458 Analyst, July, 1968, Vol. 93, $9. 458460 Species Identification of Cooked Fish by Disc Electrophoresis BY I. M. MACKIE (Ministry of Technology, Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen) The present objective methods of identifying fish species are based on the species-specific protein-separation patterns obtained on electrophoresis of the water-soluble sarcoplasmic proteins of fish muscle. As the proteins must be in their native undenatured state, electrophoretic identification of fish species has, so far, been restricted to raw fish. An extension of the electrophoretic method to the identification of cooked fish is described. The protein fragments extractable in 6 M urea from the denatured proteins of cooked muscle can also be separated by electrophoresis into species' characteristic patterns that could be used for species identifica- tion.The separation patterns obtained on polyacrylamide gel for the urea extracts of cooked herring, halibut, plaice, salmon, cod and haddock are presented. In its present form the method does not apply to canned fish. THE identity of a fish species is usually readily determined from the physical appearance of the whole fish, and it is only when the distinguishing features normally used for identification have been removed, as in a fillet, that the identity of the species can be in doubt. After further processing, such as cooking, and subsequent incorporation into fish cakes and fish pastes, identification by sensory means is often impossible. There is, therefore, a need for a reliable non-sensory method of identifying the species in fish products whenever there can be doubt as to its authenticity.This is of importance commercially because of the possibility of substituting cheaper for more expensive varieties of fish. An objective method of species identification is based on the species characteristic protein separation patterns obtained after electrophoresis of the water-soluble sarcoplasmic proteins of the muscle.1 Zone electrophoresis on starch gel2 has been used by Thompson3 as a routine analytical method of species identification, and, more recently, disc electrophoresis on poly- acrylamide4 has been used by PayneY5 Mancuso,6 Torry Research Station' and Thompson.s These methods of identifying species can only be applied to raw undenatured muscle as the separation patterns are obtained from the proteins in their native, undenatured state.If the fish is cooked or dried the proteins become denatured by heating and coagulate to form precipitates, which can no longer be examined by this method. Partially cooked products such as fish fingers can, however, be examined provided there is still sufficient undenatured flesh to give a satisfactory protein separation. This paper describes a possible extension of species identification to cooked fish products. METHODS PREPARATION OF COOKED FISH FILLETS- Heat fish fillets on a steam-bath for 30 minutes in covered casseroles. 0 SAC; Crown Copyright Reserved.(4 (b) (4 (d) (4 (f (€9 Fig. 1. Electrophoretic patterns of urea extracts of cooked fish (a to f ) of (a) herring, (b) halibut, (c) plaice, (d) salmon, (e) haddock and (f) cod; (g) is the pattern for the sarcoplasmic proteins of cod in 6 M urea.Acrylamide gel concentration is 7.5 per cent. w/v Fig. 2. Electrophoretic patterns of water extracts (the sarcoplasmic proteins) of raw fish of (a) herring, (b) halibut, (c) plaice, (d) salmon, (e) haddock and (f) cod. Acrylamide gel concentration is 6.0 per cent. w/v [To face page 458MACKIE: SPECIES IDENTIFICATION OF COOKED FISH 459 UREA EXTRACT OF FISH- urea solution. fish residue by centrifuging for 20 minutes at 6000 x g. for electrophoresis. DISC ELECTROPHORESIS- Tris - glycine bzq$er solzdiow-Weigh 28.8 g of glycine and 6.0 g of tris(hydroxymethy1) aminomethane and dissolve it in water. Make the solution up to 1 litre and adjust the pH to 8.6.Dilute 1 to 10 for use as the solvent for the gel reagents and as the electrolyte solution for electrophoresis. AcryZamide gel rods-To prepare a 7-5 per cent. w/v acrylamide gel, dissolve 3.0 g of “Cyanogum 41”” in 20 ml of tris - glycine buffer solution. Add 10 ml of 1.60 per cent. w/v /3-dimethylaminopropionitrileg and 10 ml of 0.20 per cent. w/v ammonium persulphate. Transfer the solution quickly to the gelling tubes (7.5 x 0.5 cm), fill to a depth of 6.5 cm, overlayer with water according to the procedure of Ornstein and Davis,4 and set aside to polymerise at room temperature for about 20 minutes. Prepare a 6 per cent. gel by reducing the amount of “Cyanogum 41” to 2.40g. Method of electrophoresis-After polymerisation is complete transfer the tubes to a disc electrophoresis apparatus? in a chilled room at 1” C.Carry out a pre-run for 20 minutes at 200 volts to remove any discontinuities in the gel. Apply 30 to 60 pl of the urea extracts of the fish directly to the tops of the gels by dipping a syringe through the upper electrolyte compartment. Carry out the electrophoresis for 55 minutes at 280 volts. Staining and developing the gels-When the run has been completed remove the gels from the tubes4 and stain them for half an hour in a 0.1 per cent. solution of Amido black in 7 per cent. acetic acid solution. Wash out the excess of dye with the acetic acid solution and allow to stand overnight in a methanol - acetic acid - water solvent (21 + 3 + 96). Examine the developed gels the following morning.Break up a 25-g portion of fish fillet or fish product and suspend it in 50 ml of 10 M After allowing it to stand overnight at room temperature, remove the insoluble Use the supernatant solution directly RESULTS AND DISCUSSION In Fig. 1 are given the electrophoretic separation patterns of the protein residues extracted into 6 M urea from cooked herring, halibut, plaice, salmon, cod and haddock. For comparison the patterns of the corresponding water extracts from raw fish (the sarco- plasmic proteins) are given in Fig. 2. In general, the urea-extracted protein residues have fewer slow moving components and there is a greater over-all similarity of the pattern that makes differentiation more difficult. For example, the patterns for the closely related species cod and haddock, are similar and are more likely to be confused than are the corresponding sarcoplasmic protein patterns.Nonetheless, the patterns obtained for all six species are sufficiently different to allow an unequivocal identification of the species to be made. Preliminary examination of some cooked fish products was carried out. When the urea extracts from canned herring were examined by this method there were no well identifiable protein zones as are obtained from herring steam-cooked at atmospheric pressure. A fish “chip” preparation made from a homogenised mixture of cod flesh, potato powder and starch (6 + 7 + l ) , which had been cooked in vegetable oil at 260” C, gave a pattern identifiable as that of cod. There was no contribution to the pattern from the small amount of vegetable protein present.A commercial white fish cake of unspecified fish content gave a pattern easily recognisable as that of cod. Urea is well known as an agent for splitting hydrogen and hydrophobic bonds in native ProteinslO and, as such bonds are believed to form when proteins are denatured by heating, it is not surprising that fragments of these denatured proteins are extracted with strong urea solutions. In fact, when raw fish were examined by this method the separation patterns of the urea extracts were the same as the corresponding ones from urea extracts of cooked fish, suggesting that non-covalent bonds of this type are formed on cooking and that they are subsequently broken by urea. As shown in Fig.1 (f and g), the sarcoplasmic proteins contribute to only part of the pattern of the urea extract of the fish flesh as a whole. The remainder of the zones must derive from the myofibrillar and connective tissue proteins. monomer and 5 per cent. of bisacrylamide. * “Cyanogum 41” is obtainable from British Drug Houses Ltd. t Supplied by the Shandon Scientific Company Ltd., London. It contains 95 per cent. of acrylamid460 MACKIE SPECIES IDENTIFICATION OF COOKED FISH CONCLUSION Electrophoretic examination of the protein fragments in urea extracts of fish appears to be a promising method for the identification of species in cooked products. A more extensive survey would, however, be necessary before it could be used as an objective method. In its present form it is not applicable to the identification of canned products. It is restricted to fish cooked under atmospheric pressure. Mr. B. W. Thomson assisted in the experiments described in this paper. The work described was carried out as part of the programme of the Ministry of Technology. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Connell, J. J., Biochem. J., 1953, 55, 378. Smithies, O., Ibid., 1959, 71, 585. Thompson, R. R., J . Ass. Off. Agric. Chem., 1960, 43, 763. Ornstein, L., and Davis, B. J ., “Disc Electrophoresis,’’ pre-printed by Distillation Products Payne, W. R., J . Ass. Ofl. Agric. Chem., 1963, 46, 1003. Mancuso, V. M., Ibid., 1964, 47, 841. Torry Research Station Annual Report, H.M. Stationery Office, Edinburgh, 1966, p. 53. Thompson, R. R., J . Ass. Off. Analyt. Chem., 1967, 50, 282. Barka, T., J . Histochem. Cytochem., 1961, 9, 542. MacKenzie, H. A., Smith, M. B., and Wake, R. G., Biochim. Biophys. Acta, 1963, 69, 222. Industries, Rochester, New York, U.S.A. Received November 22nd, 1967