Carmen Piñeiro

Fish species identification in seafood products

Abstract The opening up of international food markets has resulted in the establishment of new regulations that affect different aspects of labels, including ingredients lists. Many fish species sold around the world, especially those caught far away from the countries in which they are consumed, need to be processed on board ship, which may result in the subsequent removal of characteristics used for their classification (head, fins, internal organs). The biochemical characterization of fish species could be achieved using proteins or DNA sequences as species-specific markers. Since different seafood products undergo different processes, the method of analysis has to be chosen according to the modifications undergone by fish constituents during processing. As DNA molecules are more stable than proteins to various processes, including thermal treatment, DNA analysis appears to be a promising method for fish species identification.

Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study.

A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS‐containing solutions were evaluated as extractants. Several preelectrophoretic operations — such as treatment with RNase/DNase, ultrafiltration and desalting — and up to ten types of gels and three SDS‐PAGE systems were considered. The SDS‐containing solution allowed a higher protein extractability than urea. Unlike urea extraction, SDS extraction seemed not to be influenced so much by the state of the sample (raw, cooked at 60oC, cooked at 85oC). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under the optimized technical conditions described above, all the fish species tested, either raw and cooked, yielded reproducible and discriminant species‐specific protein patterns.

Denaturation of fish proteins during frozen storage : role of formaldehyde

Proteins of fish muscle undergo chemical and physical changes during frozen storage which may result in, under certain conditions (i.e. long periods of storage, poor freezing practices, temperature fluctuations, etc), loss of quality, reflected mainly by an unacceptable texture as well as an undesirable flavour, odour and colour. In frozen gadoid fish species, most of these changes are caused by the production of formaldehyde in the muscle. Formaldehyde is produced, along with dimethylamine, by the enzymatic reduction of trimethylamine oxide (TMAO). Many aspects of formaldehyde production by TMAO demethylase (TMAOase) have been studied throughout the last decade. In addition, different approaches have been used to investigate the effect of formaldehyde production on protein denaturation and the associated muscle textural changes. Some insight into the reaction between protein and formaldehyde has clarified the possible mechanism of formaldehyde-mediated denaturation. However, evidence of covalent bonding between proteins and formaldehyde, to form crosslinks, has not explained fully the changes observed in fish proteins during frozen storage. The study of cold-induced denaturation of proteins might give new clues for further investigation of the problem. The implications of formaldehyde in toxicological and nutritional issues is also reviewed, as general concern about the safety of food products is a growing field in food science. Finally, different approaches have been proposed to avoid the detrimental action of formaldehyde during frozen storage of gadoid fish; they are some of the practical applications of the knowledge acquired after years of study of different workers in the field.

Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis

The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of “hake” is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species‐specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two‐dimensional electrophoresis (2‐DE) high‐resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2‐DE analysis as potentially species‐specific, were obtained by “in‐gel” tryptic digestion, followed by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Analysis of group A polypeptides (with pI in the range of 5.0–5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass‐maps from the heat‐resistant parvalbumin fraction (pI below 4.5; molecular mass <12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M. merluccius was also partially sequenced by nanospray‐ion trap‐tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular level.

Species identification of smoked and gravad fish products by sodium dodecylsulphate polyacrylamide gel electrophoresis, urea isoelectric focusing and native isoelectric focusing: a collaborative study

Abstract A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight laboratories. With SDS-PAGE, minor changes took place in the profiles of the processed salmonid species making it impossible or very difficult to identify closely related species. With urea-IEF, there were fewer changes in the profiles due to processing and the system generally had greater species-discriminating power for the processed salmonids than SDS-PAGE. The profiles of the eel species as obtained on SDS-PAGE or urea-IEF were not affected by smoking. Urea-IEF had greater species-discriminating power than SDS-PAGE for the eel species. Native IEF was useful in providing supplementary identification on species difficult to identify by SDS-PAGE or by urea-IEF in the case of cold smoked 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.

Species identification of cooked fish by urea isoelectric focusing and sodium dodecylsulfate polyacrylamide gel electrophoresis: a collaborative study

The suitability and reliability of urea IEF and SDS-PAGE for the identification of cooked fish flesh was tested by a collaborative study among nine laboratories. Urea IEF was performed with CleanGels as well as with ImmobilineGels, and ExcelGels were used for SDS-PAGE, enabling all three types of gels to be run in the same flat bed electrophoresis chamber. By strictly following optimised standard operation procedures (SOPs), five unknown cooked samples had to be identified with each technique using a set of 10 raw reference samples. With urea IEF, only one out of 35 identifications was incorrect, and with SDS-PAGE a similar result was obtained. It was concluded that methods, as now developed, are suitable for checking the species declaration of fishery products.

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.

Trimethylamine oxide and derived compounds' changes during frozen storage of hake (Merluccius merluccius)

Changes in total volatile bases (TVB), dimethylamine (DMA), formaldehyde (FA), trimethylamine (TMA) and trimethylamine oxide (TMAO) of whole hake (Merluccius merluccius L.) were evaluated at different temperatures of storage: −5, −12 and −20 °C. Significant changes in TVB, DMA, TMA, TMAO nitrogen and FA were observed at −5 °C. Only TMAO changed significantly at −12 °C and −20 °C. TMAO decrease was not, therefore, followed by DMA and/or TMA production at those temperatures.

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96—5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water‐soluble fish muscle proteins on Servalyt Precotes 3—6.