Development of an SPME-GC-MS method for the specific quantification of dimethylamine and trimethylamine: use of a new ratio for the freshness monitoring of cod fillets

International audienceBACKGROUND: Fish is a highly perishable food, so it is important to be able to estimate its freshness to ensure optimum quality for consumers. The present study describes the development of an SPME‐GC‐MS technique capable of quantifying both trimethylamine (TMA) and dimethylamine (DMA), components of what has been defined as partial volatile basic nitrogen (PVB‐N). This method was used, together with other reference methods, to monitor the storage of cod fillets (Gadus morhua) conserved under melting ice.RESULTS: Careful optimisation enabled definition of the best parameters for extracting and separating targeted amines and an internal standard. The study of cod spoilage by sensory analysis and TVB‐N assay led to the conclusion that the shelf‐life of cod fillet was between 6 and 7 days. Throughout the study, TMA and DMA were specifically quantified by SPME‐GC‐MS; the first was found to be highly correlated with the values returned by steam distillation assays. Neither TMA‐N nor DMA‐N were able to successfully characterise the decrease in early freshness, unlike dimethylamine/trimethylamine ratio (DTR), whose evolution is closely related to the results of sensory analysis until the stage where fillets need to be rejected.CONCLUSION: DTR was proposed as a reliable indicator for the early decrease of freshness until fish rejection

Evaluation de la qualité-fraîcheur du poisson par des approches biochimiques (SPME-GC/MS) et moléculaires (qPCR)

Freshness is a key parameter for the fish quality assessment. By the past, several methods have been developed, but they displayed limits mainly in terms of subjectivity and precision. The aim of this work was to search indicators of quality based on causes and consequences of fish spoilage. Concerning causes, the spoilage flora was studied by microbiological and molecular techniques. A first work allowed characterizing Shewanella baltica strains isolated from fish; one of the strains has the particularity to be H2S negative suggesting that traditional microbiological techniques might be limited. Then, a qPCR approach has selected primers tested during a spoilage monitoring study. Results show a linear decrease of the signal for the early days of alteration, attributed to the presence of Photobacterium phosphoreum, with good anticorrelations with biochemical methods of reference. Subsequently, consequences have been studied quantifying volatile amines by a method of SPME-GC-MS by defining the concept of partial volatile basic nitrogen (PVB-N) specifically quantifying trimethylamine (TMA) and dimethylamine (DMA). This method showed for TMA an excellent correlation with a reference technique. PVB-N measurements allowed creating an indicator, DMA on TMA ratio, evolving in the early steps of spoilage. Meanwhile, two projects based of the analysis of processed fish volatilome allowed working on quality in a wider sense.La fraîcheur est un paramètre important pour l’évaluation de la qualité du poisson. Plusieurs méthodes ont été développées par le passé, mais elles possèdent des limites principalement en termes de subjectivité et de précision. Le but de ce travail a été de chercher des indicateurs de la qualité en se basant sur les causes et les conséquences de l’altération du poisson. En ce qui concerne les causes, la flore d’altération a été étudiée par des techniques de microbiologie et de biologie moléculaire. Un premier travail a permis de caractériser des souches de Shewanella baltica isolées de poisson ; une des souches possède la particularité d’être H2S négative laissant penser que les techniques microbiologiques traditionnelles pourraient être limitées. Une approche qPCR a ensuite sélectionné des amorces testées lors d’un suivi d’altération. Les résultats montrent une baisse linéaire du signal dès les premiers jours d’altération, attribuée à la présence de Photobacterium phosphoreum, avec de bonnes anticorrélations aux méthodes biochimiques de référence. Par la suite, les conséquences ont été évaluées en quantifiant des amines volatiles par une méthode de SPME-GC-MS en définissant le concept d’azote basique volatil partiel (ABVP) permettant de quantifier spécifiquement la triméthylamine (TMA) et la diméthylamine (DMA). Cette méthode a montré, pour la TMA, une excellente corrélation avec une technique de référence. Les mesures d’ABVP ont permis de créer un indicateur, le ratio DMA sur TMA, évoluant lors des premiers stades d’altération. Parallèlement, deux projets basés sur l’analyse du volatilome de poissons transformés ont permis de travailler sur la qualité au sens larg

Evaluation of fish quality-freshness by biochemical (SPME-GC/MS) and molecular (qPCR) approaches

La fraîcheur est un paramètre important pour l’évaluation de la qualité du poisson. Plusieurs méthodes ont été développées par le passé, mais elles possèdent des limites principalement en termes de subjectivité et de précision. Le but de ce travail a été de chercher des indicateurs de la qualité en se basant sur les causes et les conséquences de l’altération du poisson. En ce qui concerne les causes, la flore d’altération a été étudiée par des techniques de microbiologie et de biologie moléculaire. Un premier travail a permis de caractériser des souches de Shewanella baltica isolées de poisson ; une des souches possède la particularité d’être H2S négative laissant penser que les techniques microbiologiques traditionnelles pourraient être limitées. Une approche qPCR a ensuite sélectionné des amorces testées lors d’un suivi d’altération. Les résultats montrent une baisse linéaire du signal dès les premiers jours d’altération, attribuée à la présence de Photobacterium phosphoreum, avec de bonnes anticorrélations aux méthodes biochimiques de référence. Par la suite, les conséquences ont été évaluées en quantifiant des amines volatiles par une méthode de SPME-GC-MS en définissant le concept d’azote basique volatil partiel (ABVP) permettant de quantifier spécifiquement la triméthylamine (TMA) et la diméthylamine (DMA). Cette méthode a montré, pour la TMA, une excellente corrélation avec une technique de référence. Les mesures d’ABVP ont permis de créer un indicateur, le ratio DMA sur TMA, évoluant lors des premiers stades d’altération. Parallèlement, deux projets basés sur l’analyse du volatilome de poissons transformés ont permis de travailler sur la qualité au sens large.Freshness is a key parameter for the fish quality assessment. By the past, several methods have been developed, but they displayed limits mainly in terms of subjectivity and precision. The aim of this work was to search indicators of quality based on causes and consequences of fish spoilage. Concerning causes, the spoilage flora was studied by microbiological and molecular techniques. A first work allowed characterizing Shewanella baltica strains isolated from fish; one of the strains has the particularity to be H2S negative suggesting that traditional microbiological techniques might be limited. Then, a qPCR approach has selected primers tested during a spoilage monitoring study. Results show a linear decrease of the signal for the early days of alteration, attributed to the presence of Photobacterium phosphoreum, with good anticorrelations with biochemical methods of reference. Subsequently, consequences have been studied quantifying volatile amines by a method of SPME-GC-MS by defining the concept of partial volatile basic nitrogen (PVB-N) specifically quantifying trimethylamine (TMA) and dimethylamine (DMA). This method showed for TMA an excellent correlation with a reference technique. PVB-N measurements allowed creating an indicator, DMA on TMA ratio, evolving in the early steps of spoilage. Meanwhile, two projects based of the analysis of processed fish volatilome allowed working on quality in a wider sense

Current frontiers and recommendations for the study of microplastics in seafood

International audienceFor seventy years, mass plastic production and waste mismanagement have resulted in huge pollution of the environment, including the marine environment. The first mention of seafood contaminated by microplastics was recorded in the seventies, and to date numerous studies have been carried out on shellfish, fish and crustaceans. Based on an ad hoc corpus, the current review aims to report on the numerous practices and methodologies described so far. By examining multiple aspects including problems related to the definition of the term microplastic, contamination at the laboratory scale, sampling and isolation, and quantification and identification, the aim was to point out current limitations and the needs to improve and harmonise practices for future studies on microplastics in seafood. A final part is devoted to the minimum information for publication of microplastics studies (MIMS). Based on the aspects discussed, MIMS act as a starting point for harmonisation of analyses

Think positive: Proposal of a simple method to create reference materials in the frame of microplastics research

In the context of the harmonization of methodologies employed to isolate and count microplastics in samples or to organize ring trials tests, the use of reference materials, i.e. samples with controlled amount of particles is required. The method proposed here uses transparent, sealed capsules containing in-house generated microplastics as a convenient way to generate microplastic reference materials. This method is a simple approach for adding particles to samples without risk of loss during particle extraction or transporting/handling. • Low-cost and easy-to-use preparation of heterogeneous mix of microplastic reference particles • Possibility to control microplastic size, shape, and polymeric composition • Applicable to many protocols and wide range of applications on water, sediments and biota

Quantification of organic plastic additives in plastics with (TD) Py-GC-HRMS

International audiencePlastics were revolutionary inventions that symbolized globalization and the interconnection of economies between countries in the second half of the 20th and the early of the 21st centuries. They are widely used in various industrial sectors including food packaging, construction, automotive, electronics, textiles, household items, and toys, with the current global production reaching over 370 million tons per year. These synthetic materials are made of an organic polymer matrix and chemical additives. In total, more than 10 000 additives were identified in plastics and over 2 400 are considered as substances of potential concern as they meet one or more of toxicity criteria in the European Union (toxicity for reproduction, bioaccumulation,…).These substances may leached during the plastic life cycle (to foodstuff, environment, etc), resulting in potential human exposure.Thus, the development of analytical methods capable of identifying and quantifying additives in plastics is necessary. It has been demonstrated that the thermal desorption method using aPyrolysis coupled to GC-HRMS system can be a useful analytical tool for the rapid quantification of additives in polymer samples but method still need to be developed. For this study, additives of interest were first selected based on two main criteria: their toxicity according to the European Chemicals Agency and their migration limits set by EU Regulation No 10/2011. In total, 13 additives were selected (5 plasticizers, 6 flame retardants and 2 UV stabilizers).Then, a reflection was set up in order to consider how to evaluate the response function of additives in the context of solid-state sample direct analysis. It was chosen to develop reference material incorporating additives into a polymer matrix at specified concentration levels, to produce plastic films, using masterbatch. This process is aimed to ensure homogeneous dispersion of the additives in the polymer matrix.Finally, some preliminary analytical developments were carried out in order to perform future analyses, like acquisition of additives HRMS spectra, MS/MS patterns to select the optimum collision energies for each characteristic ion and the most abundant fragment resulting from fragmentation. A first application on solid-state plastic will be proposed

Phenotypic and genotypic characterization of H2S-positive and H2S-negative strains of Shewanella baltica isolated from spoiled whiting ( Merlangius merlangus )

International audienceFour strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4 to 30°C and also able to develop anaerobically in the presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2 S. Two strains synthetized an ornithine decarboxylase being potential putrescine producers. Results of carbon source use highlighted that the four strains were able to use citrate and d-sucrose and one strain was not able to use l-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2 S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora

Study of a fishery product process in the frame of microplastics research

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Répartition verticale et variabilité saisonnière de la contamination microplastiques dans la colonne d’eau d’une rivière en utilisant une méthode d’échantillonnage innovante

National audienceDans 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