A survey on genetically modified maize in foods commercialised in Portugal

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

Soybean DNA extraction from blended refined vegetable oils

The major genetically modified (GM) crop species is soybean (Glycine max), accounting for 53% of the total world’s GM planted area and 70% of the soybean annual production in 2008 [1]. Since the approval of Roundup Ready® (RR) soybean in EU, the production of soybean oil using GM seeds has been increasing. In EU, the doubts raised by the use of genetically modified organisms (GMO) lead to the mandatory labelling for food products containing more than 0.9% of authorised GMO. In blended edible oils prepared with mixtures of two or more different oils, it is important to verify the labelling statements concerning their constituents and the presence of GM material, since soybean oil is frequently used. The analysis of DNA coupled with polymerase chain reaction (PCR) has been the technique of choice to monitor the presence of GMO in food. However, it is very difficult to obtain amplifiable DNA from oil since most vegetable oils, like crude soybean oil, must be refined prior to its consumption

Assessing the effect of pharmaceutical excipients on the DNA extraction from plant food supplements

in the EU market as ingredients in formulations, which are sold as plant food supplements (PFS). Among the several issues that may affect the safety of PFS, the most relevant concerns aduÍterations by the illegal addition of pharmaceutical drugs and/or the swap/ misidentification ofplant material, with cases of acate toxicity already reported [l]. Owing to the high similarity and distinct therapeutic uses ofseveral medicinal plants, accurate and fast methodologies allowing their distincüon are required. For that purpose, DNA-based methods are considered fast, sensitive and highly specific tools, allowing the unequivocal identification of plant species. Up to date, most of DNA methodologies reporting the identification of plant species essentially concern medicinal plants [2], with few works being developed for the authentication of PFS.This work was supported by the projects EXPL/DTP-SAP/1438/2013 (4SaferPFS) and Pest-C/EQB/LA0006/2013 financed by FCT (Fundação para a Ciência e Tecnologia) (FEDER funds through COMPETE). Teimo J.R. Fernandes is grateful to FCT PhD grant (SFRH/BD/93711/2013) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

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.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/publishedVersio

Exploiting DNA markers for the authentication of Hypericum food supplements

During the last years. the consumption of plant food supplements CPFS) containing medicinal plants has been growing in popularity. Consequently, there has been an increasing demand for plant material that can result in a higher number of frauds (substitution of a higher cost medicinal plant for a closely related. but cheaper species) and possibility of unintentional swap/misidentiflcation of plants. In botll cases. the PFS integrity. efficacy and safety are compromised. Therefore. methodologies for the unequivocal identification of plant species in PFS are required. In this work. DNA-markers were used to specifically identify Hypericum perforatum (used in for its antidepressive properties) and H. androsaemum (used as cholagogue and hepatic protector) in several PFS samples (tablets. capsules. tintures and ampoules). Different DNA extraction protocols. including in-house methods and commercial kits were tested. Tile extracts were amplified by real-time PCR targeting reference genes (universal eukaryotic and plant rubisco genes) and using species-specific primers targeting a DNA barcode loci CmatK gene). Best results were achieved for capsules an tablets using Nucleospin Plant 11 extraction method. whi le for liquid samples using an in-11ouse method based on DNA precipitation with ethanol and centrifugation. Although labeled. three samples tested negative for H. perfuratum. For some samples. negative amplification was obtained regard less of the targeted gene and DNA extraction method. pointing to some matnx interference. possibly due to DNA adsorption phenomena to pharmaceutica l excipients.This work was supported by projects EXPL/DTP-SAP/1438/2013 (4SaferPFS) and Pest-C/EQB/LA0006/2013 financed by FCT (FEDER funds through COMPETE). T J .R Fernandes and J Costa are grateful to FCT grants SFRH/BD/93711/2013 and SFRH/BPD/102404/2014info:eu-repo/semantics/publishedVersio

Development of a polymerase chain reaction assay for the specific detection of Citrus aurantium in plant food supplements

Plant food supplements (PFS) for weight-loss are very popular, being among the most-well consumed. The aim of this study was to detect Citrus aurantium in PFS, using polymerase chain reaction (PCR)-based methods. For this purpose, teas labelled as containing C. aurantium were commercially acquired. Voucher leaves of C. aurantium and other Citrus spp. (C. sinensis, C. limon, C. medica) were gently provided by Germplasm Banks. Primers were specifically designed in three different genomic sequences, namely RGAs4-6-like gene that encodes a resistance-like protein and two anonymous marker genes of C. aurantium retrieved from NCBI database. DNA was extracted using the commercial Nucleospin Plant 11 kit. Yield, purity and integrity of extracts were evaluated by UV /Vis spectrophotometry and agarose gel electrophoresis.info:eu-repo/semantics/publishedVersio

Detection of botanical adulterations in plant food supplements by molecular biology techniques

In the last years, botanicals have become increasingly available in the EU market in the form of plant food supplements (PFS), which are legally considered as foods under Directive 2002/46/EC and consequently not submitted to safety assessment prior to commercialisation. A concern related with PFS regards its botanical composition since unintentional swap of plants has been reported and also because adulterations by the substitution of higher cost botanicals for closely related, but cheaper species, can occur. Thus, there is a need for reliable methodologies to authenticate botanicals in commercialised PFS. Recently, molecular biology techniques have been suggested for this purpose. However, difficulties in recovering DNA from some PFS samples have been described (1). Thus, as part of a study for the botanical authentication of PFS, this work aimed at assessing the interference of pharmaceutical excipients on the recovery/amplification of DNA. Different PFS (tablets and capsules) were submitted to DNA extraction and amplified by real-time polymerase chain reaction (PCR) targeting universal eukaryotic and plant genes using species-specific primers for Hypericum DNA barcode loci. However, some samples gave consistently negative PCR amplifications irrespective of the target gene or DNA extraction method used, raising the question of whether some excipients could interfere with DNA extraction from PFS. To address this question, model mixtures of pharmaceutical excipients and water as control, were spiked with known amounts of template maize DNA. Each mixture was then submitted to DNA extraction and maize DNA quantified by real-time PCR. The use of either 10% talc or 0.5 % dyes (iron oxide or titanium dioxide) completely adsorbed DNA, resulting in negative PCR amplifications. The use of 1% talc or 10% silica, both frequently used as diluents in PFS, allowed recovering very low amounts of maize DNA (7.1 % and 2.5%, respectively). The results showed a clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS explaining the inability of recovering DNA from some samples reported in previous works. Thus, a strategy to release plant DNA from excipients, allowing its extraction and further analysis was also assayed. Hypericum species were not detected in four PFS, although being described on the label.This work was supported by EU (FEDER funds through COMPETE) and National Funds (FCT) through project EXPL/DTP-SAP/1438/2013 and PEst-C/EQB/LA0006/2013. TJR Fernandes and J Costa are grateful to FCT grants (SFRH/BD/93711/2013 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

High resolution melting analysis to discriminate artichoke (Cynara scolymus) in plant food supplements

Artichoke (Cynara scolymus L.) is a medicinal plant mainly used for its antioxidant, diuretic, choleretic and hepatoprotective properties, being frequently included in weightloss plant food supplements (PFS) (Lattanzio et al, 2009). PFS are legally considered as foods under EU Directive 2002/46/EC, which means that PFS are not submitted to any safety assessment prior to their commercialisation. This can lead to adulteration issues. such as accidental swap ofplants or deliberate substitution ofhigh value plant material by other species of lower cost. In arder to ensure consumer's safety, the development of analytical methods for plant species identifícation in complex matrices hás become cmcial. Só far DNA-based methods have been reported as the most adequate tools for plant authentication (Kazi et al, 2013). Thus, the main goal of the present study was to discriminate C. scolymus from other Cynara spp. in PFS by real-time polymerase chain reaction (PCR) coupled to high resolution melting (HRM) analysis. For this purpose, differeat Cynara species (C. scolymus, C. cardunculus, C. humilis and C. syriaca} were obtained from Portuguese, Spanish and French germplasm banks. A total of eight PFS samples containing artichoke were acquired at local herbal stores. DNA fi-om plant material and PFS was exbracted using the commercial NucleoSpin Plant II kit/The specificity and sensitivity of the designed primers targeting the C. scolymus (GenBank EU744973. 1) were assayed by qualitative and real-time PCR. Prior to the specific amplification of C. scolymus, DNA extracts were positively tested targeting an universal eukaryotic sequence (18S rRNA gene). The application of the specific PCR assay was successfül for the detection ofthe genus Cynara in some ofthe PFS samples. The results of real-time PCR coupled to HRM analysis showed that different Cynara ~spp. were included in three distinct clusters with a levei of confidence above 99.4%, thus discriminating artichoke from other Cynara species. The proposed HRM analysis allowed confirming the unequivocal presence of C. scolymus in the tested PFS with high levei of confídence (>98. 8%). To our knowledge, this is the first successfül attempt for the rapid discrimination ofC. scolymus in PFS.This work was supported by European Union (FEDER fünds through COMPETE) and National Fundds (FCT Fundação para a ciência e Tecnologia) through projecïs EXPL/DTP-SAP/1438/2013 and UID/QUI/50006/2013. T.J.R. Fernandes and J. Costa are grateful to FCT grants (SFRH/BD/93711/2013 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Food authentication by molecular methods

Quality and authenticity evaluation of foods encompasses many issues, such as the entire or partial fraudulent substitution of higher commercial value constituents by others with lower value and the presence of undeclared constituents/ingredients. To protect consumers from misleading labeling, regulations issued across the world have implemented specific guidelines and listed particular foods that sh ould be declared on the label. Particularly in the case of genetically modified foods, the introduc tion of specific regulations for labeling and traceability of genetically modified organisms (GMO) aimed to protect human health and environment and to give the consumer an informed choice. To address the referred food authenticity/safety problems, several analytical methodologies have been developed, particularly in processed foods, including refined oil matrices. For the first time, monitoring of amplifiable DNA in all the stages of a chemical refining industrial unit of soyb ean oil was succeeded by PCR techniques. Mo reover, we have also proved that it is possible to detect GM soybean along the refining process of crude oil. The study was also extended with success to commercial refined oils produced from blends and pure soybean

High resolution melting analysis as a new tool to authenticate plant food supplements: the case of artichoke (Cynara Scolymus)

Artichoke (Cynara scolymus L.) is a medicinal plant mainly used for its antioxidant, diuretic, choleretic and hepatoprotective properties, being frequently included in herbal infusions and plant food supplements (PFS) marketed for weight‐loss (Lattanzio et al, 2009). Both types of products can be adulteration targets, either by the deliberate substitution of other lower‐cost plant species, or by the accidental swap of plants owing to misidentification. Therefore, to ensure consumer’s safety, analytical methods for plant species identification in complex matrices are crucial. For this purpose, DNA‐based methods have been reported as the most adequate tools for plant authentication. Genetic composition of each plant is unique and independent from the part of the plant used (Kazi et al., 2013). Moreover DNA molecules are very stable, not affected by the plant’s age, physical conditions or environmental factors, in opposition to chemical markers. In this work, a molecular approach based on real‐time PCR coupled to high resolution melting (HRM) analysis to discriminate C. scolymus from other Cynara species was developed and applied to the analysis of herbal mixtures and PFS labelled as containing artichoke as ingredient. For this purpose, different Cynara voucher species (C. scolymus, C. cardunculus, C. humilis and C. syriaca) were obtained from germplasm banks, while samples of herbal infusions (6) and PFS (8) were acquired at local herbal and dietetic stores. DNA from plant material and PFS was extracted using the commercial NucleoSpin Plant II kit. For Cynara spp. differentiation, new primers were designed on a microsatellite region of C. cardunculus (GenBank EU744973.1) for the development of qualitative polymerase chain reaction (PCR) and real‐time PCR assays. Prior to the specific PCR assays, DNA extracts were positively tested targeting a universal eukaryotic sequence (18S rRNA gene). The qualitative PCR results were specific for Cynara genus. Further development of real‐time PCR coupled to HRM analysis showed that the tested Cynara spp. were grouped in three distinct clusters with a level of confidence above 99.4%, thus enabling the discrimination of C. scolymus from the others. The analysis of commercial samples showed that, with the exception of one PFS sample, all samples were positive for the presence of the universal eukaryotic gene. All herbal infusions and three PFS were positive for the presence of Cynara spp. based on the qualitative PCR assay. The application of the proposed method of HRM analysis confirmed the unequivocal presence of C. scolymus with high level of confidence (>98.8%) in the tested samples. To our knowledge, this is the first successful attempt for the rapid discrimination of C. scolymus in PFS.This work was supported by European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project EXPL/DTP‐SAP/1438/2013 and UID/QUI/50006/2013. Telmo J. R. Fernandes and Joana Costa are grateful to FCT grants (SFRH/BD/93711/2013 and SFRH/BPD/102404/2014, respectively) financed by POPH‐QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio