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 species identification by isoelectric focusing of parvalbumins in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients (IPG) is reported for unequivocal identification of fish species. Three orders of fishes have been analyzed: salmons, flat and cod fishes. In each family, four closely related species have been analyzed. For clear-cut species identification, two strategies have been adopted: (i) to perform IPGs in very narrow (1 pH unit and less) acidic gradients, typically spanning the pH 4-5 range, where fewer proteins are present and the pattern is much clearer; (ii) to focus the analysis on the parvalbumins, since this protein class if highly species-specific and resistant to heat. Thus, not only fresh muscle could be analyzed, but also boiled fish samples. In all cases unambiguous determination of each species could be performed, either by simple visual band inspection or, in the most difficult cases, by densitometric evaluation of the Coomassie-blue stained profiles. The analysis was performed in extracts of single species and also in mixtures of the most closely related species.