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

Bifidobacterium longum Requires a Fructokinase (Frk; ATP:d-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (K(m)(fructose) = 0.739 ± 0.18 mM and K(m)(ATP) = 0.756 ± 0.08 mM), is highly specific for d-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for d-fructose

Quantification of fish partial volatile basic nitrogen by SPME-GC/MS

International audienceFreshness is a key parameter in the evaluation of the fish quality. As this matrix is highly perishable, objective tools for the freshness estimation are required. By the past, numerous methods including sensorial, chemical, microbiological and physical analysis were developed to follow evolution of odour, sight or texture throughout the spoiling process.Spoiling of fish is a complex process combining autolysis and exogenous degradations. One of the most famous examples is the degradation of trimethylamine-N-oxide (TMAO) in Gadidae by both TMAO reductases of specific spoiling organisms and TMAO demethylase, an endogenic enzyme. These reactions lead respectively to the production of trimethylamine (TMA) and dimethylamine (DMA). To date, TMA and DMA are routinely non-specifically measured among other volatile amines thanks to total volatile basic nitrogen (TVB-N) analysis; TMA can be nonetheless measured more specifically by TVB-N when formaldehyde is added. The concept of partial volatile base nitrogen (PVB-N) was implemented using SPME-GC/MS to quantify both TMA and DMA. Further, this technique allows specifically detecting and measuring TMA and DMA.Extraction (temperature, time, sample preparation) and separation (temperature, column phase, split type) parameters were optimized to obtain best chromatographic profiles. Both fresh and spoiled fish were analyzed by TVB-N and PVB-N techniques. First results show consistent conclusions. Moreover, comparison of TVB-N and PVB-N leads to the conclusion that the current TMA measurement, with addition of formaldehyde, is overestimating TMA content in the flesh of fish

Quantification de l’azote basique volatil partiel par SPME-GC-MS : Application au suivi de l’évolution de la fraîcheur de filets de cabillaud (Gadus morhua)

National audienceLa fraîcheur est un paramètre clé pour l’évaluation de la qualité du poisson. Cette matrice étant extrêmement périssable, l’utilisation d’outils objectifs est nécessaire. Par le passé de nombreuses méthodes incluant les analyses sensorielles, chimiques, microbiologiques et physiques ont été développées pour suivre l’évolution de paramètres olfactifs visuels et tex-turaux tout au long du processus d’altération.L’altération du poisson est un processus complexe combinant des dégradations autolytiques et exogènes. Un exemple connu concernant l’altération de la chair des Gadidés est la dégra-dation de l’oxyde de triméthylamine (OTMA) selon deux voies cataboliques: la première fait intervenir l’OTMA réductase synthétisée par la flore spécifique d’altération et la seconde est le résultat de l’activité d’une enzyme endogène du poisson l’OTMA déméthylase. Ces réac-tions conduisent respectivement à la production de triméthylamine (TMA) et la diméthyla-mine (DMA).Jusqu’à présent la TMA et la DMA sont mesurées en routine de façon non-spécifique avec d’autres amines volatiles grâce l’analyse des amines basiques volatiles totales (ABVT) ; la TMA pouvant néanmoins être analysée plus spécifiquement avec l’ajout de formaldéhyde lors du processus analytique de l’ABVT. Le concept d’azote basique volatil partiel a été déve-loppé pour quantifier spécifiquement la DMA et la TMA à l’aide d’une SPME-GC-MS.La méthode développée a fait l’objet d’une phase d’optimisation puis de sélection d’un étalon interne avant de s’intéresser à la plage de quantification. Suite à ce développement un suivi d’altération a été réalisé sur des filets de cabillaud stockés sous glace fondante. Aux diffé-rents jours de conservation, trois filets ont été analysés à l’aide de différentes techniques : analyse sensorielle réalisée avec la méthode du Quality Index Method (QIM), analyses chi-miques par les méthodes d’ABVT et ABVP. Les concentrations en TMA mesurées par la méthode d’ABVT et celle d’ABVP sont très bien corrélées (r=0,98). Au regard des résultats de l’ABVT et de ceux du QIM, il apparait que la limite d’acceptation/rejet des filets est située au voisinage du 7ème jour de conservation. La production de TMA en cours d’altération a pu être modélisée, néanmoins son utilisation seule n’apparait pas comme un bon indicateur du déclin de la fraîcheur lors des premiers jours d’altération. Afin de surmonter cette limite, l’utilisation d’un nouvel indicateur issu des résultats de l’ABVP, le ratio DMA sur TMA (DTR), a montré des résultats encourageants avec une anti-corrélation significative (r = - 0,87 ; p-value < 0.001) lors de la phase précoce de déclin de la fraîcheur du poisson

Quantification of partial volatile basic nitrogen by SPME-GC-MS : Application to the monitoring of the evolution of cod fillets (Gadus morhua) freshness

International audienceLa fraîcheur est un paramètre clé pour l’évaluation de la qualité du poisson. Cette matrice étant extrêmement périssable, l’utilisation d’outils objectifs est nécessaire. Par le passé de nombreuses méthodes incluant les analyses sensorielles, chimiques, microbiologiques et physiques ont été développées pour suivre l’évolution de paramètres olfactifs visuels et texturaux tout au long du processus d’altération.L’altération du poisson est un processus complexe combinant des dégradations autolytiques et exogènes. Un exemple connu concernant l’altération de la chair des Gadidés est la dégradation de l’oxyde de triméthylamine (OTMA) selon deux voies cataboliques: la première fait intervenir l’OTMA réductase synthétisée par la flore spécifique d’altération et la seconde est le résultat de l’activité d’une enzyme endogène du poisson l’OTMA déméthylase. Ces réactions conduisent respectivement à la production de triméthylamine (TMA) et la diméthylamine (DMA).Jusqu’à présent la TMA et la DMA sont mesurées en routine de façon non-spécifique avec d’autres amines volatiles grâce l’analyse des amines basiques volatiles totales (ABVT) ; la TMA pouvant néanmoins être analysée plus spécifiquement avec l’ajout de formaldéhyde lors du processus analytique de l’ABVT. Le concept d’azote basique volatil partiel a été développé pour quantifier spécifiquement la DMA et la TMA à l’aide d’une SPME-GC-MS.La méthode développée a fait l’objet d’une phase d’optimisation puis de sélection d’un étalon interne avant de s’intéresser à la plage de quantification. Suite à ce développement un suivi d’altération a été réalisé sur des filets de cabillaud stockés sous glace fondante. Aux différents jours de conservation, trois filets ont été analysés à l’aide de différentes techniques : analyse sensorielle réalisée avec la méthode du Quality Index Method (QIM), analyses chimiques par les méthodes d’ABVT et ABVP. Les concentrations en TMA mesurées par la méthode d’ABVT et celle d’ABVP sont très bien corrélées (r=0,98). Au regard des résultats de l’ABVT et de ceux du QIM, il apparait que la limite d’acceptation/rejet des filets est située au voisinage du 7ème jour de conservation. La production de TMA en cours d’altération a pu être modélisée, néanmoins son utilisation seule n’apparait pas comme un bon indicateur du déclin de la fraîcheur lors des premiers jours d’altération. Afin de surmonter cette limite, l’utilisation d’un nouvel indicateur issu des résultats de l’ABVP, le ratio DMA sur TMA (DTR), a montré des résultats encourageants avec une anti-corrélation significative (r = - 0,87 ; p-value < 0.001) lors de la phase précoce de déclin de la fraîcheur du poisson

Development of a qPCR method targeting torA gene and application for the freshness monitoring of modified atmosphere-packed chilled whiting (Merlangius merlangus)

International audienceIntroduction:Tracking the early decline of freshness is an important concern for the fishery industry to insure the best quality for foodstuffs highly liable to spoil. A lot of techniques have been developed like sensory, chemical and more slightly microbial analysis. Unfortunately, most of them have drawbacks of being subjective, or having the disadvantage of being either reliable once the freshness is lost or for the analysis of whole fish. The study presents the development of a qPCR method targeting a gene harboured by specific spoilage organisms (SSOs) of fish: torA. This gene encodes an enzyme responsible of the production of trimethylamine whose odour is characteristic of the spoiled fish.Material and methods:The study aimed to develop a degenerate primer pair able to amplify torA gene in a wide range of SSOs. For that, numerous software and algorithms were used for a maximal reliability of the in silico design process. A first selection of pairs where tested in vitro to further characterization. Finally a primer pair was conserved for efficiency and selectivity tests. The selected primer pair was tested for analysing MAP-chilled whiting along a 15 days storage study. Methods such as total volatile basic nitrogen (TVB-N) and trimethylamine (TMA) analysis or total viable count (TVC) analysis were performed simultaneously to evaluate fish overall quality.Results and discussion:A degenerate primer pair was selected after six steps of in silico design and selection. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies, ranging from 91.6 to 93 % regarding species, on 7-log DNA dilutions. The best conditions of annealing temperature and primer concentration were found to be 62°C and 1 µM. As regards with selectivity, the degenerate primer pair allows to selectively amplify torA gene of Vibrio and Photobacterium compared with other tested species. TVC study led to inconclusive results, probably because ISO standard used was not suitable for these kinds of food and storage. However, throughout the storage of fillets, the qPCR approach allows detecting an increase of torA copies. Additionally, good correlation between qPCR results and the evolution of known spoilage markers were established, such as -0.86 (TVB-N) and -0.81 (TMA). Conclusion:This study, thanks to careful steps of primers characterization, allowed designing a primer pair able to amplify torA gene of both Vibrio and Photobacterium. A very promising, sensitive and time-effective qPCR technique is thus proposed to characterize the freshness of processed whiting

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium

Bifidobacterium bifidum, in contrast to other bifido-bacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetyl-glucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism

Preliminary evidence for a serum disaccharide signature of invasive Candida albicans infection detected by MALDI Mass Spectrometry

The diagnosis of systemic Candida infections is a recognized challenge. We developed a mass spectrometry strategy to detect signals from Candida molecules in patients' sera. Pre-analytical procedures were designed to extract oligosaccharides from serum. A peak m/z of at 365 was specifically revealed in sera from patients with candidaemia with regard to healthy controls. This biomarker was identified as a disaccharide, its presence did not correlate with mannanaemia or glucanaemia. Mouse models of Candida albicans colonization and infection showed that the signal was specifically associated with tissue invasion, suggesting that clinical evaluation of its usefulness in discriminating colonized and infected patients would be worthwhile

Use of degenerate primers to detect and quantify torA gene harbored by specific spoilage organisms of fish

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

Identification of O-GlcNAcylated proteins in Plasmodium falciparum

Abstract Background Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. Methods The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). Results While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. Conclusions This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle