DNA‐Based Methods for the Identification of Commercial Fish and Seafood Species

ABSTRACT: The detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. Increases in international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species, have resulted in intentional product mislabeling. The effects of species substitution are far‐reaching and include economic fraud, health hazards, and illegal trade of protected species. To improve detection of commercial seafood fraud, a variety of DNA‐based techniques have been developed, including Multiplex PCR, FINS, PCR‐RFLP, PCR‐RAPD, PCR‐AFLP, and PCR‐SSCP, which are all based on polymorphisms in the genetic codes of different species. These techniques have been applied in the differentiation of many types of fish and seafood species, such as gadoids, salmonids, scombroids, and bivalves. Some emerging technologies in this field include the use of real‐time PCR, lab‐on‐a‐chip, and DNA microarray chips. In this review article, the major DNA‐based methods currently employed in the authentication of commercial fish and seafood species are discussed and future trends are highlighted. Examples of commercial applications and the use of online database resources are also considered.     

Application of barcoding to Amazonian commercial fish labelling

Methods for seafood authentication are increasingly demanded in food sciences. In this study we have detected mislabelling for eight out of 22 Amazonian fish species in two Brazilian markets, employing species-specific DNA markers. Mislabelling was most likely due to inadvertent errors in fish classification. These results emphasize the convenience of applying DNA methodology for seafood authentication.     

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.     

Application of DNA‐Based Methods to Identify Fish and Seafood Substitution on the Commercial Market

ABSTRACT: Fish and seafood substitution has become an important concern in domestic and international marketplaces, in part due to increased international trade, per capita seafood consumption, and production of processed foods. In many cases, seafood substitution is a form of economic deception, where highly prized species are substituted with those of lesser value. To prevent illegal species substitution, a number of DNA‐based methods have been developed to detect fish and seafood species in commercial products. These methods, along with common gene targets, have been reviewed previously in this journal. The current article is meant to build upon earlier discussions by providing a comprehensive review of the application of these DNA‐based methods to the discovery of fish and seafood substitution on the commercial market. Popular food uses, potential substitution cases, and peer‐reviewed research articles published to date are discussed for all major species groups of concern, including flatfish, gadoids, scombroids, salmonids, percoids, sturgeons, sharks, eels, and bivalves. The use of DNA‐based methods to monitor commercial whale meat products is also reviewed.     

Differentiation of Sparidae species by DNA sequence analysis,PCR-SSCP and IEF of sarcoplasmic proteins

The increasing global trade of fishery and aquaculture products makes it necessary to develop methods for species identification in case of fish fillets or other highly processed seafood with external morphological characteristics (e.g. gills, fins) of the original fish being removed.     

Methicillin‐Resistant Staphylococcus aureus in Seafood: Prevalence,Laboratory Detection,Clonal Nature,and Control in Seafood Chain

Methicillin‐resistant Staphylococcus aureus (MRSA), a versatile pathogen bearing multiple virulence determinants, is increasingly being detected in various food‐producing animals, including fish. In addition, it is a potential food poisoning agent. MRSA is not an inherent microbiota of fish; its presence is attributed to pre‐ or postharvest contamination through fish handlers, water, ice, and processing equipment. Several reviews have been written on MRSA in clinical as well as the food animal‐producing sector, but information specific to MRSA in seafood is scant. This review puts forth insights on MRSA detection in seafood, antibiotic resistance, diversity of clones in seafood, and possible control measures in seafood production chain. Emphasis has been given on assessing the variations in the protocols employed for isolation and identification in different food matrices and lay the foundation for researchers to develop optimized procedure.     

Allergen analysis and authenticity control: Development of mass spectrometry-based methods for animal-derived food products

Food allergy and food fraud involving animal-derived products are two of the most significant issues in food markets. On one hand, immunoglobulin E (IgE)-mediated allergic reactions after ingestion of fish, crustaceans, eggs, or milk are among the most prevalent and can happen even after ingestion of trace amounts. On the other hand, new rules regarding product commercialization (e.g., novel food regulation) are more and more created while fraudulent species substitution in fishery products is very common. Sensitive and accurate analytical methods for allergen quantification and species identification in commercial food products are therefore urgently required whether to help food industries inform allergic consumers, to ensure the food compliance with new regulations or to combat food fraud. In the past few years, bottom-up proteomic techniques, which rely on the detection of peptide biomarkers resulting from a tryptic digest of food proteins using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been emerging in this field. The selection of reliable allergen-specific or species-specific peptide biomarkers is one of the most crucial steps when developing such methods whether for qualitative protein detection (i.e., screening analysis) or protein absolute quantification. The first part of this dissertation relates therefore to the selection of allergen peptide biomarkers for fish, invertebrates, eggs, and milk in an experimental way using a single chaotropic urea extraction buffer. The allergenic proteins responsible for those severe reactions are mainly parvalbumin, tropomyosin, ovalbumin, and caseins. The protein extraction was first assessed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the major allergens were well extracted. After that, data-dependent MS/MS spectra which were obtained using digest samples of authentic animals or pure allergenic ingredients were processed against a matching protein database, and identified peptides were filtered according to several criteria such as the sequence length, amino acid composition, specificity, reproducibility, and sensitivity. Myosin proteins were also a target of choice for fish detection due to their high prevalence and sequence homology even if they are non-allergenic. Contrary to fish and invertebrate databases which included entries for only one specific protein (i.e., parvalbumin, myosin, or tropomyosin), egg and milk databases contained all known allergenic proteins or even additional proteins for egg yolk. Two extra selection steps were achieved for egg and milk markers based on more stringent criteria regarding their sensitivity after targeting them in the corresponding allergenic ingredients. At this point, 17, 13, 10, 10 and 12 potential markers were respectively selected for fish, invertebrates, egg white, egg yolk and milk. An alignment algorithm was used for all those markers to get an idea about their biological specificity. The biological specificity was verified experimentally for fish and invertebrate potential markers by targeted analysis in digest samples of animal species that are relevant in the food industry (vertebrates and invertebrates). This verification was not done for egg and milk markers as it was not regarded as significant due to reported cross-reactivity among avian eggs and among mammalian milk. Two potential parvalbumin markers were detected in other nonfish vertebrates, while all potential tropomyosin markers except one were specific to at least an invertebrate class belonging to the same phylum. Parvalbumin and myosin markers as well as tropomyosin markers were exclusively found in vertebrates or invertebrates. Marker detectability was checked by analyzing processed fish products as well as cooked fish for potential parvalbumin and myosin markers, while commercial insect-based food products such as cereals bars or pasta were studied for potential tropomyosin markers. All expected fish and invertebrate markers were detectable in those complex food products. Detectability of egg and milk markers was assessed by analysis of bread, cookies, and chocolate samples contaminated at different stages of the sample preparation with trace amounts (100 pg/g) of eggs and milk (i.e, fortified, spiked, and incurred digest samples). Two egg white markers and seven milk markers were detected in all those samples. The most suitable markers in terms of sensitivity and specificity were finally chosen for each allergenic product. Thus, besides two myosin fish global markers, five parvalbumin markers were retained including at least one of the investigated fish species. In addition, five tropomyosin markers were chosen, their specificity allowing us to distinguish crustacean tropomyosin from that of insects/arachnids, or mollusks. At last, two ovalbumin markers and three casein markers were confirmed to be the most suitable allergen markers respectively for egg white and milk. All those retained markers could be compiled in a single multiplex method. The automation of the sample preparation could also be a promising improvement whether for qualitative or quantitative analysis.     

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.     

Application of modified atmosphere packaging on aquacultured fish and fish products: A review

The aquaculture industry has undergone a rapid and continuous growth during the last decade due to increased demands in seafood consumption and reduced pelagic fish production. The aquaculture sector can provide products with a consistent flow and quality standards to cover the market's needs. The modified atmosphere packaging (MAP) technique has evolved over the last two decades and many reports indicate its beneficial effect on many quality parameters during fish and shellfish preservation. The use of MAP can clearly offer an advantage to the safer distribution of quality aquacultured fishery products. This article summarizes most of the experimental data of packaging techniques applied (MAP, VP, various pretreatments and packaging materials) on aquacultured fish and fish products to provide a clear view of the potential for a future commercial use.     

Safety Assessment of Fresh and Processed Seafood Products by MALDI-TOF Mass Fingerprinting

Foodborne intoxications caused by the consumption of fish and other products of marine origin contaminated with bacterial pathogens are an ever-present threat, either due to bacteria and/or its metabolites. In addition, the rapid spoilage of seafood due to microbial activity, results in high economic losses. The development of the microbiota in seafood products depends on the microbiological ambience of capture, processing and storage, and the applied preservation method. Thus, pathogenic and spoilage bacterial species in seafood may come from the indigenous microbiota of the aquatic ambience or are introduced by contamination during processing. Rapid and accurate bacterial species identification is essential for an effective control program to ensure safety and quality of either processed or minimally processed seafood. In the present work, matrix-assisted laser desorption ionization-time of flight mass spectrometry was successfully applied to identify 26 bacterial strains isolated from fresh fish and processed seafood samples. The approach was based on the comparison of unknown spectra to a reference spectral library and demonstrated to be a fast and accurate technique for bacterial species differentiation, which can be used for the rapid identification of foodborne pathogens and spoilage bacteria potentially present in products of marine origin.     

Implementation of quality control methods (physico-chemical, microbiological and sensory) in conjunction with multivariate analysis towards fish authenticity

Nowadays authenticity of foods and fish in particular has become of crucial importance because of high number of adulteration cases. Authenticity control has gained ground thanks to the development of several rapid physico‐chemical and microbiological methods aiming at distinguishing one species from another based on solid scientific evidence. It has been proven that despite the precision and accuracy of robust analytical and protein and DNA‐based techniques, detection of authenticity could not be claimed without resorting to multivariate analysis. This review summarizes both the most advanced and state of the art used techniques for detecting fish and seafood authenticity (both in terms of species and geographical origin). Another issue reported in this review is the preservation of fish and seafood through the implementation of old and novel techniques (ice, modified atmosphere packaging). Several informative tables were included in this paper referring to the employed quality control and sensory analysis methods and multivariate analysis for fish and seafood.     

Quality evaluation of fish and other seafood by traditional and nondestructive instrumental methods: Advantages and limitations

Although being one of the most vulnerable and perishable products, fish and other seafoods provide a wide range of health-promoting compounds. Recently, the growing interest of consumers in food quality and safety issues has contributed to the increasing demand for sensitive and rapid analytical technologies. Several traditional physicochemical, textural, sensory, and electrical methods have been used to evaluate freshness and authentication of fish and other seafood products. Despite the importance of these standard methods, they are expensive and time-consuming, and often susceptible to large sources of variation. Recently, spectroscopic methods and other emerging techniques have shown great potential due to speed of analysis, minimal sample preparation, high repeatability, low cost, and, most of all, the fact that these techniques are noninvasive and nondestructive and, therefore, could be applied to any online monitoring system. This review describes firstly and briefly the basic principles of multivariate data analysis, followed by the most commonly traditional methods used for the determination of the freshness and authenticity of fish and other seafood products. A special focus is put on the use of rapid and nondestructive techniques (spectroscopic techniques and instrumental sensors) to address several issues related to the quality of these products. Moreover, the advantages and limitations of each technique are reviewed and some perspectives are also given.     

Identification of Species Origin of Meat and Meat Products on the DNA Basis: A Review

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.     

A quantitative sandwich ELISA for the detection of <Emphasis Type="Italic">Anisakis simplex</Emphasis> protein in seafood

There is a growing awareness by consumers and food safety authorities regarding the possible presence of parasites or parasite-related potentially hazardous substances in seafood. Anisakis simplex is among the most frequently occurring parasites in wild-caught marine fish. Except for various visual inspection techniques and PCR-based methods for the detection of more or less intact worms or parasite DNA, respectively, there are at present no validated methods for the quantification of A. simplex proteins in processed fish products. This work describes the development and validation of a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification and analysis of proteins from A. simplex in seafood products. The ELISA is based on a polyclonal rabbit anti-A. simplex antibody for capture and a biotinylated conjugate of the same antibody for detection. The ELISA is specific for A. simplex and does not cross-react with other species. Recoveries ranged from 72–101% in typical food matrixes, while intra- and inter-assay precisions were <11 and <25%, respectively. With a limit of detection of 1.1 μg A. simplex protein/g of sample, the sensitivity of the A. simplex sandwich ELISA appears to be sufficient to detect even low levels in seafood products.     

Application of random amplified polymorphic DNA (RAPD) analysis for identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets

A random amplified polymorphic DNA (RAPD) method was developed for the specific identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. Using two different reaction primers (S1 and L1), RAPD analysis produced clear fingerprints from which the three fish species could be easily identified. This approach is rapid and reliable and offers the potential to detect fraudulent or unintentional mislabeling of these species in routine seafood authentication analysis.     

Challenges in the Traceability of Seafood

The increasing global trade of foods and animal feeds have to be channelled in right directions to guarantee product safety, transparency and the protection of consumer health. According to this fact the European Regulation (EC) No 178/2002 lays down the common principle for implementing traceability systems in product supply chains. However, seafood is a highly perishable food item which is increasingly global traded so that a lot of particular conditions and difficulties more have to be taken into account compared to other food products. This review discusses the relevant regulations for traceability in seafood and gives a survey about the most important analysis techniques for characterizing seafood.     

Fast identification of scallop adductor muscles using species-specific microsatellite markers

Scallop adductor muscles are very popular delicious seafood distributed in the international markets. Identification of commercial adductor muscles to the species level is an important issue to assure the correct labeling for their distribution. In the present study, we established the species-specific microsatellite markers for four main economic scallop species to identify commercial adductor muscles. A total of 140 adductors derived from three different food processes, the frozen, the dried and the canned, were collected from markets and analyzed using the species-specific microsatellite markers. Although the DNA extracted from the commercial samples was highly degraded, all samples were well identified using the method established in the present study. This method well demonstrated high precision and high sensitivity, and more competence in the identification of mixed commercial samples. Moreover, the results in this study gave the experimental evidences for the potential use of species-specific microsatellite markers for species identification in the market management.     

Nutrients and Chemical Pollutants in Fish and Shellfish. Balancing Health Benefits and Risks of Regular Fish Consumption

Dietary patterns and lifestyle factors are clearly associated with at least five of the ten leading causes of death, including coronary heart disease, certain types of cancer, stroke, non-insulin insulin-dependent diabetes mellitus, and atherosclerosis. Concerning specifically fish and seafood consumption, its beneficial health effects in humans are clearly supported by an important number of studies performed in the last 30 years. These studies have repeatedly linked fish consumption, especially those species whose contents in omega-3 fatty acids are high, with healthier hearts in the aging population. The nutritional benefits of fish and seafood are also due to the content of high-quality protein, vitamins, as well as other essential nutrients. However, a number of studies, particularly investigations performed in recent years, have shown that the unavoidable presence of environmental contaminants in fish and shellfish can also mean a certain risk for the health of some consumers. While prestigious international associations as the American Heart Association have recommended eating fish at least two times (two servings a week), based on our own experimental results, as well as in results from other laboratories, we cannot be in total agreement with that recommendation. Although a regular consumption of most fish and shellfish species should not mean adverse health effects for the consumers, the specific fish and shellfish species consumed, the frequency of consumption, as well as the meal size, are essential issues for adequately balancing the health benefits and risks of regular fish consumption.     

Risk‐Benefit Analysis of Seafood Consumption: A Review

Abstract: Seafood, defined here as marine and freshwater fish and shellfish, is recognized as a healthy food choice because it is a low‐fat protein source that provides long‐chain omega‐3 fatty acids important for early development along with eye and heart health. However, seafood is also known to contain certain contaminants, such as methylmercury and persistent organic pollutants, which can have harmful effects on human health and development. In order to limit exposure to contaminants while maximizing the benefits of seafood consumption, a number of quantitative and qualitative risk‐benefit analyses have been conducted for seafood consumption. This review paper provides a brief background on risk‐benefit analysis of foods, followed by a discussion of the risks and benefits associated with fish consumption. Next, risk‐benefit analyses are reviewed in an historical context. While risk‐benefit analysis consists of three main elements (that is, assessment, management, and communication), this review will primarily focus on risk‐benefit assessments. Overall, most studies have found that the benefits far outweigh the risks among the general population, especially when a variety of fish is consumed at least twice per week. However, for certain populations (for example, pregnant women and young children) a more targeted approach is warranted in order to ensure that these groups consume fish that are low in contaminants but high in omega‐3 fatty acids. The potentially harmful unintended consequences of risk‐benefit communication on the general population and certain groups are also discussed.     

Challenges in the Traceability of Seafood

The increasing global trade of foods and animal feeds have to be channelled in right directions to guarantee product safety, transparency and the protection of consumer health. According to this fact the European Regulation (EC) No 178/2002 lays down the common principle for implementing traceability systems in product supply chains. However, seafood is a highly perishable food item which is increasingly global traded so that a lot of particular conditions and difficulties more have to be taken into account compared to other food products. This review discusses the relevant regulations for traceability in seafood and gives a survey about the most important analysis techniques for characterizing seafood. Received: September 17, 2007