Telmo J.R. Fernandes

Tracing transgenic maize as affected by breadmaking process and raw material for the production of a traditional maize bread, broa.

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.

Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources

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.

DNA barcoding coupled to HRM analysis as a new and simple tool for the authentication of Gadidae fish 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.

An overview on fish and shellfish allergens and current methods of detection

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 representative analytical methods for their detection.

DNA extraction from plant food supplements: Influence of different pharmaceutical excipients

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.

Novel Strategies for Genetically Modified Organism Detection

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.

Genetically Modified Organism Analysis as Affected by DNA Degradation

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.

Advances on the Molecular Characterisation, Clinical Relevance and Detection Methods of Gadiform Parvalbumin Sllergens

ABSTRACT 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.

Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches

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.