Rapid identification of pufferfish in roast fish fillet by real-time PCR.

The purpose of this study was to develop a rapid method based on a real-time PCR assay designed to identify the presence of pufferfish in roasted fish fillet. Specific primers and probes were designed targeting Takifugu spp. and Lagocephalus spp., the most common genera in China. Specificity and sensitivity of this assay design were tested by using artificially spikes of pufferfish mixed in with other fish, such as Gadus and Thamnaconus septentrionalis,among others. Fifteen samples of retail roasted fish fillet and six samples from a 1999 poisoning event that occurred in Fujian province China were analysed for pufferfish. When the assay design was validated, no cross-reaction was observed between pufferfish and other species of fish. The limit of detection (LOD) was determined to be 0.001 ng pufferfish template, and the sensitivity of the method was 1%. Lagocephalus lunari was detected in six samples assayed from 1999 and no pufferfish was detected in the 15 retail roasted fish fillet samples tested. These results showed that the method was efficient for screening for pufferfish contamination in the roasted fish fillet and it could benefit public health protection by reducing the risk of tetrodotoxin poisoning.

Composition of nutrients, heavy metals, polycyclic aromatic hydrocarbons and microbiological quality in processed small indigenous fish species from Ghana: Implications for food security.

The triple burden of malnutrition is an incessant issue in low- and middle-income countries, and fish has the potential to mitigate this burden. In Ghana fish is a central part of the diet, but data on nutrients and contaminants in processed indigenous fish species, that are often eaten whole, are missing. Samples of smoked, dried or salted Engraulis encrasicolus (European anchovy), Brachydeuterus auritus (bigeye grunt), Sardinella aurita (round sardinella), Selene dorsalis (African moonfish), Sierrathrissa leonensis (West African (WA) pygmy herring) and Tilapia spp. (tilapia) were collected from five different regions in Ghana. Samples were analyzed for nutrients (crude protein, fat, fatty acids, several vitamins, minerals, and trace elements), microbiological quality (microbial loads of total colony counts, E. coli, coliforms, and Salmonella), and contaminants (PAH4 and heavy metals). Except for tilapia, the processed small fish species had the potential to significantly contribute to the nutrient intakes of vitamins, minerals, and essential fatty acids. High levels of iron, mercury and lead were detected in certain fish samples, which calls for further research and identification of anthropogenic sources along the value chains. The total cell counts in all samples were acceptable; Salmonella was not detected in any sample and E. coli only in one sample. However, high numbers of coliform bacteria were found. PAH4 in smoked samples reached high concentrations up to 1,300 µg/kg, but in contrast salted tilapia samples had a range of PAH4 concentration of 1 µg/kg to 24 µg/kg. This endpoint oriented study provides data for the nutritional value of small processed fish as food in Ghana and also provides information about potential food safety hazards. Future research is needed to determine potential sources of contamination along the value chains in different regions, identify critical points, and develop applicable mitigation strategies to improve the quality and safety of processed small fish in Ghana.

Fish-Based Baby Food Concern-From Species Authentication to Exposure Risk Assessment.

In this work, two different but complementary approaches were used to evaluate the reliability of fish-based baby foods as a source of safe nourishment for babies. More specifically, barcoding analysis based on the Cytochrome Oxidase I sequences was used for fish species authentication and an analysis of metal/metalloid levels was performed to estimate the exposure risk assessment derived from consumption of selected fish-based baby food in infants and toddlers. COI DNA barcoding revealed that in three samples the species detected did not match the common name of the species shown on the label. In particular, G. chalcogrammus and M. australis were found in place of M. merluccius and O. mykiss was found in place of S. salar. The analysis of exposure risk assessment indicated a low risk for developing chronic systemic and carcinogenic effects in infants and toddler, under an exposure scenario based on daily consumption of a single box of fish-based baby food. However, it is important to highlight that in order to provide a comprehensive risk assessment it would be important to supplement the levels of exposure resulting from the total diet. Overall, our results suggest that more attention should be paid by authorities to ensure the safety of food for infants and toddlers.

Visualization of the Distance among Fishes by MALDI MS for Rapid Determination of the Taxonomic Status of Fish Fillets.

Taxonomic research plays an important role in the classification of organisms. Molecular techniques provide useful tools for the determination of the taxonomic status of species, although often time-consuming and not cost-efficient. Herein, we developed a strategy to analyze fish samples in a rapid mode. Experimentally, fish fillet samples were pretreated with trifluoroacetic acid aqueous solution, and the obtained protein fraction was analyzed by matrix-assisted laser desorption/ionization mass spectrometry. Principal component analysis of mass spectrometric datasets was used to visualize the taxonomical distance among the analyzed 13 seafood species. The results were illustrated using treemaps where the fish relationship distance can be visualized. The obtained mass spectral results can be taken as reference and successfully used for the identification of unknown fish fillet samples. It is promising to utilize the present strategy to provide clues for the taxonomy study among ambiguous species and identify fish species.

DNA Barcoding Revealed Mislabeling and Potential Health Concerns with Roasted Fish Products Sold across China.

HIGHLIGHTS: 75.5% of products were identified as species outside the expected family. Six products were identified as containing multiple species from distinct families. Species from distinct families were verified in products of same brand for six groups. Identification of potentially toxic pufferfish species highlighted health concerns.

Investigation of sensory profiles and hedonic drivers of emerging aquaculture fish species.

BACKGROUND: The aquaculture sector needs to increase the diversity fish species and their processed products to cover rising consumer demands. Candidates for this diversification have been identified to be meagre, greater amberjack, pikeperch and wreckfish. Yet scientific knowledge on their sensory profiles and consumer hedonic responses is scarce. The aim of the current study was to investigate these aspects, since they are essential for product development and market targeting. RESULTS: Species exhibited different sensory profiles with the exception of the odor/flavor profiles of meagre and greater amberjack, which were similar. Texture was more important than odor/flavor in explaining interspecies differences. Yet the hedonic responses were equally related to texture and odor/flavor. None of the species received negative hedonic scores. Both positive and negative hedonic drivers were identified within the odor/flavor and texture modalities. CONCLUSION: The distinct profiles of meagre, greater amberjack, pikeperch and wreckfish make these fish species valuable first materials for new product development and for covering markets with different sensory preferences. Differences in fish texture are more easily perceivable, yet small variations in fish odor/flavor can have a great impact on consumers' hedonic responses. © 2017 Society of Chemical Industry.

Authentication of Closely Related Fish and Derived Fish Products Using Tandem Mass Spectrometry and Spectral Library Matching.

Proteomics methodology has seen increased application in food authentication, including tandem mass spectrometry of targeted species-specific peptides in raw, processed, or mixed food products. We have previously described an alternative principle that uses untargeted data acquisition and spectral library matching, essentially spectral counting, to compare and identify samples without the need for genomic sequence information in food species populations. Here, we present an interlaboratory comparison demonstrating how a method based on this principle performs in a realistic context. We also increasingly challenge the method by using data from different types of mass spectrometers, by trying to distinguish closely related and commercially important flatfish, and by analyzing heavily contaminated samples. The method was found to be robust in different laboratories, and 94-97% of the analyzed samples were correctly identified, including all processed and contaminated samples.

Determination of polycyclic aromatic hydrocarbons in smoked fish samples by a new microextraction technique and method optimisation using response surface methodology.

Microwave-assisted extraction coupled with dispersive liquid-liquid microextraction as a recently introduced method was applied to determine polycyclic aromatic hydrocarbons in Iranian smoked fish. The results showed that the interaction of hydrolysing solution volume with ethanol ratio, volumes of extracting and disperser solvent is significant in the obtained model. Optimized conditions were: a hydrolysing solution volume 10 ml with 50% ethanol, a pH of 5, and extracting and disperser solvent volumes of 150 and 500 µl respectively. The level of 16 polycyclic aromatic hydrocarbons (PAHs) was determined in 80 smoked fish consisting of four species. The contamination of benzo[a]pyrene in all samples except three was below the European Commission's maximum level of 2 µg kg(-1) for smoked fish, while the ∑4 PAHs (benzo[a]pyrene, chrysene, benzo[a]anthracene and benzo[b]fluoranthene) were between 3 and 12 µg kg(-1) wet weight in all samples. Of the species examined, Hypophthalmichthys molitrix had the highest PAHs (∑16).

DNA barcoding unveils a high rate of mislabeling in a commercial freshwater catfish from Brazil.

BACKGROUND AND AIMS: Molecular markers have contributed to species authentication by flagging mislabeling and the misidentification of commercial landings. Such tools are of great value since the market substitution of fish of lower value for highly commercialized species is expected to become more pronounced due to a shortage of natural stocks. MATERIALS AND METHODS: Here we report on the molecular identification 4results from processed fish products (i.e. fillets) and whole fishes sold in Brazilian markets under the common name surubim (Pseudoplatystoma spp.). RESULTS: DNA barcoding revealed the incorrect labeling of around 80% of all samples analyzed, with mislabeling being more pronounced within fillets rather than whole fish. CONCLUSION: To our knowledge, this is the first report correlating the rate of fraud with processed fish products. The establishment of an official list of acceptable common names for freshwater fish and seafood is urgently needed in Brazil for further trade regulations to take place.

DNA barcoding of commercially important salmon and trout species (Oncorhynchus and Salmo) from North America.

The present study investigated the ability of DNA barcoding to reliably identify the seven commercially important salmon and trout species (genera Oncorhynchus and Salmo ) in North America. More than 1000 salmonid reference samples were collected from a wide geographic range. DNA extracts from these samples were sequenced for the standard 650 bp barcode region of the cytochrome c oxidase subunit I gene (COI). DNA barcodes showed low intraspecies divergences (mean, 0.26%; range, 0.04-1.09%), and the mean congeneric divergence was 32-fold greater, at 8.22% (range, 3.42-12.67%). The minimum interspecies divergence was always greater than the maximum intraspecies divergence, indicating that these species can be reliably differentiated using DNA barcodes. Furthermore, several shorter barcode regions (109-218 bp), termed "mini-barcodes", were identified in silico that can differentiate all eight species, providing a potential means for species identification in heavily processed products.

Chemical characterization and DNA tracking of Sardinian botargo by Mugil cephalus from different geographical origins.

The chemical composition of the Sardinian botargo by Mugil cephalus from different geographical origins was investigated. Fat ( approximately 20%), proteins ( approximately 50%), moisture ( approximately 22%), and salt ( approximately 7%) were measured in ground (G) and whole (W) commercial products. Among the nutritional compounds, omega-3 fatty acids were approximately 8%, squalene was approximately 15 mg/100 g, vitamin E was approximately 8.5 mg/kg, and cholesterol was approximately 300 mg/100 g, on average in both products. Antioxidant properties, assessed by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test and expressed as Trolox equivalent antioxidant capacity (TEAC), showed quite good activity in extracted oil (0.8-1.1 mmol of Tolox/L). Major constituents in the samples varied noticeably, but only few statistical differences were evidenced between G or W products or between samples from different origins. Principal component analysis (PCA) of random amplified polymorphic DNA (RAPD) and proteins, coupled with both, did not differentiate samples from different origins. On the basis of our results, chemical and molecular data exclude the differentiation of samples from diverse origins.

[Mineral elements content in smoked fish]. / Zawartosc skladników mineralnych w rybach wedzonych.

The content of macroelements (phosphorus, calcium, magnesium potassium and sodium) and microelements (copper, zinc, iron, manganese, chromium, selenium, fluorine and iodine) in the following smoked fish: sprat, mackerel, salmon, smoked herring and trout were determined. The most of calcium, phosphorus, iron, zinc, copper and manganese contain smoked sprat. The amount of calcium in fish's remaining species is considerably lower, and phosphorus approximated to his content in sprat. The selenium and the fluorine occur on approximate level in all examined fish, however the amount of iodine was diverse but high. Smoked salmon and trout contain the most iodine, and the least smoked herring, sprat and mackerel. Contribution of fish in the recommended daily intake for mineral elements was estimated.

Fish species identification in surimi-based products.

Whole fish morphologically identified as belonging to Theragra chalcogramma, Merluccius merluccius, Merluccius hubbsi, and Merluccius capensis and 19 fish products commercialized as surimi with different commercial brands and labeled as T. chalcogramma were analyzed by direct sequence analysis of the cytochrome b gene. A phylogenetic analysis of surimi products was performed as well. Results demonstrated that mislabeling is a large-scale phenomenon, since 84.2% of surimi-based fish products sold as T. chalcogramma (16/19) were prepared with species different from the one declared. In fact, only three samples (samples 15-17) were found to belong to T. chalcogramma. In the remaining samples, Merluccidae (samples 4-14), Gadidae (samples 18 and 19), Sparidae (sample 1), and Pomacentridae (samples 2 and 3) families were detected. A phylogenetic tree was constructed, and the bootstrap value was calculated. According to this methodology, 11 samples were grouped in the same clade as Merluccius spp.

[Macro- and microelements in canned sprats]. / Makro i mikroelementy w konserwach ze szprotów.

The content of macro- and microelements and toxic metals in the most popular canned sprat was described in this paper. The research included the following canned sprat: sprat in tomato, smoked and steamed sprat in oil. The following analyses were carried out: content of calcium, phosphorus, potassium, magnesium, copper, zinc, iron, manganese, chromium, selenium, fluorine, iodine, cadmium, lead, mercury and arsenic. Fluorine, iodine, selenium, and calcium and phosphorous are provided to customer organism in large amount by canned sprat, however canned sprat cannot be considered as a source of copper, chromium, and manganese. On the base of assessment data one canned sprat (weight 170 g) provides to customer organism more than 50% recommended daily intake of calcium and phosphorus, 85-233% fluorine, 62.5% iodine, 43% recommended selenium, more than 25% zinc, about 15% daily intake of magnesium, potassium and iron. It was found that all of the analyzed canned sprat contained relatively low content of cadmium, lead, mercury and arsenic, thus confirming the established safety standards.

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.

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.

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products.

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.

Applying DNA techniques to the identification of the species of dressed toasted eel products.

To differentiate the species of processed eel products, the gene identification of four fresh eel species was first established and the species of eel products collected from markets were investigated. Polymerase Chain Reaction (PCR) and sequence analysis were used to determine the genetic variation in a 362-nucleotide region of the mitochondrial cytochrome b gene in four fresh eels including Anguilla japonica, Anguilla anguilla, Anguilla rostrata, and Muraenesox cinereus. It was found that each eel species had a unique genotype, which was no different among fresh, frozen, and sterilized meats. The restriction enzyme HinfI could differentiate the species of A. japonica and A. rostrata but could not differentiate A. anguilla and M. cinereus. Another restriction enzyme, Sau96 I, was valuable in the differentiation of M. cinereus from the other three species of Anguilla. By applying PCR and restriction enzymes, the species of 12 commercial eel products were identified as A. japonica (9 samples), A. anguilla (2), and A. rostrata (1). This indicated that the sequence and restriction enzyme cutting site analyses were very usable to authenticate species of different processed eel products.

Identification of Atlantic hake species by a simple PCR-based methodology employing microsatellite loci.

Three Atlantic hake species (Merluccius merluccius, M. bilinearis, and M. hubbsi) were PCR typed for two microsatellite loci. A blind survey of the markers in samples of the three species determined the suitability of microsatellite loci for identification of hake product. All the analyzed samples were correctly assigned to the corresponding species. Typing of processed products (fish fingers, preprocessed frozen pieces) employing an automated sequencer was successful. This simple (PCR + fragment size) automated determination method is faster than any other method yet described for identification of hake commercial products.