Comparison of crabmeat protein patterns by isoelectric focusing

Isoelectric focusing (IEF) of the water and urea extracts of raw or cooked blue crabmeat showed no difference in protein banding patterns with respect to sex, body part (body lump and claw), time of year (January, April, June, August and October) or locations (Florida, Maryland, Louisiana and South Carolina) of harvesting, though differences in intensities of some bands occurred. However, the cooked Taiwanese crabmeat was found to differ in IEF profiles from the US blue crab and the two frozen crabmeat blocks from China. IEF and two-dimensional gel electrophoresis (2-DGE) were not effective for differentiating pasteurized or canned crabmeat from different countries of origin. Heat treatment during pasteurization and canning drastically affected protein composition and thus the profiles.     

Species identification in anchovy pastes from the market by PCR-RFLP technique

The wide variety of marketed fishery products sometimes makes the recognition of species difficult, especially in processed food. So the consumer can be exposed to several commercial and health frauds. The European law obliges to indicate the species by specific commercial names only in fresh and prepared fishery products, but not in processed ones, in which the species can be named using generic denominations. In the present study, the use of a PCR-RFLP technique for species identification in anchovy paste, a widespread product in Italy, is described. After the PCR amplification of a common 272 bp fragment of mitochondrial cytochrome b gene from different fish species used as standards (European anchovy, European pilchard, European sprat, twaite shad, round sardinella, Atlantic horse mackerel and Mediterranean horse mackerel), the amplicons were digested using four restriction enzymes (MspI, HincII, Eam1104I and Alw26I). These patterns were compared with patterns from DNA extracted from anchovy pastes purchased from the market enabling to detect the presence of species different from anchovy (Engraulis encrasicolus) in some of them. The suitability of extending the obligation to indicate the species using specific commercial denominations even to processed fishery products is also discussed.     

Isoelectric focusing of sarcoplasmic proteins to distinguish swordfish,blue marlin and Mediterranean spearfish

In the present study we show that the method of isoelectric focusing (IEF) is a simple and reliable one for detecting the fraudulent substitution of cold-smoked fillets of swordfish (Xiphias gladius) with those of lower value blue marlin (Makaira mazara) and for detecting substitution of blue marlin steaks with those of Mediterranean spearfish (Tetrapturus belone). The cold smoking process does not alter the sarcoplasmic proteins of the fish species, so cold smoked fillets of the species investigated can be identified in the same was as fresh and frozen samples.     

Protein changes in post mortem large yellow croaker (Pseudosciaena crocea) monitored by SDS-PAGE and proteome analysis

This study was devoted to the identification of proteins and specific peptides which could be used as indicators of freshness and quality in fish. The post mortem evolution of protein patterns in large yellow croaker muscle was monitored by SDS-PAGE and two-dimensional electrophoresis (2-DE) after cold storage for 0, 5, 10, 15, 20, 25 days. SDS-electrophoresis revealed the gradual degradation of protein bands of 16–17 kDa, 22–23 kDa, 35–36 kDa, 76–78 kDa, as well as the increase of protein bands of 32–34 kDa and 67 kDa. The analysis of 2-DE showed that five spots of interest, including 3 structural proteins, namely myosin heavy chain (MHC), LIM domain-binding protein 3 (LDB3) and troponin T, as well as 2 metabolic proteins α-enolase and glyceraldehyde 3-phosphate dehydrogenase, changed progressively, which could be used as the potential freshness markers of large yellow croaker. Further research is required to evaluate their expression and post mortem evolution in another fish species.     

Substitution of high-priced fish with low-priced species: Adulteration of common sole in German restaurants

High-priced fish and seafood prepared as restaurant dish is especially prone to fraudulent substitution due to the lack of morphologic characters and less stringent labelling requirements. Common sole (Solea solea) is considered one of the most valuable and tasty fish species on the European markets and fraudulent substitutions of common sole in restaurant stores have been reported by official German food authorities in the past (from 2005 to 2011). The aim of the present study was to assess the substitution of common sole prepared as dishes in restaurants and to identify species used as substitutes for common sole. Furthermore, it was investigated whether substitution takes place at the level of the restaurants or earlier within the food supply chain. Altogether, 47 common sole dishes were ordered incognito in 24 restaurants and 98 whole fish specimens or fish fillets were purchased from 35 different shops (including wholesale dealers and speciality markets). Species identities were determined by cytochrome b gene sequencing and isoelectric focusing of sarcoplasmic proteins, which are official German methods for fish species identification afforded by 464 of the German Food and Feed Code (LFGB). 50% of the restaurant samples were shown to be substituted by species of lower commercial values, such as catfish species (Pangasidae), Senegalese tonguesole (Cynoglossus senegalensis) or Portuguese sole (Synaptura lusitanica). As almost all samples from the retail were shown to be authentic – only one sample was identified as lemon sole (Microstomus kitt) – we presume that fraudulent substitution is performed by restaurant owners and staff rather than by persons responsible for fishing, manufacturing or retail.     

Development of primer set for the identification of fish species in surimi products using denaturing gradient gel electrophoresis

The purpose of this study was to develop a DNA marker for the identification of fish species in processed surimi products. A DNA marker was designed based on the mitochondrial cytochrome c oxidase subunit I gene, and fish species in surimi products were identified using a molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE); the results were subjected to sequence-based analysis. The DGGE profiles indicated the presence in surimi products of a greater diversity of fish species than reported previously: 20 species belonging to 16 genera were identified. Therefore, our method facilitates the simple and rapid detection and identification of fish species in seafood products produced from minced fish.     

Textural properties of raw Atlantic salmon (Salmo salar) at three points along the fillet,determined by different methods

The textural properties of raw Atlantic salmon (Salmo salar) were measured at three different points along the fillet using different instrumental methods employing blade, sphere and cylinder probes. The variables examined were the force, energy and the slope of the force–deformation curve. Texture profile analysis (TPA test) was also undertaken. The tail region was firmer than the rest of the fillet. The most appropriate method for differentiating among the three locations was the compression test with a cylindrical probe. The slope of the force–deformation curve is the most appropriate variable to measure.     

Study of the microbial diversity of Oreochromis niloticus of three lakes of Cameroon by PCR-DGGE: Application to the determination of the geographical origin

The new European regulation 178/2002 imposes the determination of the geographical origin in the traceability process of the foodstuffs at the moment of commercial transactions. In practice, it is difficult to determine with accuracy the geographical origin of foodstuffs. For this purpose, the total analysis of the bacterial communities in samples is used. In the present study the molecular technique using 16S rDNA profiles generated by PCR-DGGE was used in order to detect the variation in bacterial community structures of Tilapia fish from three different lakes of the north of Cameroon and the effect of the season and fish species on these bacteria profiles. The V3 region of bacterial rDNA from fish was amplified by PCR and was analyzed by DGGE. When the 16S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The bands profiles obtained from different lakes were different and specific for each location and could be used as a bar code to certify the origin of the fish. The fish specie did not have an influence on microbial profiles of fish contrarily to the season.     

DNA barcoding as a tool for detecting mislabeling of fishery products imported from third countries: An official survey conducted at the Border Inspection Post of Livorno-Pisa (Italy)

The importation of fishery products into the European Union (EU) is constantly rising. The aim of this study was to conduct a survey on labeling non-compliances on fishery products imported from extra-European countries, in collaboration with the veterinary staff of the Border Inspection Post of Livorno-Pisa (BIP), of the Italian Ministry of Health. The correspondence between the products' identity and the scientific denominations reported on the accompanying certificates was checked using the DNA barcoding method. Overall, 277 products belonging to different categories (fish, cephalopods, crustaceans, bivalves, amphibian) were submitted to analysis for species identification. The comparison of the molecular results with the scientific names declared on the accompanying documents showed that 22.5% (95%CI 17.8–28.0) of the analyzed products were mislabeled. The highest percentage of mislabeling was observed on cephalopod based products (43.8%, 95% CI 32.3–55.9), followed by crustaceans (17.0%, 95% CI 9.2–29.2) and fish (14.0%, 95% CI 8.7–21.9). A higher rate was found in products imported from China, Vietnam and Thailand. The present study is the first survey on mislabeling in products sampled at BIPs in Italy. The results highlight the need of implementing analytical checks, based on DNA analysis, on incoming fishery products.     

The use of imported pangasius fish in local restaurants

Pangasius fish, primarily Pangasius hypophthalmus (tra/swai) and Pangasius bocourti (basa) belonging to the Pangasiidae family of catfish, are an imported farm-raised freshwater fish. The labels “tra/swai” and “basa” seldom appear on restaurant menus, so it is unclear how and to what extent pangasius is used in restaurants. In this study, we investigated 47 different fish products served at 37 restaurants in a city in the southeastern United States. A commercial rapid lateral flow (LF) assay (EZ Pangasius™ kit) was used to identify pangasius fish and the results were verified by enzyme-linked immunosorbent assay (ELISA) using a pangasius-specific monoclonal antibody (mAb) T7E10. Isoelectric focusing (IEF) was then employed to examine the protein patterns in the ready-to-eat fish samples. The results showed that 26.7% of the domestic catfish (Ictaluridae family) and 22.2% of the grouper dishes served were actually pangasius. A high percentage (66.7%) of dishes displayed under the general name of “fish” on the menu were also identified as pangasius, revealing the widespread but economically favorable and/or fraudulent use of this fish in the restaurant industry. The IEF results revealed that the pangasius positive samples were exclusively tra/swai. There was no significant difference in the prices charged to restaurant customers between pangasius negative and positive samples, indicating that price is not a good indicator of fish authenticity. These findings highlight the need for stringent enforcement of the existing regulations to discourage the fraudulent use of pangasius fish, either tra/swai or basa.     

Differential detection of small pelagic fish in Tunisian canned products by PCR-RFLP: An efficient tool to control the label information

Correct identification of fish species including their origin requires a compilation of recognized material to verify the conformity of the product with the labelling requirement. Sardine is among the internationally disputed example of species identification conflicts, with the lack of genetic reference material at the Southern part of the Mediterranean. Therefore a method was developed to discriminate between Tunisian small pelagic fish: Sardina pilchardus, Sardinella aurita and Engraulis encrasicolus. DNA extraction from fresh fish and from 12 canned sardine and 2 anchovy-type products, was followed by a PCR method that specifically amplifies a 252 bp fragment of the cytochrome b gene. The new sequences which were deposited in the NCBI GenBank, were searched against a nucleotide sequence database (GenBank) and phylogenetically analyzed with the MEGA software. Multiple alignments of 3 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned anchovy and sardine (<0.05), whereas mean interspecific variability was 0.23. A phylogenetic tree was constructed, and the calculated bootstrap values (BP, 71-100%) were used as indicators of the correct assignment of unknown canned samples to reference species. Postamplification digestion with HaeIII and ALuI restriction enzymes, yielded specific profiles that enabled anchovy to be distinguished from either of the sardine species. This PCR-RFLP was found to be reliable for species identification of canned fish products.     

White fish authentication by COIBar-RFLP: Toward a common strategy for the rapid identification of species in convenience seafood

The global trade and the increased demand for seafood products have encouraged the common practice of replacement of valuable species with species of lower value worldwide. The species of the genus Merluccius are often subject to fraudulent substitution due to their high commercial interest. The present investigation of labeling accuracy on 54 samples taken from 20 convenience seafood products collected from Southern Italy markets, allowed the identification of four species through DNA barcoding: Gadus chalcogrammus, Merluccius merluccius, Merluccius productus and Merluccius paradoxus. Mislabeling was observed in seven of 20 (35%) products (frozen breaded steaks and fish fingers), six of which (30%) were labeled as hake (M. merluccius). To reduce analysis time of fish species authentication, a COIBar-RFLP, using DNA barcoding in combination with PCR-RFLP methods, was performed for species discrimination. The restriction enzyme HinfI yielded differential digestion patterns suitable for unveiling inconsistencies between product labels and genetic species identification. The COIBar-RFLP represents an effective tool for fish authentication in convenience seafood and responds to the emerging interest in molecular identification technologies that reduce processing time and eliminate the need for lab-based DNA sequencing.     

Effect of Anisakis simplex (sl) larvae on the spoilage rate and shelf-life of fish mince products under laboratory conditions

Wild caught marine fish are commonly infected with anisakid nematodes lodging in the intestinal linings or in the fish muscle. One of the most commonly found nematode parasites in marine fish is Anisakis simplex. During production of mince from the muscle of wild caught Anisakis-infected fish, the larvae would be disrupted during mince production. Any bacteria within or on the surface of such larvae are during the mincing process evenly distributed throughout the mince, and could thus possibly affect the spoilage rate of the final products. To explore if or how any bacteria associated with muscle-invading Anisakis larvae may affect the spoilage rate of fish mince, a controlled storage trial was conducted. Fillets of farmed Atlantic cod (Gadus morhua), exclusively fed on dried and heat-treated compound feed and hence expectably free from Anisakis larvae, were aseptically collected and homogenised. Fish mince aliquots were added different volumes of Anisakis homogenate based on larvae which were freshly sampled from the visceral cavity of NE Atlantic blue whiting (Micromesistius poutassou). The volumes of added parasite homogenate (parasite(+)-samples) reflected different infection intensities from 15 (low) to 50 (high) larvae per 100 g fish fillet, representing an actual Anisakis intensity range in the flesh of blue whiting. The samples were kept at 4 °C for 15 days and subjected to microbiological, sensory and chemical evaluation at 3 days intervals. Upon visual examination and plate count measurements (PC) on Iron Agar Lyngby (IAL), the samples without any parasite additives (no[parasite]) spoiled differently and more rapidly than any of the parasite(+)-samples. However, H₂S-producing bacteria were only recorded in the latter samples, which were also the only ones that showed increased levels of the spoilage indicator substance trimethylamine (TMA). Moreover, the parasite(+)-samples changed their sensory characteristics at a later stage compared to the no[parasite]-samples. Although some cultures of H₂S-producing bacteria were found on IAL, molecular identification by PCR-DGGE of the actual bacteria was not conclusive. Psychrobacter sp. which has no or only little spoilage activity, was identified in all samples until trial day 9, but was probably outgrown by the stronger spoilers Pseudomonas fluorescence/fragi and Photobacterium phosphoreum. Thus, and somewhat unexpected, our findings indicate that – under the present trial conditions – fish mince contaminated with bacteria which originate from Anisakis larvae, spoiled less rapidly than samples without any parasite-related bacteria present. Moreover, the shelf-life of fish mince was apparently not reduced by the presence of bacteria transferred to the mince by Anisakis larvae.     

A sandwich ELISA for the detection of fish and fish products

Fish is classified as one of the major allergenic foods. However, a reliable assay for detecting the presence of fish protein is not available. In this study, a sandwich enzyme-linked immunosorbent assay (sELISA) was developed using polyclonal antibodies raised against a 36 kDa thermal-stable fish muscle protein. The sELISA was able to detect both raw and cooked (100 °C, 8 min) fish samples of all 63 common species tested without any cross-reaction with non-fish samples. The assay can also detect fish products with different processing (salting, smoking and canning). The detection limit of the assay was 0.1 ppm for both raw and cooked fish (whiting, pollock and basa) in crab meat. The assay exhibited low intra- (CV ≤ 8.9%) and inter-assay variability (CV ≤ 9.3%). The rates of false-positive and false-negative were 0%. This fish-specific sELISA is, therefore, an effective assay for the detection of fish muscle protein in food for the protection of fish-allergic individuals.     

Inactivation of Anisakis simplex larvae in raw fish using high hydrostatic pressure treatments

The nematode larvae of the Anisakis genus are one of the main health hazards for consumers of raw fish or similar, such as “carpaccio”, and cold smoked or marinated products.This study evaluated the efficacy of high hydrostatic pressure treatments on Anisakis larvae in mackerel (Scomber scombrus) from the North Sea. From the treatment parameters studied, a combination of 300 MPa for 5 min resulted as being capable of inactivating 100% of the larvae present in the tissues of the fish treated. Biomolecular examination confirmed their main belonging to the species Anisakis simplex sensu stricto.The use of this technology is for the treatment of other fatty fish such as sardines and anchovies prior to marinating.     

Traceability system in cod fishing

The objective of this research was to examine how various factors in Icelandic cod fishing can influence the quality of the raw material, using traceability systems to link these factors, and how transfer that knowledge and techniques to the Brazilian seafood industry. Data were collected in 2007 and analysed, to find a functional relationship between various quality factors. The analysis, showed, that there is a correlation between the number of parasites in the fillets and location of the fishing ground. It also showed that fishing ground and volume in haul can influence gaping, and that fillet yield differs between fishing grounds. These conclusions could only be drawn because of the ability to trace the fish from catch and all the way through processing. Recommendations drawn from this research to the Brazilian Competent Authority are to revise the countries fisheries legislation in order to enable the implementation of a traceability system that could be used as a tool to improve the quality of the raw material.     

THE DISTRIBUTION OF APTIAN SANDSTONES IN THE CENTRAL AND NORTHERN NORTH SEA (UK SECTOR) - A LOWSTAND SYSTEMS TRACT PLAY

In this paper, we discuss the distribution in the Central and Northern North Sea (UK sector) of the Late Aptian sandstones of reservoir potential which are assigned to members of the Sola Formation. An exploration strategy for these sandstones is proposed, based on sequence-stratigraphic and palaeogeographic models derived from the examination of numerous wells. The sandstones were deposited by mass-flow processes as a consequence of a major, tectonically-induced or enhanced, Late Aptian fall in relative sea-level. The distribution of these lowstand sandstones, and the facies developed, was controlled by the structure and palaeogeography that existed in the study area both before and after this sea-level fall.
The pattern of faulting in the study area during Sola Formation deposition is related to Jurassic and older tectonism, which is associated with the development of the proto-North Atlantic and the thermal subsidence of the North Sea rift system. Faults controlled the areas of sandstone provenance, and also determined the routes by which reworked sediments were transported into depocentres.
We have mapped the distribution of these sandstones using both well and seismic data. Maps of fault patterns, basinal and high areas, and facies distributions have been generated for the top- Valhall Formation level (i. e. immediately beneath the Sola Formation), and also for the Sola Formation itself. These maps were used to model the distribution of Aptian lowstand sandstones and prospects in the UK Central and Northern North Sea. This modelling exercise is of relevance for the identification of lowstand sandstones with reservoir potential elsewhere on the NE Atlantic margin.     

High contents of cadmium,lead, zinc and copper in popular fishery products sold in Turkish supermarkets

Selected toxic (cadmium, lead) and essential (zinc and copper) trace metals were determined by means of differential pulse stripping anodic voltammetry (DPSAV) in some different brands and kinds of fishery products purchased from the popular supermarkets of Turkey. Among the fishery products, the highest concentration of cadmium, lead, zinc and copper were found in the frozen anchovy (494.2 μg/kg, 314.2 μg/kg, 566 mg/kg, 45.7 mg/kg, respectively). While the canned anchovy fillet had the lowest cadmium (25.1 μg/kg), zinc (33.8 mg/kg) and copper (7.1 mg/kg) concentrations, canned tuna fish (Brand A) had the lowest lead (76.1 μg/kg). The concentrations of all toxic and essential elements in the selected products were high and often exceeded legal limits set by health authorities. Therefore these products must be monitored more often.     

Molecular methods for the differentiation of species used in production of cod-fish can detect commercial frauds

A molecular approach was used to differentiate eight species commonly used in the production of cod-fish. Since visual identification can only be applied easily on whole fish, we used the PCR method to obtain a short fragment of the cytochrome B (cytB) gene that was then analyzed by RFLP, SSCP and DGGE. While RFLP and SSCP resulted in differentiation of only some of the species tested, DGGE was able to produce patterns that made possible the identification of the species considered. The application of molecular methods to the identification of the species in this study was found to be useful, fast and reliable.     

High prevalence of Anisakidae larvae in marketed frozen fillets of pink salmons (Oncorhynchus gorbuscha)

The wild pink salmon (Oncorhynchus gorbuscha) has a great economic importance having a meat quality higher than that of breeding salmons; however, the consumption of wild pink salmons is a public health concern due to the high risk of infection with the third-stage nematode larvae (L3) of the family Anisakidae. Only L3 of Anisakis and Pseudoterranova genera are the causative agents of anisakidosis and allergies, which can be even fatal. The aims of the present work were to investigate the infection prevalence and species identity of Anisakidae larvae in fillets of North Pacific wild pink salmons. A total of 244 samples of frozen fillets, collected between 2009 and 2013, were examined by the artificial digestion method. L3 were detected in 177 (72.5%) fillets. Out of 708 recovered L3, 705 were identified as Anisakis type I and 3 as Pseudoterranova sp. by morphological examination; of these, one L3 from each infected fish, was identified as Anisakis simplex sensu stricto (#174) or as Pseudoterranova decipiens (#3) by PCR-RFLP. Despite the risk of acquiring human anisakidosis is efficiently prevented by fish fillet freezing, our findings highlight how the high prevalence of Anisakidae larvae in frozen fillets of pink salmon, may be the source of allergic reactions in sensitized persons. The increased interest of consumers in the pink salmon may pose considerable food safety problems that merits to be systematically investigated.