Hartmut Rehbein

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.

Application of PCR‐SSCP to Species Identification of Fishery Products

A method of DNA analysis has been developed to verify authenticity of labelled raw material of canned fish or in products made from closely related fish species (tuna, eel, salmon, trout and sturgeon). Short segments (123–358 bp) of the mitochondrial cytochrome b gene were amplified by the polymerase chain reaction (PCR) and analysed by single strand conformation polymorphism (SSCP) to get species-specific patterns of single-stranded DNA (ssDNA). DNA strands were separated by polyacrylamide gel electrophoresis and visualised by silver staining. Differentiation between four eel species was possible. Each of three types of sturgeon caviar gave a characteristic pattern of ssDNA. Canned sardine, herring, tuna and other species expressed specific bands of ssDNA.

Electrophoretic techniques for species identification of fishery products

ZusammenfassungZur Zeit erfolgt die Bestimmung der Tierart in Fischereiprodukten nahezu ausschließlich mit elektrophoretischen Methoden, vorzugsweise durch die isoelektrische Focussierung (IEF). In der vorliegenden Übersichtsarbeit werden die Anwendungsmöglichkeiten der IEF und anderer Elektrophoreseverfahren zur Analyse roher, getrockneter, gesalzener, geräucherter, gereifter, gegarter oder sterilisierter Fischereiprodukte diskutiert. Es wird aufgezeigt, in welchem Ausmaß die Proteinmuster durch die Art der Muskulatur (hell oder dunkel), den Frischegrad der Fische bzw. Filets und durch die Gefrierlagerbedingungen der Produkte beeinflußt werden. In vielen Fällen kann auf Referenzproben nicht verzichtet werden; es wird ein Proteinpräparat vorgestellt, das aus der sarkoplasmatischen Fraktion zahlreicher Fischarten isoliert wurde und Spezies-spezifische Proteinmuster lieferte. Das Präparat ist bei Raumtemperatur stabil und kann daher ohne Aufwand verschickt werden.SummaryAt the present time species identification of fishery products is mainly performed by electrophoresis; in most cases isoelectric focusing (IEF) is given preference over other electrophoretic techniques. In this review the possibilities of application of IEF and other electrophoretic methods for analysis of raw, dried, salted, smoked, ripened, cooked or canned fish are discussed. It is shown that the protein patterns may be influenced by the type of muscle (light or dark), the freshness of fish or fillet, and by the conditions of frozen storage. Reference samples must often be used to obtain unequivocal results. A protein dry powder is introduced, which has been prepared from the sarcoplasmic fraction of many fish species yielding species-specific protein patterns. The powder is stable at room temperature and can be shipped without cooling.

Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns

Analysis of single strand conformation polymorphism (SSCP) of an amplicon (123 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrome b gene was used to identify the fish species in canned tuna. Single-stranded DNA (ssDNA) was separated by polyacrylamide gel electrophoresis, and visualised by silver staining. The reliability of the method was tested by a collaborative study in which eight European laboratories participated. Seven unknown samples (five from individual species and two mixtures of two tuna species) of canned tuna had to be identified by comparison with reference material. From a total of 72 cases, 65 (90.3%) were assigned correctly. Intra-species variability of SSCIP patterns was found in the case of Katsuwonus pelamis and Sarda sarda. As specimens from various fishing grounds gave two or three different patterns of ssDNA, the possibility of some variability of the DNA patterns has to be considered in SSCP analysis of these species.

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.

Influence of variation in methodology on the reliability of the isoelectric focusing method of fish species identification

Abstract The reliability of isoelectric focusing (IEF) of sarcoplasmic proteins for fish species identification was evaluated by a collaborative study among eight European laboratories. Each laboratory used its own method of IEF to identify 10 unknown samples of raw muscle by means of reference material. In 93% of cases the assignment between sample and reference was correct. In a second study, the influence of extradant (water, low ionic strength buffer, or detergent) and the position of sample application on the protein pattern was examined. Working with light muscle of rainbow trout (Oncorhynchus mykiss), it was found that the type of extractant did not influence the protein pattern. Comparison of the patterns of samples, which had been applied near the anode, in the middle, or near the cathode, revealed differences in the number and position of the protein bands under the experimental conditions applied by most laboratories. This effect was not observed with the Phast System.

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.

A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: A collaborative study

A urea‐isoelectric focusing (urea‐IEF) method of identifying fish species in processed fishery products was investigated as an interlaboratory collaborative study. The technique was optimized with respect to (i) protein extraction conditions, composition of the extraction solution (urea and SDS solutions), determination of protein concentrations of the fish extracts (five tested methods); (ii) nature of gel (with carrier ampholytes and Immobilines), conditions of rehydration of commercial dry gels, urea concentration; (iii) staining conditions, Coomassie blue and silver staining. The results of various experiments were compared to select the most appropriate methodology, with respect to the discrimination power of differentiating species with the minimal influence of heat processing, reproducibility, speed, and ease of application. The method recommended meets the requirements of food control and customs laboratories.

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.