USE OF A MODIFIED UREA GEL ISOELECTRIC FOCUSING METHOD FOR SPECIES IDENTIFICATION OF RAW OR BOILED WHITE,PINK, AND ROCK SHRIMP1

Isoelectric focusing (IEF) polyacrylamide gel containing an 80% pH 4–6.5 and 20% pH 3–10 ampholyte mixture greatly improved protein banding pattern for species identification of water extracts of raw pink, white and rock shrimp compared with the system using only the pH 3–10 range ampholyte. Identification of a specific species in mixture samples was achieved by the detection of water-extractable shrimp specific protein bands present in the gel. Sodium dodecyl sulfate (SDS) was a better protein extractant than water for cooked shrimp. Both water and SDS extracts of cooked shrimp showed specific protein banding patterns and improved resolution for species identification.     

Preliminary Studies on SDS-PAGE and Isoelectric Focusing Identification of Sturgeon Sources of Caviar

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) analyses of caviar were used to identify species of American, European and Asian sturgeon. Caviar samples of American and Gulf of Mexico sturgeon showed distinctive protein banding patterns on SDS-PAGE. IEF gels containing 20% pH 3-10 and 80% pH 4-6.5 ampholyte enabled differentiation of sturgeon species. IEF was more reliable than SDS-PAGE for identification of sturgeon species.     

Rapid Species Identification of Cooked Red Snapper Using Isoelectric Focusing

A rapid method using isoelectric focusing (IEF) was developed to identify species of cooked snapper. The IEF protein band patterns of red snapper as impacted by two extraction systems (water and 8M urea), various cooking times (raw, 5, 10, 15, 20, 25 and 30 min) and cooking methods (microwave, bake, steam, broil, deep fry, and pan fry) were investigated. Samples were desalted and cleaned using a 10-sec rinsing procedure. The characteristic IEF protein pattern of red snapper was reproducible for various cooking conditions. Both 8M urea and water could be used as extractant of snapper sarcoplasmic proteins. IEF protein bands in the acidic region of the polyacrylamide gel (pH 3.5-9.5) were useful as markers for species identification.     

Identification of commercial prawn and shrimp species of food interest by native isoelectric focusing

Native isoelectric focusing (IEF) of water-soluble sarcoplasmic proteins was applied to the identification of 14 shrimp species of food interest belonging to the order Decapoda. These species have different commercial values, but due to their phenotypic similarities and carapace removal in their industrial processing, incorrect food labelling and deliberate or inadvertent adulteration can occur. Each of the 14 tested species showed species-specific protein band profiles and intra-specific polymorphism was low, not preventing the correct identification of the species. Therefore, IEF of water-soluble sarcoplasmic proteins allowed the differentiation of the 14 species considered. In addition, sarcoplasmic calcium-binding proteins (SCPs) were identified by tandem mass spectrometry (MS/MS) as the major species-specific proteins in these species, opening the way to further studies focusing on their potential use as specific biomarkers.     

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.     

Electrophoretic Identification of Raw and Cooked Shrimp using Various Protein Extraction Systems

Proteins were extracted from raw or cooked pink (Penaeus duorarum), white (Penaeus setiferus) or rock shrimp (Sicyonia brevirostris) with five different solutions: water, water homogenate adjusted to pH 8.0, 0.1M NaCl, 1% SDS, or 8M urea. Each extract from each species was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Water extraction showed highly species-specific banding patterns for raw shrimp, while patterns for SDS extracts were not as species-specific. However, SDS extracts provided the greatest information on species variation of cooked shrimp. SDS-PAGE was useful in distinguishing shrimp of different genus. This technique was tested and proven in a blind study to be useful for species identification and detection (within 10% of actual amount) of fabricated products.     

SPECIES SUBSTITUTION OF RESTAURANT FISH ENTREES

Fish species substitution has been a common problem in the retail markets. However, this problem has not been documented in restaurant-cooked fish entrees. This study investigated fish substitution in restaurant-cooked snapper and swordfish entrees from four major cities in the U.S. The species of 16 snappers including 13 red snapper and 21 swordfish samples were verified as presented on the menu using isoelectric focusing (IEF). Fish samples were cleaned and desalted by rinsing with distilled water before protein extraction. Protein separation was carried out in a polyacrylamide gel with a wide pH gradient range of 3.5–9.5. Distinctive IEF protein band patterns of unknown samples were compared with the authentic standards. Six out of 13 (46%) red snapper samples and two out of 21 (10%) swordfish samples were misrepresented. The method used in this study adequately and efficiently verified the species of restaurant-cooked snapper and swordfish samples.     

Quantification of Shrimp in Shrimp-Surimi Mixtures using Urea Gel Isoelectric Focusing

A method of quantitate the percent content by weight of shrimp in surimi-pink shrimp mixtures was developed by comparing peak areas of two Alaska pollock surimi-specific (pI 7.11 and 7.17) and two shrimp-specific (pI 5.46 and 5.52) protein bands on isoelectric focusing (IEF) gels. An equation describing the linear relationship between peak areas and protein from the standard samples of surimi, shrimp and surimi-pink shrimp mixtures was also developed. Using this method, unknown mixtures containing 10.0, 12.4 and 5.7% pink shrimp, respectively, were determined in blind studies to contain 8.3, 12.0 and 4.4% shrimp and 85.7, 86.8, and 92.7% surimi, respectively.     

Identification of freshwater fish commercially labelled “perch” by isoelectric focusing and two-dimensional electrophoresis

Isoelectric focusing (IEF) and two-dimensional electrophoresis (2-DE) were used to distinguish four freshwater fish species which are sold under the generic label of “perch”: Perca fluviatilis (European perch), Lates niloticus (Nile perch), Stizostedion lucioperca (European pikeperch) and Morone chrysops x saxatilis (sunshine bass). These species have different commercial values but are easily “interchangeable” because they are sold already filleted, in view of the numerous bones of the whole fish. IEF of the water-soluble proteins extracted from fish muscle resolved in species-specific patterns. Intra-species polymorphism was low, and did not concern the bands identified as characteristic of the species. As well, 2-DE maps showed numerous species-specific protein spots. Interestingly, while none of the IEF bands was common to all four species, several major 2-DE spots were similar. Therefore, IEF of water soluble sarcoplasmic proteins is sufficient to unambiguously discriminate among the four species considered. Analysis by 2-DE, which has a higher resolution power but it is more expensive and time consuming, may be applied to obtain further knowledge of the proteome of poorly characterized species.     

Identification of Red Snapper (Lutjanus campechanus) using Electrophoretic Techniques

Isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional (2-D) gel electrophoresis were employed to produce protein profiles for species identification of red snapper and 11 other fish species. Comparing the distinctive patterns of water-soluble sarcoplasmic proteins for each species on IEF and SDS gels, red snapper could be identified. IEF gels of ampholyte mixture of 20% pH 3–10 and 80% pH 4–6.5 resolved better than gels with ampholyte ranging from pH 3–10, 4–6.5, 6–8, or 5–6 for species identification. The 10 and 12.5% SDS-PAGE gels produced more distinctive protein profiles for identification than 7.5 and 15% gels. Thus, these techniques could be applied to identify fish species.     

A databank able to be used for identifying and authenticating commercial flatfish (Pleuronectiformes) products at the species level using isoelectric focusing of native muscle proteins

Summary A computerized databank of IEF protein patterns for use in identifying flatfish species (in total 17 species, including 15 commercial ones) is presented. The databank includes all species regulated by the Belgian Law (22 May 1996) on the use of official names for fishes and seafood products. It was found that interspecimen similarity of the IEF patterns, as processed by digitization, was always larger than interspecies similarity, which allows for unequivocal authentication of unknown samples, as long as the authentic pattern is available in the databank. The databank was used to authenticate 17 commercial fish fillets.     

Electrophoretic Identification of Fish Species Used in Surimi Products

Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and urea gel isoelectric focusing (IEF) were used to identify species-specific protein bands of raw and cooked fish and surimi samples from Alaska pollock (Theragra chalcogramma) and red hake (Urophycis chuss). In raw samples, species-specific bands were found in the water extracts, while in cooked samples 1% SDS and 8M urea extracts were more effective for species identification in both fish and surimi.     

Donkeys’ milk protein fraction investigated by electrophoretic methods and mass spectrometric analysis

Donkeys' milk has attracted a growing interest in human nutrition, particularly in the dietotherapy of patients affected by cows' milk protein allergy. In this paper, donkeys' milk protein fractions were analysed by isoelectric focusing (IEF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four different IEF patterns were observed out of 51 individual milk samples. Thirty-six samples presented a common pattern, used as a reference, whereas 15 were characterized by the presence or absence of protein bands. A single sample, lacking two bands of the casein fraction, showed an apparent reduced amount of caseins, by SDS-PAGE as well as by chromatographic analysis. Mass spectrometry and high-performance liquid chromatography were used to characterize the IEF patterns. Compared with the reference, one pattern lacked two α_s1-casein bands, another lacked two β-lactoglobulin II bands and one accounted for a different β-lactoglobulin II variant. No signals related to putative α_s2- and κ-casein were observed.     

Electrophoretic techniques for species identification of fishery products

Summary At 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.

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Elektrophoretische Methoden zur Bestimmung der Tierart in Fischereiprodukten

Zusammenfassung Zur 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.

     

Identification of some Aegean fish species by native isoelectric focussing

In this study, native protein patterns and band analysis of 20 Aegean and 2 North Atlantic fish species were obtained by isoelectric focussing (IEF) by determining their protein (in extract), TMA and TVB-N contents. Band specifications of the samples have been determined for comparing the different species of fish used as raw material. Aegean Sea has around 40 commercial fish species and 20 of them were taken into investigation. Building up a database for characterization of these species may help to find a way for further studies.     

Isoelectric focusing and sodium dodecylsulphate electrophoresis of buffer soluble seed proteins of twelve varieties of winged bean (Psophocarpus tetragonolobus L.)

Buffer-soluble seed proteins of 12 varieties of winged bean (Psophocarpus tetragonolobus L.) were separated by ultrathin-layer isoelectric focusing (IEF) and sodium dodecylsulphate electrophoresis(SDS-PAGE). Whereas the molecular weight spectra of the proteins obtained by SDS-PAGE were identical for all varieties studied, the IEF patterns showed variety specificity for each sample. Most of the variety specific protein bands were identified as glycoproteins. The zymograms of the isoelectically focused trypsin inhibitors were variety specific in both activity and isoelectric point distribution.     

The use of two-dimensional electrophoresis in the characterization of the water-soluble protein fraction of commercial flat fish species

 The water-soluble proteins of nine flat fish species of high commercial value, belonging to the Pleuronectidae, Scophtalmidae, and Soleidae families, were analyzed by two-dimensional (2D) electrophoresis, this being carried out by nondenaturing isoelectric focusing (IEF) in the 3.5–9.5 pH range and gradient SDS-PAGE in the 12–14% range. Most of the major proteins fell in the 3.5–6.9 pH range. From these, the most specific proteins were in the acidic fraction (pI<5.2; MW<16 kDa). Species-specific 2D protein patterns were observed in all the nine species, and more than 25 proteins could be individualized. The combination of nondenaturing IEF and gradient SDS-PAGE, both in the absence of urea, allowed detailed characterization of both the isoelectric points and molecular weights of the major water-soluble proteins and proved to be a valuable tool for the differential characterization of the flat fish species studied.     

New Fish on the German Market: Consumer Protection against Fraud by Identification of Species

Protein- and DNA-based methods have been developed to identify fish species recently introduced into the German market. By means of isoelectric focusing (IEF) of sarcoplasmic proteins, characteristic protein patterns were obtained for pangasius (P. hypophthalmus), yellow fin sole (Limanda aspera) and arrowtooth flounder (Atheresthes stomias). In case of pangasius and yellow fin sole a ~460 base pair segment of the cytochrome b gene was amplified by PCR and sequenced. Restriction fragment length polymorphism (RFLP)- and single strand conformation polymorphism (SSCP)-analysis of three genes (cytochrome b, immunoglobulin heavy chain constant region, trypsinogen) demonstrated the possibilities of rapid identification of P. hypophthalmus.     

New Fish on the German Market: Consumer Protection against Fraud by Identification of Species

Protein- and DNA-based methods have been developed to identify fish species recently introduced into the German market. By means of isoelectric focusing (IEF) of sarcoplasmic proteins, characteristic protein patterns were obtained for pangasius (P. hypophthalmus), yellow fin sole (Limanda aspera) and arrowtooth flounder (Atheresthes stomias). In case of pangasius and yellow fin sole a ~460 base pair segment of the cytochrome b gene was amplified by PCR and sequenced. Restriction fragment length polymorphism (RFLP)- and single strand conformation polymorphism (SSCP)-analysis of three genes (cytochrome b, immunoglobulin heavy chain constant region, trypsinogen) demonstrated the possibilities of rapid identification of P. hypophthalmus. Received: September 10, 2007     

A Novel Approach to Quantify the Amount of Formaldehyde Added to Milk in Grana Padano Cheese Production

Little information is available on the reaction products of formaldehyde (FA) with milk components. An isoelectric focusing (IEF) method was developed which detected a unique protein band with pi = 6.7 in defatted Grana Padano cheese samples prepared with FA. The quantity of FA (ppm) added to milk may be extrapolated by calculating the amount of protein associated with this band. The IEF method was rapid, simple, highly specifiC., sensitive, reproducible, inexpensive and easy to utilized in routine controls.