Iciar Martinez

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.

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.

Estimation of freezing storage time and quality changes in hake (Merluccius merluccius, L.) by low field NMR.

The potential of low field NMR (LF NMR) as a fast monitoring technique to estimate the quality of hake (Merluccius merluccius) frozen stored at -10°C for up to 6months was evaluated. LF NMR clearly detected three populations of water: water strongly bound to macromolecules (T(2b)), trapped water (T(21)) and free water (T(22)). As storage time increased, and concomitant with an increase in the T(22) and a decrease in the T(21) water populations, the water holding capacity (WHC) and apparent viscosity values decreased and the shear strength increased, reflecting the characteristic loss of juiciness and tougher texture developed by hake during frozen storage. Two mathematical models were constructed: a simple regression using the biexponential analysis of the relaxation times (T(21), T(22)) and amplitudes (A(21), A(22)) and a partial least square regression (PLS) of CONTIN analysis. Both models seemed suitable to estimate the quality of the product.

Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods

Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.

Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing

RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants. The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units. There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers. The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them. None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases. Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples. In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora. Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources. We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L. monocytogenes must be treated as potentially harmful.

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.

Species identification of formed fishery products and high pressure-treated fish by electrophoresis: a collaborative study

The suitability and reliability of three electrophoretic methods of fish species identification, urea isoelectric focusing (IEF), sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and native IEF, were evaluated on formed fish fillets and high pressure fish flesh by a collaborative study among four institutes. By following optimized standard operation procedures, the protein patterns of processed fish were compared to patterns of raw reference samples. The method to use depended of the effect of processing on the protein pattern. The proteins obtained from formed products were not denatured and therefore any of the three methods proved to be adequate, with a preference for native IEF which had a better discriminatory power for the species used. The high pressure process altered the proteins, and so only urea IEF and SDS-PAGE methods could be used. For these products, the chosen method should then be the one with the better discriminating power for the species being examined.

Effects of T3 and rearing temperature on growth and skeletal myosin heavy chain isoform transition during early development in the salmonid Salvelinus alpinus (L.)

Abstract Rearing of 1-year-old Arctic charr (Salvelinus alpinus) at 12°C, as well as the administration of 50 or 75 mgT3/kg feed, accelerated the neonatal to adult fast myosin heavy chain transition, but the effect of temperature was more dramatic than the effect of T3 administration. The endogenous plasma levels of T3 in charrs reared at 12°C were higher than those of analogous groups reared at natural temperature, which in the period under study was between 0.5 and 12°C. As in other species, T3 seemed to play a role in the regulation of the neonatal to adult fast myosin isoform transition by down-regulating the levels of the neonatal and increasing the levels of an adult fast myosin heavy chain. Temperature seemed to accelerate this transition at least, but not only, by inducing an increase in the endogenous levels of T3 in the Arctic charr.

Myofibrillar proteins in skeletal muscles of parr, smolt and adult atlantic salmon (Salmo salarl.). Comparison with another salmonid, the arctic charr Salvelinus alpinus (l.)

Abstract The heavy and light subunits of myosin from white and red muscles of Atlantic salmon parr, smolt and adult individuals were analyzed by SDS-PAGE and two-dimensional electrophoresis. Tropomyosin was identified by comigration with rat tropomyosins in two-dimensional gels in the presence and absence of urea. These myofibrillar proteins were compared to those of Arctic charr. 1. 1. The myosin heavy chain from Atlantic salmon red muscles was associated with two types of light chain, 1S and 2S, that comigrated with the light chains 1S and 2S of Arctic charr. 2. 2. As in the Arctic charr, four myosin light chain spots were detected in white muscles: two fast myosin light chains type 1, one of which comigrated with its analogous in the Arctic charr; one fast myosin light chain type 2, differing slightly in isoelectric point from that of Arctic charr; and one fast myosin light chain type 3 with higher electrophoretic mobility than that of Arctic charr. 3. 3. Three tropomyosin spots were detected. White muscles contained only one type of β-tropomyosin and red muscles two types of α-tropomyosin. These three tropomyosin spots comigrated with those of Arctic charr. 4. 4. Two myosin heavy chain bands were observed in red muscles of salmon parrs but only one in the rest of the red muscles analyzed. 5. 5. Only one myosin heavy chain band was detected in white muscles by SDS-glycerol-polyacrylamide gel electrophoresis. Alfa-chymotryptic peptide mapping of these white myosin heavy chain bands revealed differences attributed to the presence of a new type of myosin heavy chain first detected several months after smoltification.

Electrophoretic study of myosin isoforms in white muscles of some teleost fishes

1. White skeletal muscle myosin of four marine teleost fish species (cod, blue whiting, Norway haddock, and spotted wolf-fish) was analyzed by native, SDS-PAGE, and 2-dimensional electrophoresis. 2. Four types of native myosin were present in cod, blue whiting and Norway haddock. The second fastest migrating form was predominant. 3. Myosin from spotted wolf-fish also resolved into four forms. The fastest migrating form was hardly noticeable. The other three were present in apparently similar amounts. 4. In the myosin from each species there were three types of light chains. The pattern of light chains was species specific. 5. Apparently, there was only one type of heavy chain in myosin from cod, Norway haddock and spotted wolf-fish. One preparation of cod showed an extra band of higher electrophoretic mobility than the main band. In blue whiting we found two bands present in approximately equal amounts.