Guillaume Duflos

Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua)

Collagenase activity and changes in muscular protein of iced Atlantic cod stored for 9 days were studied. The crude fish muscle extract showed maximum collagenase-like activity against bovine insoluble tendon collagen at 48 h of incubation at 37 °C. Collagenase activity against synthetic substrate increased (P<0.05), especially for fish in initial and advanced stages of decomposition. These results suggest that endogenous collagenases and other proteases may be responsible for the destruction of fine collagenous fibrils in the skeletal muscle of cod. The content of titin 1 decreased when decomposition was advanced. Moreover, a progressive degradation of sarcoplasmic proteins with a molecular weight of 100, 94, 85 and 80 kDa was observed. Results suggest that softening of cod muscle during iced storage is caused more by collagenase activity than by proteolysis of myofibrils.

Occurrence and effects of plastic additives on marine environments and organisms: A review

Plastics debris, especially microplastics, have been found worldwide in all marine compartments. Much research has been carried out on adsorbed pollutants on plastic pieces and hydrophobic organic compounds (HOC) associated with microplastics. However, only a few studies have focused on plastic additives. These chemicals are incorporated into plastics from which they can leach out as most of them are not chemically bound. As a consequence of plastic accumulation and fragmentation in oceans, plastic additives could represent an increasing ecotoxicological risk for marine organisms. The present work reviewed the main class of plastic additives identified in the literature, their occurrence in the marine environment, as well as their effects on and transfers to marine organisms. This work identified polybrominated diphenyl ethers (PBDE), phthalates, nonylphenols (NP), bisphenol A (BPA) and antioxidants as the most common plastic additives found in marine environments. Moreover, transfer of these plastic additives to marine organisms has been demonstrated both in laboratory and field studies. Upcoming research focusing on the toxicity of microplastics should include these plastic additives as potential hazards for marine organisms, and a greater focus on the transport and fate of plastic additives is now required considering that these chemicals may easily leach out from plastics.

Freshness characterisation of whiting (Merlangius merlangus) using an SPME/GC/MS method and a statistical multivariate approach

BACKGROUND The freshness of whiting was studied at five stages of ice storage by comparing the analysis of volatile compounds obtained through solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) with two sensory methods. RESULTS Of the volatile compounds identified, 38 were analysed using a statistical multivariate approach and classified according to their role in the estimation of freshness during storage as markers of freshness or spoilage. Regarding the evolution of the presence or absence of individual compounds, three categories were defined. For example, the volatile compounds propanal, hexanal, 1-penten-3-ol, pentanal, 2,3-pentanedione, 1-penten-3-one, heptanal, (E)-2-pentenal, 2,3-octanedione, (Z)-2-penten-1-ol, 1-pentanol, butanal, octanal, 3,5,5-trimethyl-2-hexene, 1-hexanol and 4,4-dimethyl-1,3-dioxane appeared highly relevant, because they are found throughout storage and can be divided into several categories that are directly related to the quality of fish. CONCLUSION SPME/GC/MS combined with a statistical multivariate approach may be a useful method to identify volatile compounds and characterise fish freshness during storage.

Sensory and physicochemical evolution of tropical cooked peeled shrimp inoculated by Brochothrix thermosphacta and Lactococcus piscium CNCM I-4031 during storage at 8 °C

This study investigated the sensory quality and physicochemical evolution (pH, glucose, l-lactic acid, biogenic amine, free amino-acids and volatile compounds) during storage at 8°C of cooked peeled shrimp inoculated with the specific spoilage bacteria Brochothrix thermosphacta alone or mixed with the protective strain Lactococcus piscium CNCM I-4031. Growth of both bacteria was monitored at regular intervals during storage by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. Bacterial counts showed that L. piscium and B. thermosphacta inoculated at 7 log CFU/g and 3 log CFU/g were well adapted to shrimp, reaching a maximum level of 9 log CFU/g after 4days and 10days respectively. In mixed culture, the growth of B. thermosphacta was reduced by 3.2±0.1 log CFU/g. The TTGE technique allowed monitoring the colonisation of the strains on the shrimp matrix and confirming the dominance of L. piscium in mixed culture throughout the experiment. Sensory analysis confirmed that B. thermosphacta spoiled the product after 11days, when its cell number attained 8 log CFU/g with the emission of strong butter/caramel off-odours. This sensory profile could be linked to the production of 2,3 butanedione, cyclopentanol, 3-methylbutanol, 3-methylbutanal, 2-methylbutanal, 4-methyl-3-chloro-3-pentanol and ethanol, which were produced in more significant quantities in the B. thermosphacta batch than in the batches in which the protective strain was present. On the contrary, TVBN and TMA were not suitable as quality indicators for B. thermosphacta spoilage activity. In the products where the protective L. piscium strain was present, no adverse effect on sensory quality was noted by the sensory panels. Moreover, biogenic amine assessment did not show any histamine or tyramine production by this strain, underlining its safety profile. Both strains produced lactic acid (1850mg/kg in L. piscium and B. thermosphacta batch on days 3 and 10 respectively; 3830mg/kg on day 7 in mixed culture) and the pH decrease from 6.6±0.0 to 5.9±0.1 was similar in all batches. Lactic acid production or competition for free amino-acid was not involved in the inhibition mechanism; however rapid glucose consumption by L. piscium could partially explain the growth limitation of the spoilage micro-organism. This study demonstrated the spoilage characteristic of B. thermosphacta and the usefulness of L. piscium as a bioprotective culture for tropical cooked peeled shrimp without any adverse effect on the sensory quality of the product.

Differentiation of fresh and frozen/thawed fish, European sea bass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), cod (Gadus morhua) and salmon (Salmo salar), using volatile compounds by SPME/GC/MS

BACKGROUND A simple method based on solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) was applied for studying the volatile profiles of whole fish samples of European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) and fillets of cod (Gadus morhua) and salmon (Salmo salar) during frozen storage in order to be able to differentiate a fresh product from one that has been frozen. Analysis of volatile compounds was performed on these two product types, fresh and after freezing/thawing following storage at - 20 °C for 30 and 90 days. RESULTS More than a hundred volatile compounds were found by SPME/GC/MS. Statistical processing by principal component analysis and ascending hierarchical classification was used to classify the samples into categories and verify the possibility of separating fresh samples from those that had been frozen and thawed. The compounds to be used as differentiators were identified. Four compounds were common to all species: dimethyl sulfide, 3-methylbutanal, ethyl acetate and 2-methylbutanal. Not only were they found in larger quantities after thawing but they also increased with the duration of storage at - 20 °C. CONCLUSION These four compounds can therefore be considered as potential markers of differentiation between a fresh product and one that has been frozen.

Amino acid decarboxylase activity and other chemical characteristics as related to freshness loss in iced cod (Gadus morhua).

Biogenic amine levels and other biochemical indicators were measured to study the safety of and the loss of freshness in iced Atlantic cod. Biogenic amine content exhibited high variability during iced storage of Atlantic cod. Ornithine and lysine decarboxylase activity apparently increased at the end of the storage period. Amino acid activity was probably generated by endogenous amino acid decarboxylases of raw fish. No statistical differences were observed in the total volatile base fraction or in the ammonia or monomethylamine contents during iced storage. However, trimethylamine contents showed a significant exponential relationship with time and sensory score. Cod formed inosine as the major metabolite of IMP. The H and G indices showed a linear relationship with time and sensory score and served as good indicators of cod freshness quality. However, the K, Ki, and P indices showed a logarithmic relationship with time and sensory score. IMP, K, Ki, and P served as indicators of freshness lost during the early stages of chilled storage of cod.

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

Four 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 H2S. 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 H2S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora.

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.

BACKGROUND 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.

Differentiation between fresh and frozen–thawed sea bass (Dicentrarchus labrax) fillets using two-dimensional gel electrophoresis

This study aimed to identify a protein marker that can differentiate between fresh skinless and frozen-thawed sea bass (Dicentrarchus labrax) fillets using the two-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Distinct gel patterns, due to proteins with low molecular weight and low isoelectric points, distinguished fresh fillets from frozen-thawed ones. Frozen-thawed fillets showed two specific protein spots as early as the first day of the study. However, these spots were not observed in fresh fillets until at least 13days of storage between 0 and 4°C, fillets were judged, beyond this period, fish were unfit for human consumption as revealed by complementary studies on fish spoilage indicators namely total volatile basic nitrogen and biogenic amines. Mass spectrometry identified the specific proteins as parvalbumin isoforms. Parvalbumins may thus be useful markers of differentiation between fresh and frozen-thawed sea bass fillets.

Quantitative PCR Method for Evaluating Freshness of Whiting (Merlangius merlangus) and Plaice (Pleuronectes platessa)

We have developed a method for rapid quantification of fish spoilage bacteria based on quantitative PCR with degenerated oligonucleotides that hybridize on the torA gene coding for trimethylamine N-oxide reductase, one of the major bacterial enzymes in fish spoilage. To show the utility of this gene, we used a regular PCR with DNA extracts from whiting (Merlangius merlangus) and plaice (Pleuronectes platessa) stored in ice. Quantitative PCR showed that the number of copies of the torA gene, i.e., the number of spoilage bacteria, increases with length of storage. This approach can therefore be used to evaluate freshness for the two fish species studied (whiting and plaice).