Protein Signatures to Trace Seafood Contamination and Processing.

This review presents some applications of proteomics and selected spectroscopic methods to validate certain aspects of seafood traceability. After a general introduction to traceability and the initial applications of proteomics to authenticate traceability information, it addresses the application of proteomics to trace seafood exposure to some increasingly abundant emergent health hazards with the potential to indicate the geographic/environmental origin, such as microplastics, triclosan and human medicinal and recreational drugs. Thereafter, it shows the application of vibrational spectroscopy (Fourier-Transform Infrared Spectroscopy (FTIR) and Fourier-Transform Raman Spectroscopy (FT Raman)) and Low Field Nuclear Magnetic Resonance (LF-NMR) relaxometry to discriminate frozen fish from thawed fish and to estimate the time and temperature history of frozen fillets by monitoring protein modifications induced by processing and storage. The review concludes indicating near future trends in the application of these techniques to ensure seafood safety and traceability.

Proteomic Strategies to Evaluate the Impact of Farming Conditions on Food Quality and Safety in Aquaculture Products.

This review presents the primary applications of various proteomic strategies to evaluate the impact of farming conditions on food quality and safety in aquaculture products. Aquaculture is a quickly growing sector that represents 47% of total fish production. Food quality, dietary management, fish welfare, the stress response, food safety, and antibiotic resistance, which are covered by this review, are among the primary topics in which proteomic techniques and strategies are being successfully applied. The review concludes by outlining future directions and potential perspectives.

Commented checklist of marine fishes from the Galicia Bank seamount (NW Spain).

A commented checklist containing 139 species of marine fishes recorded at the Galician Bank seamount is presented. The list is based on nine prospecting and research surveys carried out from 1980 to 2011 with different fishing gears. The ichthyofauna list is diversified in 2 superclasses, 3 classes, 20 orders, 62 families and 113 genera. The largest family is Macrouridae, with 9 species, followed by Moridae, Stomiidae and Sternoptychidae with 7 species each. The trachichthyd Hoplostethus mediterraneus and the morid Lepidion lepidion were the most abundant species. Biogeographically, the Atlantic group, with 113 species (81.3%) is the best represented, followed by the Lusitanian one with 17 species (12.2%). Data on species abundance, as number of individuals caught, size and depth are reported. Habitat, distribution and vulnerability status are commented. Moreover, biometric data and meristic counts are also reported for several species. The results obtained showing a high fish biodiversity and a sensible number of threatened species, strongly support the future declaration of the Galicia Bank as a Marine Protected Area.

Production of Hyaluronic Acid by Streptococcus zooepidemicus on Protein Substrates Obtained from Scyliorhinus canicula Discards.

This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs.

Contrasting evolutionary demography induced by fishing: the role of adaptive phenotypic plasticity.

Mounting evidence now shows that fishing activity modifies both heritable life-history traits and ecological processes in harvested populations. However, ecological and evolutionary changes are intimately linked and can occur on the same time scale, and few studies have investigated their combined effect on fish population dynamics. Here, we contrast two population subunits of a harvested fish species in the Northeast Atlantic, the European hake (Merluccius merluccius), in the light of the emerging field of evolutionary demography, which considers the interacting processes between ecology and evolution. The two subunits experienced similar age/size truncation due to size-selective fishing, but displayed differences in key ecological processes (recruitment success) and phenotypic characteristics (maturation schedule). We investigate how temporal variation in maturation and recruitment success interactively shape the population dynamics of the two subunits. We document that the two subunits of European hake displayed different responses to fishing in maturation schedules, possibly because of the different level of adaptive phenotypic plasticity. Our results also suggest that high phenotypic plasticity can dampen the effects of fisheries-induced demographic truncation on population dynamics, whereas a population subunit characterized by low phenotypic plasticity may suffer from additive effects of ecological and life-history responses. Similar fishing pressure may thus trigger contrasting interactions between life history variation and ecological processes within the same population. The presented findings improve our understanding of how fishing impacts eco-evolutionary dynamics, which is a keystone for a more comprehensive management of harvested species.

Identification of the major ACE-inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate from cuttlefish wastewater.

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 µg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 µg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A-D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 µg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.

Optimization of the pepsin digestion method for anisakids inspection in the fishing industry.

During the last 50 years human anisakiasis has been rising while parasites have increased their prevalence at determined fisheries becoming an emergent major public health problem. Although artificial enzymatic digestion procedure by CODEX (STAN 244-2004: standard for salted Atlantic herring and salted sprat) is the recommended protocol for anisakids inspection, no international agreement has been achieved in veterinary and scientific digestion protocols to regulate this growing source of biological hazard in fish products. The aim of this work was to optimize the current artificial digestion protocol by CODEX with the purpose of offering a faster, more useful and safer procedure for factories workers, than the current one for anisakids detection. To achieve these objectives, the existing pepsin chemicals and the conditions of the digestion method were evaluated and assayed in fresh and frozen samples, both in lean and fatty fish species. Results showed that the new digestion procedure considerably reduces the assay time, and it is more handy and efficient (the quantity of the resulting residue was considerably lower after less time) than the widely used CODEX procedure. In conclusion, the new digestion method herein proposed based on liquid pepsin format is an accurate reproducible and user-friendly off-site tool, that can be useful in the implementation of screening programs for the prevention of human anisakiasis (and associated gastroallergic disorders) due to the consumption of raw or undercooked contaminated seafood products.

Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring.

A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers.

De novo mass spectrometry sequencing and characterization of species-specific peptides from nucleoside diphosphate kinase B for the classification of commercial fish species belonging to the family Merlucciidae.

The characterization by de novo peptide sequencing of the different protein nucleoside diphosphate kinase B (NDK B) from all the commercial hakes and grenadiers belonging to the family Merlucciidae is reported. A classical proteomics approach, consisting of two-dimmensional gel electrophoresis, tryptic in-gel digestion of the excised spots, MALDI-TOF MS, LC-MS/MS, and nanoESI-MS/MS analyses, was followed for the purification and characterization of the different isoforms of the NDK B. Fragmentation spectra were used for de novo peptide sequence. A high degree of homology was found between the sequences of all the species studied and the NDK B sequence from Gillichthys mirabilis, which is accessible in the protein databases. Particular attention was paid to the differential characterization of species-specific peptides that could be used for fish authentication purposes. These findings allowed us to propose a rapid and effective classification method, based in the detection of these biomarker peptides using the selective ion reaction monitoring (SIRM) scan mode in mass spectrometry.

Hypoploidy defines patients with poor prognosis in breast cancer.

The clinicopathological features currently used in breast cancer prognosis often fail to characterize the clinical heterogeneity of the disease accurately. Our study is aimed to investigate the predictive value of DNA flow cytometry in breast cancer. Previously untreated breast carcinoma samples (584) were snap frozen for flow-cytometry. Tumors were classified into three DNA index (DI) categories: i) tumors showing a DI =0.96-1.15 (diploid and near-diploid); ii) tumors with a DI >or=1.16 (hyperdiploid, tetraploid, multiploid and/or those with more than one diploid population); and iii) tumors with a DI

Trends in sample preparation for classical and second generation proteomics.

Sample preparation is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extraction and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein separation by 2-DE, protein digestion, mass spectrometry analysis, multidimensional peptide separations and quantification of protein expression level are described.

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction.

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.