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The world's seafood supply and trade have increased in the last decades, as well as the potential for marketed species substitution. Currently, seafood safety and authenticity assessment have become central issues, directly related with the identification of improper labeling of processed foods. To detect and prevent mislabeling issues, species identification using DNA barcodes has been widely used as effective molecular markers. Therefore, this review intends to present the current status on the application of DNA barcodes to seafood species authentication. In this regard, the barcode regions, reference databases and related methodologies are described, while applications are listed and summarized. Cytochrome c oxidase subunit I (COI) gene has been the preferential targeted DNA region in animal species identification, including fish and shellfish, though other mitochondrial (cytb, 12S rRNA, 16S rRNA) and nuclear genes have been used. DNA barcoding relying on Sanger's sequencing has been the most used approach for seafood authentication. Nevertheless, in recent years, noteworthy progresses have been advanced toward DNA barcoding strategies, involving next generation sequencing. Methods relying on real-time PCR using species-specific primers and probes or followed by high resolution melting analysis combined with DNA barcodes represent alternative and promising approaches for simple, cost-effective and high-throughput species discrimination in processed seafood. Still, polymerase chain reaction with restriction fragment length polymorphism detection, targeting DNA barcodes, continues to be a well-established and broadly accepted method in seafood authentication.

Allergy to crustaceans is an increasingly important food safety issue. To protect people from experiencing adverse allergic reactions, reliable methodologies are necessary to verify the labelling of processed seafood. In the present work, two new DNA-based approaches targeting the 16S rRNA mitochondrial gene are proposed to detect crustaceans in foods using a qualitative PCR assay specific for crustaceans (shrimps, lobsters and crabs) and a quantitative real-time PCR assay specific for shrimp crustaceans. The real-time PCR system allowed the detection and quantification down to 0.1 pg and 0.0001% (w/w) of shrimp DNA and shrimp in model mixtures, respectively. The method exhibited high performance for quantitative analysis in the range of 0.0001% to 50% as inferred by the calibration curve parameters being effectively validated with blind mixtures. The qualitative PCR assay can provide a simple, fast and high throughput tool for screening the presence of crustaceans in processed foods, while the proposed real-time PCR method proved to be a useful tool for the accurate detection and quantification of shrimp in foods at trace levels.

Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001–50%) than the EvaGreen (0.05–50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices.

Hake species of Merluccius genus represent an important group of fish commonly sold all over the world. Therefore, they are highly prone to be adulterated, particularly the substitution of M. merluccius by other species with lower market value. The present work intended the development of a highly sensitive methodology for the rapid detection and differentiation of hake species based on mini-barcoding of cytochrome c oxidase subunit I (COI) gene combined with high resolution melting (HRM) analysis. The method allowed the full discrimination of M. merluccius, M. productus, M. hubbsi, M. capensis and M. paradoxus with high levels of confidence. Real-time PCR assay targeting COI mini-barcode provided a high sensitive tool to detect hake species down to 0.2–20 pg of DNA with adequate performance parameters. The application of the COI-HRM approach to 45 fish-containing foods showed that two samples did not comply with the declared species, suggesting mislabelling or species substitution. These findings highlight the need of controlling processed fish-containing foods and the feasibility of the proposed tool for their authentication at trace levels.

Penaeidae family includes shrimp species of high commercial value, sharing noticeable morphological similarities, which makes them potential targets of adulteration. Therefore, mechanisms for authentication and certification of such crustaceans, frequently included in processed foods, constitute a benefit for the food industry. Litopenaeus vannamei, Penaeus monodon, Fenneropenaeus indicus, Metapenaeus affinis and Melicertus kerathurus are some of the most relevant penaeid shrimps, being their differentiation of high importance. This work intended to develop a new approach for the specific detection and differentiation of those five closely related shrimp species based on high resolution melting (HRM) analysis targeting a cytochrome oxidase subunit I (COI) mini-barcode. The method enabled the differentiation of the five species with high levels of confidence (>99%), being successfully applied to analyse processed seafood samples. F. indicus and L. vannamei were the main identified species in the commercial products. When verifying labelling compliance, four samples suggest adulterations based on the complete or partial substitution of declared species. The proposed method proved to be a potential tool for the rapid and cost-effective differentiation of penaied shrimp species.

This work aimed to exploit the use of DNA mini-barcodes combined with high resolution melting (HRM) for the authentication of gadoid species: Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Theragra chalcogramma) and saithe (Pollachius virens). Two DNA barcode regions, namely cytochrome c oxidase subunit I (COI) and cytochrome b (cytb), were analysed in silico to identify genetic variability among the four species and used, subsequently, to develop a real-time PCR method coupled with HRM analysis. The cytb mini-barcode enabled best discrimination of the target species with a high level of confidence (99.3%). The approach was applied successfully to identify gadoid species in 30 fish-containing foods, 30% of which were not as declared on the label. Herein, a novel approach for rapid, simple and cost-effective discrimination/clustering, as a tool to authenticate Gadidae fish species, according to their genetic relationship, is proposed.

Soybean (Glycine max L.) is the most important genetically modified (GM) crop, from which the glyphosate tolerant soybean, also known as Roundup Ready (RR), is one of the most representative. The present work intends to assess the presence of two RR soybean lines, namely GTS 40-3-2 and MON89788, in a wide range of processed foods commercialised in Portugal. For this purpose, two quantitative real-time PCR approaches were proposed using available and newly designed primers. The results showed that, from 90 food samples, 73 confirmed the presence of soybean, while 15 others suggest their incompliance with labelled soybean. RR soybean was detected in 9 samples, from which 5 were identified as being the GTS-40-3-2 event, 3 the MON89788 event and one contained both events. The developed real-time PCR methods were successfully validated through the participation in an interlaboratory proficiency program and applied to processed foods. The estimated RR soybean contents of 8 samples ranged between 0.01 and 0.39%, but the sample containing both events accounted for 23.9% of GM ingredients. Such high level above the threshold for labelling regulations suggests the need of more strict control of GMO in foods.

A vitamin E extraction method for rainbow trout flesh was optimized, validated, and applied in fish fed commercial and Gracilaria vermiculophylla-supplemented diets. Five extraction methods were compared. Vitamers were analyzed by HPLC/DAD/fluorescence. A solid-liquid extraction with n-hexane, which showed the best performance, was optimized and validated. Among the eight vitamers, only α- and γ-tocopherol were detected in muscle samples. The final method showed good linearity (>0.999), intra- ( 96%). Detection and quantification limits were 39.9 and 121.0 ng/g of muscle, for α-tocopherol, and 111.4 ng/g and 337.6 ng/g, for γ-tocopherol, respectively. Compared to the control group, the dietary inclusion of 5% G. vermiculophylla resulted in a slight reduction of lipids in muscle and, consequently, of α- and γ-tocopherol. Nevertheless, vitamin E profile in lipids was maintained. In general, the results may be explained by the lower vitamin E level in seaweed-containing diet. Practical Applications: Based on the validation results and the low solvent consumption, the developed method can be used to analyze vitamin E in rainbow trout. The results of this work are also a valuable information source for fish feed industries and aquaculture producers, which can focus on improving seaweed inclusion in feeds as a source of vitamin E in fish muscle and, therefore, take full advantage of all bioactive components with an important role in fish health and flesh quality. Muscle samples from rainbow trout fed a commercial diets were used to select, optimize, and validate an extraction method for vitamin E. The final method was used to extract α and γ-tocopherol in muscle from rainbow trout fed a control (CTRL) and a seaweed-supplemented diet (GRA).

Food-induced allergies are considered an important problem of public health with special impact in the quality of life of the sensitised/allergic individuals. As highly consumed foods, fish and shellfish represent a valuable source of proteins for the general population. In spite of their economical and nutritional importance, these foods are known to induce hypersensitivity reactions in sensitised/allergic individuals. So far, parvalbumins (fish) and tropomyosins (crustaceans and molluscs) have been considered major allergens in seafood allergy, being responsible for most of the reported cases of adverse immunological responses. More recently, other proteins such as arginine kinases, myosin light chains, troponins and sarcoplasmic calcium-binding proteins have been regarded as relevant allergens in fish, crustaceans and molluscs. This review focuses on seafood allergens, reporting an updated and compiled list of allergens from fish, crustaceans and mollusc species, with an overview on the most representa...

The present study evaluates the long term (5 months) effects of feeding vegetable oil-based diets to Senegalese sole (Solea senegalensis) until market size. Extruded isonitrogenous and isoenergetic diets (56% crude protein and 20 kJ/g) were formulated to substitute 0 (CTRL), 50 (VO50) and 100% (VO100) of FO by a VO blend (rapeseed oil, RO, soybean oil, SO, and linseed oil, LO). A concomitant replacement of 50% FM and FO (VO50PP50) by plant protein sources (pea, soybean meal, potato concentrate, corn and wheat gluten) and oils was also evaluated. After the growth trial (140 days), fish were fed a fish oil-based diet (CTRL) over a period of 26 days and growth, flesh quality and organoleptic properties were determined. Results show that it seems possible to substitute up to 100% of FO by a VO blend, as well as concomitantly substituting 50% FO and FM by vegetable sources in on-growing Senegalese sole diets, without compromising growth performance and feed utilization. This species selectively retains highly unsaturated fatty acids (ARA and DHA) and seems to adapt well to a low dietary supply of these FAs. At the end of the growth out experiment fish fed VO50 diet showed a similar n − 3 HUFA profile to the CTRL fed fish. Total FO substitution resulted in a strong reduction of muscle EPA content that was totally recovered after 26 days of re-feeding with a FO based diet. Despite the observed changes in fatty acid composition, sole fillets from fish fed 100% VO are very well accepted by fish consumers and still are good nutritional value end-products for human consumption, providing 1.5 times the RDI level (0.4 g per 100 g of muscle) of EPA + DHA.

Maize, the second most important genetically modified (GM) crop, has the highest number of authorised GM events for food and feed in the EU. To provide consumer’s information, labelling for food products containing more than 0.9% of GM material is demanded by the actual EU legislation. Analysis of foods is then essential to detect and quantify GM maize material and verify the compliance with labelling information. The aim of the present work was to assess the presence of GM maize in a range of processed foods commercialised in Portugal between 2007 and 2010. For this purpose, screening of GM material was carried out by qualitative PCR targeting the 35S promoter and the NOS terminator, followed by the specific detection of Bt11, MON810, Bt176, GA21, MON863, NK603, TC1507 (also known as DAS1507), DAS59122 and MIR604 events. The identified maize events were confirmed and quantified by real-time PCR with hydrolysis probes. The overall results of GMO screening were 30% for 35S promoter, 10% for NOS terminator and 25% for identified events. The most frequently detected events were MON810, TC1507 and NK603, with one sample containing GA21, while the other events were not detected in any of the analysed foods. The quantitative results suggest the need for a more severe control since 4% of the analysed foods contained more than the threshold for labelling and none of them declared the presence of GMO.

The replacement of fish meal (FM) by increasing levels of plant protein (PP) sources 50% (PP50), 75% (PP75) and 100% (PP100) was compared to a control diet (FM) containing fish meal as the main protein source in ongrowing Senegalese sole (mean initial body weight:106 g ± 15). These four diets were isonitrogenous and isolipidic (54% crude protein and 9% crude fat on a dry matter basis). By the end of the growth trial, groups fed PP50 and PP75 had a final body weight and daily growth index (DGI) similar to the FM fed group (200 g and 0.8–0.9, respectively), whereas PP100 diet induced a significantly lower DGI (0.4). PP100 showed the highest feed conversion ratio (FCR) (2.4 vs 1.5) and the lowest protein gain (0.5 vs 0.9 g kg− 1ABWday− 1). Nutrient intake of PP groups was statistically similar to the FM group. Apparent digestibility coefficients (ADCs) of nutrients did not differ significantly among treatments (p > 0.05) and all PP diets induced lower fecal P losses than the FM group (33%). The highest PP levels (PP75 and PP100) resulted in decreased whole body lipid contents, thought PP100 showed the highest HSI and liver total lipids. In all groups, liver was the major lipid storage organ (13.96–28.57% WW). Muscle lipid content was low (1.3–1.6% WW) and did not vary significantly among treatments. Polyunsaturated fatty acid (PUFA) levels in muscle were also similar amongst dietary treatments and the two highest replacement levels (PP75 and PP100) were still effective in producing an n-3 PUFA rich product (29–30% of total fatty acids (WW)) similar to the FM group (33% of total fatty acids (WW)). Docosahexaenoic acid (DHA) was selectively retained in muscle (17–21% of total fatty acids (WW)) and neither DHA/EPA (Eicosapentaenoic acid) nor EPA/ARA (Arachidonic acid) ratios were significantly affected (p > 0.05) by dietary treatments. The sensory evaluation of cooked slices showed that the replacement of marine protein sources (FM) by PP blends did not have a significant impact on the majority of the descriptors, though visual freshness and whiteness of the PP75 flesh yielded the highest scores. Our results indicate that ongrowing Senegalese sole can effectively use diets with high levels of PP sources, up to 75% of FM replacement, without impairing feed intake, growth performance and protein utilisation whilst preserving high n−3 PUFA and DHA flesh content.

A great effort has been dedicated to the development of new methods for the analysis of genetically modified organisms (GMO) in foods. DNA quality and integrity are critical parameters for the successful detection and quantification of transgenic crops using DNA-based methods. Food processing, such as mechanical, thermal treatment, and oil refining, may cause DNA degradation, which may impair GMO detection and quantification by polymerase chain reaction. Also, DNA extraction methods have a key role in the efficiency of DNA recovery, particularly in processed matrices. Several studies have investigated the extent of DNA degradation from several food matrices as affected by processing treatments for GMO analysis. The majority of the reports revealed that DNA degradation occurs in a time-dependent manner, but the relative quantification of the transgenic content is still possible. However, care should be taken regarding mechanical processing of plant material due to a predominant effect on GMO quantification.

Gadiform order includes several fish families, from which Gadidae and Merlucciidae are part of, comprising the most commercially important and highly appreciated fish species, such as cod, pollock, haddock, and hake. Parvalbumins, classified as calcium-binding proteins, are considered the main components involved in the majority of fish allergies. Nine and thirteen parvalbumins were identified in different fish species from Gadidae and Merlucciidae families, respectively. This review intends to describe their molecular characterization and the clinical relevance, as well as the prevalence of fish allergy. In addition, the main protein- and DNA-based methods to detect fish allergens are fully reviewed owing to their importance in the safeguard of sensitized/allergic individuals.

All rights reserved. Interest in DNA biosensors for genetically modified organism (GMO) testing is been growing due to their possibility for automation and microfabrication based on simple and portable detection systems. Biosensors can provide rapid, low-cost, sensitive, real-time, and high-throughput measurements suitable for in-field analysis. The efficiency of GMO diagnostics could be improved by simultaneously analyzing several targets, which is presently being exploited using the microarray platforms. As promising alternatives to conventional polymerase chain reaction, isothermal amplification strategies, such as loop-mediated isothermal amplification (LAMP), are low-cost and suitable in-field, enabling visual and electrochemical detection. This chapter intends to provide an overview on the advances in novel biosensing technologies, namely optical, piezoelectric, and electrochemical biosensors, as well as microarrays and LAMP applied to GMO testing. This work was supported by Marie Curie Actions FP7-PEOPLE-2013-IRSES, project no. 612545 entitled “GMOsensor—Monitoring Genetically Modified Organisms in Food and Feed by Innovative Biosensor Approaches” and by Fundação para a Ciência e a Tecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2013 financed by POPH–QREN (subsidized by FSE and MCTES). A. Plácido, J. Costa, and T.J.R. Fernandes are grateful to FCT grants SFRH/BD/97995/2013, SFRH/BPD/102404/2014 and SFRH/BD/93711/2013, respectively. info:eu-repo/semantics/publishedVersion

The consumption of plant food supplements (PFS) has been growing globally, with an increase of misleading labeling and fraudulent practices also being reported. Recently, the use of molecular biology techniques has been proposed to detect botanical adulterations, one of the possible frauds in PFS. However, difficulties in recovering DNA from some PFS samples have been described. Aiming at using DNA-based methods for the unequivocal identification of plant species in PFS, adequate DNA isolation is required. However, PFS often contain pharmaceutical excipients known to have adsorbent properties that might interfere with DNA extraction. Thus, the aim of this work was to assess the effect of different excipients (talc, silica, iron oxide and titanium dioxide) on the recovery/amplification of DNA. For that purpose, known amounts of template maize DNA were spiked either to PFS or to model mixtures of excipients and quantified by real-time PCR. The tested excipients evidenced clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS. The use of either 10% talc or 0.5% dyes completely adsorbed DNA, resulting in negative PCR amplifications. For the first time, pharmaceutical excipients were shown to affect DNA extraction explaining the inability of recovering DNA from some PFS samples in previous studies. This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundaç~ao para a Ci^encia e Tecnologia) through project EXPL/DTPSAP/ 1438/2013 (Safety of plant food supplements: searching for adulterant pharmaceutical drugs and plants) and UID/QUI/50006/ 2013. Joana Costa and Telmo J.R. Fernandes are grateful to FCT grants SFRH/BPD/102404/2014 and SFRH/BD/93711/2013, respectively, financed by POPH-QREN (subsidized by FSE and MCTES). This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundaç~ao para a Ci^encia e Tecnologia) through project EXPL/DTPSAP/ 1438/2013 (Safety of plant food supplements: searching for adulterant pharmaceutical drugs and plants) and UID/QUI/50006/ 2013. Joana Costa and Telmo J.R. Fernandes are grateful to FCT grants SFRH/BPD/102404/2014 and SFRH/BD/93711/2013, respectively, financed by POPH-QREN (subsidized by FSE and MCTES). info:eu-repo/semantics/publishedVersion

Among aminoacidopathies, phenylketonuria (PKU) is the most prevalent one. Early diagnosis in the neonatal period with a prompt nutritional therapy (low natural-protein and phenylalanine diet, supplemented with phenylalanine-free amino acid mixtures and special low-protein foods) remains the mainstay of the treatment. Data considering nutrient contents of cooked dishes is lacking. In this study, fourteen dishes specifically prepared for PKU individuals were analysed, regarding the lipid profile and iron and zinc contents. These dishes are poor sources of essential nutrients like Fe, Zn or n-3 fatty acids, reinforcing the need for adequate supplementation to cover individual patients’ needs. This study can contribute to a more accurate adjustment of PKU diets and supplementation in order to prevent eventual nutritional deficiencies. This study contributes to a better understanding of nutrient intake from PKU patients’ meals, showing the need for dietary supplementation.

Nutritional management is essential for Phenylketonuria (PKU) treatment, consisting in a semi-synthetic and low phenylalanine (Phe) diet, which includes strictly controlled amounts of low protein natural foods (essentially fruits and vegetables) supplemented with Phe-free protein substitutes and dietetic low-protein products. PKU diet has to be carefully planned, providing the best ingredient combinations, so that patients can achieve good metabolic control and an adequate nutritional status. Hereupon, it is mandatory to know the detailed composition of natural and/or cooked foodstuffs prepared specifically for these patients. We intended to evaluate sixteen dishes specifically prepared for PKU patients, regarding the nutritional composition, Phe and tyrosine (Tyr) contents, fatty acids profile, and vitamins E and B 12 amounts. The nutritional composition of the cooked samples was 15.5–92.0 g/100 g, for moisture; 0.7–3.2 g/100 g, for protein; 0.1–25.0 g/100 g, for total fat; and 5.0–62.0 g/100 g, for total carbohydrates. Fatty acids profile and vitamin E amount reflected the type of fat used. All samples were poor in vitamin B 12 (0.3–0.8 μg/100 g). Boiled rice presented the highest Phe content: 50.3 mg/g of protein. These data allow a more accurate calculation of the diet portions to be ingested by the patients according to their individual tolerance.

International audience; A growth trial was conducted to test the growth potential and nutrient utilization of Senegalese sole fed diets with increasing substitution of supplemental fish oil (FO) by vegetable oil (VO) blends. Triplicate groups of twenty Senegalese sole juveniles (12 g) were fed to satiation over a period of 12 weeks with 6 extruded diets containing 570 g protein/kg DM and 90g lipid/kg DM. Two blends of VO were tested (A and B) with two FO substitution rates 50% (VO50A and VO50B) and 100% (VO100A and VO100B). A concomitant replacement of 50% fish meal and 50% FO (VO50PP), and a control diet (CTR) containing only FO, were also evaluated. After 12-weeks feeding the dietary treatments did not affect growth performance and final body composition. Muscle eicosapentaenoic acid (EPA) was reduced in all treatments compared to CTR, but docosahexaenoic acid (DHA) was only reduced in the VO50PP group. FO substitution led to a general increase of muscle linoleic acid (18:2 n−6, LOA) with VO50PP inducing maximal levels (15% vs 6% in FO diet). Lipogenic enzymes (FAS, ME and G6PD) as well as long chain fatty acid elongation (elov5) and desaturation (Δ 4 desaturase) were not affected by dietary treatments. Results suggest that Senegalese sole can cope with high levels of VO without compromising growth performance or nutrient utilization. Despite differences in muscle fatty acid profile, fish fillet had good nutritional value

Broa is a maize bread highly consumed and appreciated, especially in the north and central zones of Portugal. In the manufacturing of broa, maize flour and maize semolina might be used, besides other cereals such as wheat and rye. Considering the needs for genetically modified organism (GMO) traceability in highly processed foods, the aim of this work was to assess DNA degradation, DNA amplification and GMO quantification along breadmaking process of broa. DNA degradation was noticed by its decrease of integrity after dough baking and in all parts of bread sampling. The PCR amplification results of extracted DNA from the three distinct maize breads (broa 1, 2 and 3) showed that sequences for maize invertase gene and for events MON810 and TC1507 were easily detected with strong products. Real-time PCR revealed that quantification of GMO was feasible in the three different breads and that sampling location of baked bread might have a limited influence since the average quantitative results of both events after baking were very close to the actual values in the case of broa 1 (prepared with maize semolina). In the other two maize breads subjected to the same baking treatment, the contents of MON810 maize were considerably underestimated, leading to the conclusion that heat-processing was not the responsible parameter for that distortion, but the size of particle and mechanical processing of raw maize play also a major role in GMO quantification.

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5–10.8% of fat) followed by fins (3.4–6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4–4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n -6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n 6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish – the muscle.