness.

International audience

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.

International audience; Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

International audience; The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A–C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain.

International audience; In the recent years, research on microplastics (MP) and nanoplastics (NP) has largely developed with better insights on the way analyses have to be conducted. It is now clear that research in this topic necessitates huge investment in terms of expertise, infrastructure and materials in order to insure the best quality for results.In the framework of a French regional project, CPER Marco, different laboratories from Universities, CNRS and Anses decided to create a common platform (3PAC) in order to share knowledge, expertise, working methodologies and analysis devices. This platform presently gather different equipment: monitored laboratory aquaria for controlled exposition studies, a laminar flow cabinet to avoid contaminations, stereomicroscopes to characterize particles and a µRaman, a Py-GC/MS and a Py-GC/HRMS for the identification of polymers. In the future, acquisitions of new analytical equipment are planned including light scattering technology for NP characterization and µFT-IR for polymer identification. By now, expertise of the different partners allows to cover a large field of applications from both laboratory and field studies. Recently investigations have been carried out on sediment, organisms from the environment and their microbiote, but also caging of marine organisms, exposition of bivalves in controlled conditions. The ambition of 3PAC is to answer national and transnational call for projects, making available an ecosystem gathering expertise, technologies and infrastructures.

International audience; Vibrio harveyi is a particularly problematic Gram-negative bacterium because it can form biofilms on aquaculture facility surfaces, leading to resistance of bacteria against antibiotics and water sanitizers. A SYBR Green I quantitative real-time PCR method was developed to detect V. harveyi directly from environmental samples, including seawater and biofilm. Specific primers targeting the mreB gene were designed. The exclusivity and inclusivity of the newly designed primers were evaluated using a panel of 85 bacteria: 58 V. harveyi from multiples origins and 27 non-V. harveyi isolates, and compared with two pairs of primers targeting the topA and toxR genes that were designed previously. All sets of primers were able to distinguish V. harveyi from closely related species belonging to the Harveyi clade. However, the mreB primers showed better inclusivity and were thus used to develop the real-time PCR assay. A quantification curve was obtained from pure culture of V. harveyi and exhibited excellent efficacy with detection levels as low as 5 genome copies in the PCR reaction. After selection of the extraction kit allowing the best DNA quantity and purity, validation was performed on both seawater and biofilm samples collected from a fish farm. The presence of inhibitors in the DNA templates was evaluated and a 10-fold dilution of template DNA was recommended in order to avoid their effects. The assay was able to detect V. harveyi from environmental samples, confirming the validity of the method. This real-time PCR method will help to evaluate the dynamics of V. harveyi in aquaculture facilities. Suitable prophylactic control measures could be designed using this method, instead of the use of curative methods such as antibiotics

Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called “EPA decorations” essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci., All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the “decorations” of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called “EPA decorations” consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.

International audience; One of the major threats to the seabass (Dicentrarchus labrax) aquaculture sector is the development of vibriosis, a disease caused mainly by bacteria from the family Vibrionaceae, especially Vibrio harveyi. This disease can result in important economic losses or severe public health problems. Furthermore, bacteria can form biofilms on the walls of aquaculture tanks, leading to the persistence of potentially pathogenic bacteria on fish farms. Given the relationship between abundance in the surrounding environment and prevalence in fish, monitoring the presence of V. harveyi in rearing water and biofilms over time may help better understand the dynamics of the pathogen and factors that increase the risk of vibriosis outbreaks. In this study, the dynamics and spatial patterns of cultivable Vibrionaceae and V. harveyi in water and biofilms from rearing tanks and in the cold-and hot-water inlets supplying the farm were monitored for 7 months. Several potential seabass pathogens were isolated: Photobacterium damselae , Vibrio alginolyticus , V. harveyi , Vibrio owensii and Vibrio rotiferianus. However, V. harveyi remained the most problematic pathogen. Its relatively constant presence in water and biofilm samples throughout the sampling campaign represents a risk for vibriosis outbreaks. Moreover, the correlation between water temperature and the abundance of V. harveyi suggests that the risk fluctuates throughout the year. Obtaining qualitative data regarding the relative diversity of viable culturable Vibrionaceae and quantitative data on the abundance of V. harveyi over time in a seabass aquaculture facility is the essential first step for designing new solutions to manage vibriosis.

International audience; The aim of this study was to evaluate the potential of using front face fluorescence spectroscopy (FFFS) to discriminate fresh refrigerated (up to 13 days at 4 °C) sea bass (Dicentrarchus labrax) fillets (F group) from: i) frozen-thawed (3 months at −18 °C), then refrigerated up to 9 days fillets at 4 °C (F-T R group), and ii) refrigerated (up to 9 days at 4 °C), then frozen-thawed (3 months at −18 °C) fillets (R F-T group). The evolution of emission fluorescence spectra after excitation set at 290, 340, and 380 nm, and excitation spectra after emission set at 410 nm were acquired throughout storage. Some traditional measurements based on color, textural, and chemical parameters were performed on the same fillets. Canonical correlation analysis (CCA), applied to the traditional and fluorescence data sets, showed high correlations between the two data sets, since squared canonical coefficients were found higher than 0.8 for canonical variates 1 and 2. This was confirmed by the factorial discriminant analysis (FDA) applied to the concatenated emission spectra recorded after excitation sets at 340 and 380 nm since 72 out of 78 samples were correctly classified. Finally, a multi-block analysis method based on common components and specific weights analysis (CCSWA) was applied to the whole data sets. A reliable discrimination between fresh from frozen/thawed fish was found suggesting that FFFS could be used as a promising tool in routine inspection for differentiating between fresh and frozen-thawed fish.

International audience; Cell wall polysaccharides (CWPS), which are usually covalently bound to the peptidoglycan and are closely associated with the cell wall, are considered as ubiquitous components of the cell envelope of gram-positive bacteria and play an important role as mediators of bacterial interactions with the environment. Here, we describe a simple method for purifying CWPS by extraction of bacterial cells with consecutive acid treatments. Purified CWPS are obtained by gel-filtration chromatography following treatment with HF. We also provide the methodology to easily assess the integrity of CWPS using high-resolution magic-angle spinning (HR-MAS) NMR.

International audience; To investigate egg storage capacity of the copepod Acartia bilobata for aquaculture interest, we tested hatching success rate (HSR) of inclusive eggs (mixture of all egg types) after 4°C storage. The HSR peaked after 14 days storage when incubating at 28°C for 48 hr (85.8 ± 1.6%) and 72 hr (87.6 ± 0.9%), then gradually declined until 1 year (48 hr: 7 ± 0.6%; 72 hr: 19.4 ± 3.9%). Reallocation of fatty acid profile suggests that docosahexaenoic acid (DHA) is correlated with the HSR of A. bilobata eggs. Additionally, we investigated the HSR of diapausing eggs (unhatched eggs after 72 hr incubation of the inclusive eggs) after 4°C storage. Their HSR peaked after 14 days storage (48 hr:75.3 ± 3.5%; 72 hr:78.2 ± 2.1%), then gradually declined until 60 days (48 hr HSR:42.1 ± 2.3%; 72 hr HSR:53.0 ± 3.2%). Overall, we illustrated the hatchability of diapausing and quiescent eggs of A. bilobata after 4°C storage. The cold storage capacities were low (

International audience; Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [6GlcpNAc14ManpNAc14GlcpNAc1] highly substituted with single residues of Glcp, Galp and GlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-/IFN- cytokine-mediated epithelium impairment.

International audience; Food authenticity is one of the major issues in the mind of today's consumers. The sale of frozen-thawed fish fillets under the "fresh fillet" label is considered as a commercial fraud. However, their close sensory properties complicate the differentiation. This study focused on analyzing the composition of exudate (pressured flesh juice) in order to rapidly differentiate between fresh and frozen-thawed skinless fillets of sea bass (Dicentrarchus labrax). Protein concentration, alpha-D-glucosidase specific activity, nucleotides and related compounds (NRCs) concentration, and free calcium concentration were measured in exudates corresponding to fresh or frozen-thawed sea bass fillets. Significant increases of these four parameters were observed in exudates from frozen-thawed fillets, especially with a twofold increase in NRCs and free calcium concentrations. These results suggest that NRCs and free calcium concentrations can be promising indicators to rapidly detect mislabeling of fresh fillets.

International audience; A single strain of Vibrio parahaemolyticus and a single strain of Vibrio cholerae were isolated from shrimps sourced from seafood processing plants. The isolated strains were evaluated for their ability to adhere to stainless steel surfaces, under conditions encountered in seafood industries. In this way, the impact of different environmental factors such as temperature (8 degrees C or 37 degrees C) and culture media (tryptic soy broth (TSB) 2% NaCl, artificial sea water (ASW) or industrial brine) were studied. The viability of these strains was analyzed by three different methods: the enumeration of cultivable bacteria on agar media, and two PCR methods, i.e. a qPCR and a PMA-qPCR method in order to distinguish between viable and dead cells. The proportion of viable but non-cultivable (VBNC) cells was evaluated by comparing the results of propidium monoazide real-time PCR (PMA-qPCR) with the enumeration results. Cultivable bacteria were not detected in brine, regardless of the conditions tested. However, V. cholerae in the VBNC state was detected in brine at 8 degrees C until 48 h of incubation. V. cholerae exhibited higher viability at 8 degrees C compared to 37 degrees C. V. cholerae also exhibited higher viability compared to V. parahaemolyticus. In contrast, for V. parahaemolyticus, temperature and media (except industrial brine) had little influence on cell viability and adhesion behavior.

International audience; The present work was carried out to quantify microplastics (MP) from three sandy beaches along the Côte d’Opale coastline located in the Hauts-de-France region of northern France. Three different study sites located along the English Channel were investigated due to different levels of anthropopression and hydrodynamic conditions. Sediments were collected at three different tide lines: high tide line (HTL), middle of the intertidal zone (IZ), and low tide line (LTL), to investigate the effects of tide line on microplastic contamination. Particles and fibers were counted and colors were recorded; polymer identification was then performed using pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Particle and fiber abundances ranged from 23.4 ± 18.9 to 69.3 ± 30.6 items kg−1 dry weight sediment, with a trend towards fiber predominance, were observed. No difference in particle and fiber abundance was found between the different beaches and tide lines, except for Boulogne-sur-Mer, where the particle number was significantly different between tide lines. Major polymers identified were polyethylene (36.6%) and polypropylene (10.7%). This citizen science project provided preliminary data about the abundance and polymeric nature of MP along the Côte d’Opale coastline.

International audience; Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.