PCR-SSCP: a simple method for the authentication of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets

A method of DNA analysis has been developed to verify the authenticity of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. A short fragment (208 bp) of the mitochondrial 12S rRNA gene was amplified by the polymerase chain reaction and analyzed by single-strand conformation polymorphism to get species-specific patterns of single-stranded DNA (ssDNA). DNA strands were separated by native polyacrylamide gel electrophoresis and visualized by silver staining. Discrimination among the three fish species studied was possible, because each one expressed a specific ssDNA pattern.     

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.     

Evaluation and comparison of the species-specificity of 3 antiparvalbumin IgG antibodies

Parvalbumin is a pan-allergen in fish and frogs that triggers IgE-mediated reactions in fish-allergic individuals. Previous studies demonstrated that antibodies raised against fish and frog parvalbumins displayed varying specificity for different fish species, and thus, the applicability of these antibodies for potential use in immunoassays to detect fish residues were limited. We aimed to determine the specificity of 3 IgG antibodies for various fish species. Indirect enzyme-linked immunosorbent assay (ELISA) and IgG-immunoblotting were used to compare the reactivity of the polyclonal anticod parvalbumin antibody and the commercially available, monoclonal antifrog and monoclonal anticarp parvalbumin antibodies against raw muscle extracts of 29 fish species. All antibodies demonstrated varying specificities for different fish species. Of the 3 antibodies, the polyclonal anticod parvalbumin antibody is the most suitable for the detection of fish parvalbumins as it showed reactivity to the widest range of species, including herring, pilchard, carp, pike, cod, pollock, haddock, cusk, hake, bluegill, tilapia, bass, grouper, trout, catfish, and perch, although detection was still limited for several key fish species.     

PCR-ELISA for the semiquantitative detection of Nile perch (Lates niloticus) in sterilized fish muscle mixtures

A PCR-ELISA technique was developed for the semiquantitative detection of Nile perch (Lates niloticus) in experimentally sterilized fish muscle mixtures. Specific oligonucleotides derived from the 5S rDNA gene of Nile perch were selected. A forward primer, together with a reverse digoxigenin-labeled primer, permitted the amplification of specific 185 bp DNA fragments showing DNA intensities proportional to the contents of Nile perch muscle tissue in the fish mixtures. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labeled fragments that were detected with peroxidase antidigoxigenin conjugate. Subsequent enzymatic conversion of substrate gave distinct absorbance differences when assaying fish binary mixtures containing different percentages of Nile perch muscle.     

Characterization of phospholipid molecular species in the edible parts of bony fish and shellfish

The phospholipid molecular species of freshwater (pangasius, Nile perch, trout), marine fish fillets (horse mackerel, European hake, common sole, European anchovy, European pilchard, Atlantic mackerel) and the edible muscle foot of bivalves (clam, mussel, oyster) commonly available in the Italian market during spring and summer were characterized by means of normal-phase high performance liquid chromatography coupled online with positive electrospray ionization ion-trap tandem mass spectrometry. From principal component analysis (PCA), it was observed that the total fatty acid profile was not suitable to differentiate among the shellfish genera. The fatty acid molecular combinations of phosphatidylcholine, the main phospholipid class, as well as phosphatidylinositol and phosphatidylethanolamine allowed for the differentiation of shellfish from the bony fishes. Phosphatidylserine and phosphatidylethanolamine plasmalogen profile allowed for the discrimination of each bony fish or shellfish genus since PS and pPE classes included a large number of fatty acid combinations that were specific for a fish genus or group.     

Effects of roasting,blanching, autoclaving,and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins

Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.     

Procedure for radiolabeling gizzerosine and basis for a radioimmunoassay.

A method for the labeling of gizzerosine (GZ), a biogenic amine found in fish meal, is described. The labeling procedure with (125)I using a water-soluble Bolton-Hunter reagent and a mild water-insoluble oxidant (Iodogen) reagent is rapid and reproducible. The (125)I-GZ hapten was demonstrated to be immunologically active in a radioimmunoassay developed with polyclonal antibodies to GZ absorbed with a histamine-Sepharose column. The curves were linear in the range of 0.0001 and 0.1 microgram/mL. Samples of fish meal previously extracted of histamine with methanol and submitted to acid hydrolysis were contaminated with known amounts of GZ and submitted to the assay. The fish meal samples contaminated with GZ show a dose-response effect similar to the standard curve, and apparently the other component present in the sample did not interfere with the binding of the antibodies to (125)I-GZ. These data indicate the suitability of the radioimmunoassay to determine specifically GZ in fish meal.     

Usefulness of near-infrared reflectance (NIR) spectroscopy and chemometrics to discriminate fishmeal batches made with different fish species

Near-infrared reflectance (NIR) spectroscopy combined with chemometrics was used to identify and authenticate fishmeal batches made with different fish species. Samples from a commercial fishmeal factory (n = 60) were scanned in the NIR region (1100-2500 nm) in a monochromator instrument in reflectance. Principal component analysis (PCA), dummy partial least-squares regression (DPLS), and linear discriminant analysis (LDA) based on PCA scores were used to identify the origin of fishmeal produced using different fish species. Cross-validation was used as validation method when classification models were developed. DPLS correctly classified 80 and 82% of the fishmeal samples. LDA calibration models correctly classified >80% of fishmeal samples according to fish species The results demonstrated the usefulness of NIR spectra combined with chemometrics as an objective and rapid method for the authentication and identification of fish species used to manufacture the fishmeal.     

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.     

PCR-RFLP authentication of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus)

PCR-RFLP analysis has been applied to the identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus). PCR amplification was carried out using a set of primers flanking a conserved region of approximately 712 base pairs from the mitochondrial 12S rRNA gene. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of MseI, MboII, BslI, and ApoI endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all domestic and game meat species analyzed in the present work.     

Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA

Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.     

Factors affecting the bioaccessibility of methylmercury in several marine fish species

Bioaccessibility refers to the maximum bioavailability of pollutant ingested with food, and its measurements can lead to a more accurate risk assessment as compared to the measurements of total concentrations of pollutant in food. This study examined the factors affecting the bioaccessibility of methylmercury (MeHg) in nine species of marine fish with an aim to identify ways of reducing MeHg bioaccessibility. MeHg bioaccessibility without any treatment in the nine species of marine fish ranged from 16.0 to 67.7%. Steaming, grilling, and frying reduced MeHg bioaccessibility by 29.4-77.4% for rabbitfish and 74.6-95.8% for grouper. Co-consumption of phytochemical-rich foods such as green tea decreased the bioaccessibility of MeHg by 72.2% for rabbitfish and 74.0% for grouper, whereas meso-2,3-dimercaptosuccinic acid increased it by 39.2-108% for rabbitfish and 45.3-75.7% for grouper. The bioaccessibilities of both MeHg and inorganic mercury were independent of the total Hg concentration and the exposure route (dietary vs dissolved). In eight of the nine species studied, bioaccessibility was negatively correlated with the extent to which MeHg was partitioned into the metal-rich granule fraction and the trophically available fraction. It was positively correlated with partitioning into the cellular debris fraction. This study demonstrated the important control of subcellular distribution in MeHg bioaccessibility.     

Hemoglobin-mediated lipid oxidation and compositional characteristics of washed fish mince model systems made from cod (Gadus morhua), herring (Clupea harengus), and salmon (Salmo salar) muscle

The use of washed cod light muscle minces in mechanistic studies of hemoglobin (Hb)-mediated fish lipid oxidation has largely increased in the past 5 years. Although cod light muscle has a low level of intrinsic lipid oxidation catalysts, a prerequisite for a good oxidation model system, we believe it cannot fully mimic the oxidation kinetics taking place in other fish species being more susceptible to lipid oxidation. The aim of this study was to systematically investigate whether washed mince model systems useful in Hb-mediated oxidation studies could be prepared also from herring (Clupea harengus) and salmon (Salmo salar) light muscles. The kinetics of oxidation in the washed models was measured during ice storage (+/-Hb), and the results were related to compositional differences. Minces from cod, herring, and salmon light muscles were washed 3 times with 3 volumes of water and buffer. A 20 microM portion of Hb and 200 ppm streptomycin was then added, followed by adjustment of pH and moisture to 6.3 and 86%, respectively. Samples with or without Hb were then stored on ice, and oxidation was followed as peroxide value (PV), rancid odor, redness (a*) loss and yellowness (b*). Prior to storage, all minces and models were also analyzed for total lipids, fatty acids, alpha-tocopherol, proteins, Hb, Fe, Cu, and Zn. Hb-mediated lipid oxidation appeared within 2 days on ice in all models. Small differences in the oxidation rates ranked the models as herring > cod > salmon. These differences were ascribed to more preformed peroxides and trace elements in the herring model, and more antioxidants in the salmon model. Controls, without Hb, stayed stable in all cases except herring, where a very slight oxidation appeared, especially if the herring raw material had been prefrozen. In conclusion, fattier fish like dark muscle species and salmonoids are useful for making washed mince model systems and would be a better choice than cod if there is an interest in the oxidation kinetics of such species.     

Pepsinogens and pepsins from mandarin fish (Siniperca chuatsi)

Four pepsinogens (PG-I, PG-II, PG-III(a), and PG-III(b)) were highly purified from the stomach of the freshwater fish mandarin fish (Siniperca chuatsi) by ammonium sulfate fractionation, anion exchange, and gel filtration. The molecular masses of the four purified PGs were 36, 35, 38, and 35 kDa, respectively. All the pepsinogens converted into their active form pepsins within a few minutes under pH 2.0. The optimum pH and temperature of the four enzymes were 3.0-3.5 and 45-50 degrees C, using hemoglobin as a substrate. The N-terminal amino acid sequences of PG-I and PG-II were determined to the 12th and 17th amino acid residues, respectively. Western blot analysis using antisea bream polyclonal antibodies cross reacted with PG-I, PG-II, and PG-III(b) while no cross reaction with PG-III(a) was detected, suggesting the diversity of pepsinogens in fish.     

Ship noise and cortisol secretion in European freshwater fishes

Underwater noise pollution is a growing problem in aquatic environments and as such may be a major source of stress for fish. In the present study, we addressed the effects of ship noise and continuous Gaussian noise on adrenal activity in three European freshwater species. Underwater ship noise recorded in the Danube River and two Austrian lakes was played back to fish at levels encountered in the field (153 dB re 1 μPa, 30 min). Post exposure cortisol secretion was compared with control situations. Cortisol was measured with enzyme immunoassay techniques (EIA, ng cortisol/l water/g fish) in extracted aquarium water with corrections for fish mass. In the first series, two hearing specialists, the common carp (Cyprinus carpio) and the gudgeon (Gobio gobio) and one hearing generalist, the European perch (Perca fluviatilis) were exposed to ship noise. The noise level was well above hearing thresholds in these species. In a second series, fish were exposed to continuous Gaussian noise at a similar level (156 dB) which is known to induce temporary hearing loss in hearing specialists. All three species responded with increased cortisol secretion when exposed to ship noise. Interestingly, no elevation was observed when fish were exposed to continuous Gaussian noise. Our results indicate that ship noise characterized by amplitude and frequency fluctuations, constitutes a potential stressor in contrast to continuous noise. Surprisingly, the data also demonstrate no apparent differences between species possessing excellent hearing abilities (hearing specialists) and species with poor hearing abilities like perch.     

Fish species identification by thin layer isoelectric focusing.

Conventional electrophoretic techniques generally lack the resolution and reproducibility needed for the reliable identification of fish species. Variations in stabilizing media composition, sample application technique, separation time, applied voltage or current, and the analyst's skill all affect the protein pattern. Thin layer polyacrylamide gel isoelectric focusing (TLIEF), a high resolution protein separation technique, has been applied to the identification of fish species. Sarcoplasmic proteins are separated according to their isoelectric points in a stable, reproducible pH gradient. Protein patterns for 12 species of fish are compared in 4.0% polyacrylamide gels with pH 4.0--6.0 and pH 3.5--10 gradients. Similar patterns are shown in commercially prepared 5.0% polyacrylamide gels with pH 4.0--6.5 and pH 3.5--9.5 gradients (LKB PAG plates). The protein patterns are reproducible in each pH gradient and also correlate well between user-prepared and commercially prepared gels. The inherent high resolution and excellent reproducibility of TLIEF should allow the positive identification of fish species without the costly procedure of using known species as standards.     

Losses and recoveries of fish populations in acidified lakes of southern Finland in the last decade

Acid-induced fish damage in small lakes in southern Finland was studied in a fish status survey of eighty lakes from 1985–1987. Later, twenty of these lakes were selected for further monitoring. A sampling of these lakes from 1988–1989 showed that the decrease in some perch (Perca fluviatilis L.) and roach (Rutilus rutilus L.) populations still continued. The results from the same lakes in 1992 showed that successful reproduction had taken place with many of the perch populations that had been close to extinction in 1985. In contrast, no signs of recovery in the roach populations were detected. The explanation for the appearance of new cohorts of perch could have been the decrease in acid deposition but the exceptional hydrological conditions of winters in the early 1990s may also have affected them. The different responses of the perch and roach populations were interpreted as a consequence of the different sensitivity of these two species to acidification. Even a slight improvement in the water quality has resulted in the appearance of strong new year-classes of perch, but not of roach. Therefore, more improvement in water quality is needed until a sensitive species like roach can reproduce again.     

Trace element accumulation in the tissues of fish from lakes with different pH values

Two species of fish, omnivorous Catostomus commersoni (white suckers) and carnivorous Perca flavescens (yellow perch) were collected from three natural lakes with different pH ranges (circumneutral, pH 6.5 to 6.8; variable, pH 5.8 to 6.7; and acidic, pH 4.9 to 5.4). The lakes are located in the North Branch of the Big Moose River drainage system in the New York State Adirondack Park Preserve. Concentrations of potentially toxic elements (Al, Cd, Cu, Ph, and Se) were measured by electrothermal atomic absorption spectrophotometry in water, sediment and fish (bone, gill, kidney, liver and muscle) from each lake. The results showed that concentrations of Pb and Cd were significantly higher (P < 0.05) in some of the tissues of the fish collected from the acidic lake. Also, the yellow perch from the acidic take had significantly higher (P<0.05) Se concentrations in their muscle and livers than fish from the other lakes. The concentrations of Al were elevated in the gill tissues of both fish species from the acidic lake relative to fish from the other lakes. Possible mechanisms contributing to these differences in tissue concentration are discussed.     

Identification of trout and salmon bloods by simple immunological technique and by electrofocusing patterns of red cell enzyme superoxide dismutase

Species identification of animal bloods is readily achieved by immunological tests. Differentiation among fish species on this basis is more difficult although considerable success has been achieved on the basis of both inter- and intra-specific differences in their serum proteins. This report describes a method for the identification of the different species of fish within the Salmonidae family and some coarse fish families on the basis of an immunological test and electrofocusing patterns of the enzyme superoxide dismutase from the red cell. The immunological technique relies on the development of a specific anti-trout (Salmonidae) serum which is used initially to differentiate the blood of a Salmonidae from other freshwater fish. Further discrimination, within the Salmonidae, is made on the basis of the different polymorphic forms of the enzyme superoxide dismutase separated in a pH 2.5 to 8 gradient. Using this technique, it is possible to differentiate among salmon, sea/brown trout, char, cheetah trout, and a number of varieties of rainbow trout.