Authentication of ginkgo biloba herbal products by a novel quantitative real-time PCR approach

Ginkgo biloba is a widely used medicinal plant. Due to its potential therapeutic effects, it is an ingredient in several herbal products, such as plant infusions and plant food supplements (PFS). Currently, ginkgo is one of the most popular botanicals used in PFS. Due to their popularity and high cost, ginkgo-containing products are prone to be fraudulently substituted by other plant species. Therefore, this work aimed at developing a method for G. biloba detection and quantification. A new internal transcribe spacer (ITS) marker was identified, allowing the development of a ginkgo-specific real-time polymerase chain reaction (PCR) assay targeting the ITS region, with high specificity and sensitivity, down to 0.02 pg of DNA. Additionally, a normalized real-time PCR approach using the delta cycle quantification (ΔCq) method was proposed for the effective quantification of ginkgo in plant mixtures. The method exhibited high performance parameters, namely PCR efficiency, coefficient of correlation and covered dynamic range (50-0.01%), achieving limits of detection and quantification of 0.01% (w/w) of ginkgo in tea plant (Camellia sinensis). The quantitative approach was successfully validated with blind mixtures and further applied to commercial ginkgo-containing herbal infusions. The estimated ginkgo contents of plant mixture samples suggest adulterations due to reduction or almost elimination of ginkgo. In this work, useful and robust tools were proposed to detect/quantify ginkgo in herbal products, which suggests the need for a more effective and stricter control of such products.This work was supported by FCT (Fundação para a Ciência e Tecnologia) under the Partnership Agreements UIDB 50006/2020 and UIDB 00690/2020. L. Grazina is grateful to FCT grant (SFRH/BD/132462/2017) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Tracing Styphnolobium japonicum (syn: Sophora japonica) as a potential adulterant of ginkgo-containing foods by real-time PCR

The rising demand for ginkgo-containing products and their high economic value make them desirable targets for adulteration, particularly by the partial substitution with other plant species. Styphnolobium japonicum (plant rich in flavonol glycosides) is known as a potential adulterant of ginkgo-based foods. Therefore, this work aimed at developing a species-specific real-time polymerase chain reaction (qPCR) method for the identification/quantification of S. japonicum as an adulterant of ginkgo-containing products. The method used the EvaGreen dye, targeting the internal transcribed spacer 2 (ITS2) region of S. japonicum, providing acceptable performance parameters and a sensitivity down to 0.02 pg of DNA. Moreover, a qPCR assay was established using binary mixtures of S. japonicum in G. biloba, covering the dynamic range of 50−0.05% (w/w) of added adulterant. After trueness evaluation with blind samples, the approach was applied to 21 commercial herbal infusions, from which one was positive to S. japonicum, but below the limit of quantification (0.05 %), suggesting its inadvertent contamination rather than adulteration. To the best of our knowledge, for the first time, a specific method was proposed to quantify potential adulterations of G. biloba products with S. japonicum, providing an accurate and cost-effective tool to authenticate ginkgo-containing herbal foods.The work was supported through the projects UIDB/50006/2020 and UIDB/00690/2020, funded by FCT/MCTES (Fundação para a Ciˆencia e Tecnologia and Minist´erio da Ciˆencia, Tecnologia e Ensino Superior) through national funds. L. Grazina thanks FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for her PhD grant SFRH/BD/132462/2017. J. Costa thanks FCT for funding through program DL 57/2016 – Norma transitória (SFRH/BPD/102404/2014).info:eu-repo/semantics/publishedVersio

Towards authentication of Korean ginseng-containing foods: differentiation of five Panax species by a novel diagnostic tool

Panax ginseng C.A. Meyer (Korean ginseng) is one of the most valuable medicinal plants, recognised for its neuroprotection and other beneficial health effects. It is present in a wide range of food products, namely plant food supplements (PFS) and herbal infusions. However, other Panax species, having distinct therapeutic effects, are also known as ginseng, pointing out the need of authenticating such products. The present work aims at proposing a new high-resolution melting (HRM) method to differentiate five Panax species (P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus and P. trifolius), targeting the gene encoding the dammarenediol synthase, involved in the biosynthesis pathway of ginsenosides. A Panax-specific real-time PCR assay was successfully developed with high analytical performance parameters (Efficiency = 100.5 %, R2 = 0.995, dynamic range 10 ng-1 pg of ginseng DNA). Panax DNA was detected in 17 out of 23 ginseng-containing commercial foods, including herbal infusions and PFS. For the first time, HRM analysis differentiated five Panax species with high level of confidence (>98 %), which corroborated sequencing data. Fourteen products were successfully clustered, being all except one in accordance with their labelling statements. Therefore, the present work proposes a reliable and high-throughput tool to authenticate ginseng products that could be useful for control laboratories.The authors acknowledge the support of project UIDB 00690/2020 funded by FCT/MCTES through national funds. The authors are grateful for the supply of leaves from the Botanical Garden of Edinburgh (Edinburgh, Scotland), Botanical Garden of University of Porto (Porto, Portugal), Botanical garden of UTAD (Vila Real, Portugal), as well as to the voucher seeds from the RBG (Kew, Ardingly, West Sussex, UK), USDA-ARS Germplasm (Beltsville, MD, USA) and NCRPIS/Iowa State university (Ames, IA, USA).info:eu-repo/semantics/publishedVersio

Botanical authentication of globe artichoke-containing foods: Differentiation of Cynara scolymus by a novel HRM approach

Cynara scolymus L., known as globe artichoke, is a medicinal plant widely used in plant food supplements (PFS) and herbal infusions due to its beneficial health properties. The high demand for artichoke-containing products can lead to adulteration practices. In this work, a real-time polymerase chain reaction (PCR) system coupled to high-resolution melting (HRM) analysis was proposed to differentiate C. scolymus from other Cynara species. Hence, a Cynara-specific real-time PCR assay was successfully developed with high analytical performance, achieving a sensitivity of 0.4 pg of globe artichoke DNA. HRM analysis enabled the discrimination of C. scolymus, with a high level of confidence (>98%), corroborating sequencing data. Application results to artichokecontaining PFS and mixed herbal infusions allowed confirming the presence of C. scolymus in 38% of the samples, suggesting the substitution/mislabelling of globe artichoke in 2 samples and the need for further efforts to increase DNA amplifiability of PFS.The authors are grateful to the University of Lisbon and Jardim Botˆanico de Coimbra (Portugal), Real Jardin Bot´anico Juan Carlos (Spain), Seed Conservation Department of Royal Botanic Garden (UK) and Jardin des plantes et Botanique (France) for the supply of plant material. The authors acknowledge the support of the project UIDB/00690/2020, subsidised by FCT/MCTES (Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through national funds.info:eu-repo/semantics/publishedVersio

Authentication of carnaroli rice by HRM analysis targeting nucleotide polymorphisms in the Alk and Waxy genes

Carnaroli is a high quality and priced variety, being considered as one of the finest Italian rice varieties due to its sensorial and rheological properties and, thus being a potential adulteration target. The present work aimed at exploiting polymorphisms in the Alk (A/G and GC/TT in exon 8) and Waxy ((CT)n and G/T in intron 1) genes by HRM analysis to differentiate Carnaroli rice from closely related varieties. The HRM method targeting the Alk gene did not allow gathering the Carnaroli subgroup genotypes in the same cluster. The HRM approach targeting Waxy gene successfully discriminated the varieties sold as Carnaroli from all the others with high level of confidence (>98%), which corroborated sequencing data. Its applicability to commercial rice samples was successful. Therefore, the proposed new HRM method can be considered a simple, specific, high-throughput and cost-effective tool for the authentication of Carnaroli rice, contributing to valorise such premium variety.This work was funded by the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, under grant agreement No 613688), the project UIDB/50006/2020 subsidised by FCT/MCTES (Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through national funds and the project NORTE-01-0145- FEDER-000052. L. Grazina thanks FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for her PhD grant (SFRH/BD/132462/2017). J. Costa thanks FCT for funding throughinfo:eu-repo/semantics/publishedVersio

Towards honey authentication: Differentiation of Apis mellifera subspecies in European honeys based on mitochondrial DNA markers

Honey is the natural sweet substance produced by Apis mellifera honeybees in Europe. Depending on the country/region, the A. mellifera subspecies native to Europe belong to three different lineages: A (A. m. iberiensis), M (A. m. iberiensis and A. m. mellifera) and C (A. m. ligustica and A. m. carnica). In this work, two DNAbased approaches were developed with the aim of entomological authentication of European honeys. A cytb specific PCR assay was proposed to identify A-lineage honeybees, while a second method based on real-time PCR coupled to high resolution melting analysis targeting the COI gene was developed to differentiate C- and Mlineages honeybees. The proposed methodologies were validated successfully with honeys of known origin and applied to the entomological authentication of 20 commercial samples from different European countries. The results highlight the predominance of honeys from C-lineage honeybees in Europe, except in Iberian Peninsula countries (honey from A-lineage honeybees).The authors are grateful to Dora Henriques for assembling the mitogenomes and to Pilar de la Rua and António Pajuelo for supplying authentic honey samples. This work was supported by FCT (Fundação para a Ciência e Tecnologia) through project UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and by the projects NORTE-01-0145- FEDER-000011 and BeeHappy – POCI-01-0145-FEDER-029871 (financed by FEDER through the COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation (POCI) and FCT). S. Soares, L. Grazina and J. Costa are grateful to FCT grants (SFRH/BD/75091/2010, SFRH/BD/132462/2017 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Applicability of HRM analysis for carnaroli rice authentication based on polymorphisms of the waxy gene

Rice (Oryza sativa L.) is a staple food and one of the most important cereals in the worldwide. Italy, the leading rice producer in Europe, holds nearly 200 different varieties in the available germplosm [1]. The Carnaroli rice is a high quality and priced variety belonging to the group of ja ponica ecotype, produced mainly in Piedmont. it is considered one of the finest Italian rice varieties due to its excellent cooking resistance, given by a low tendency to lose starch and a good ability to absorb liquid while creaming, being, thus, ideal for the preparation of traditional risotto. Italian rice varieties hove different characteristics, from which the starch composition is a highly relevant parameter. Together with amylopectin, amylose is the main component of starch, whose ratio is determinant for the rice cooking properties. After cooking, varieties with high amylose content have dry, firm and separate groins, while low amylose ones usually hove tender, cohesive and glossy texture [2]. Amylose synthesis is catalysed by the granule bound starch synthase (GBSS) that is encoded by the Waxy gene (Wx), being located on the chromosome 6. Various nucleotide polymorphisms have been associated with the Wx gene, namely (CT)n repeats and several single nucleotide polymorphisms (SNP) [2]. The aim of this work was to propose a new method based on high resolution melting (HRM) analysis, exploiting those polymorphisms to differentiate Carnaroli rice from other closely related varieties.This work has been supported by the European project FOODINTEGRITY (FP7-KBBE- 2013-single-stage, No 613688), by FCT (Fundação para a Ciencia e Tecnologia) through project UID/QUI/50006/2013 - POCI/ 01/0145/FEDER/ 007265 with financial support from FCT /MEC through national funds and eo-financed by FED ER. under the Partnership Agreement PT2020 and by the project NORTE-01-0145-FEDER-000011. L. Grazina and J. Costa ore grateful to grants (SFRH/ BD/132462/2017 and SFRH/BPD/102404/2014, respectively) from FCT financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Differentiation of production method and geographical origin of salmon based on the analysis of fatty acid composition

Currently, aquaculture production supplies almost 50% of the global fish market, as a response to the increasing global demand for fish [1]. However, several consumers prefer wild over farmed fish, with the former generally attaining higher prices when the some species is considered. Therefore, there is the need to assure correct information, not only about the species, but also about the production method (farmed vs. wild) and the catch origin of fish. Salmon, a high-trophic - level carnivorous species with high economic value due to its popularity, is among the fish species that is frequently produced in aquoculture. Although the feed given to farm-raised salmon is designed to meet its nutritional requirements, it can present differences compared to the diet of wild salmon that con be reflected on the muscle composition of formed versus wild solmons. Therefore, in this work, the use of fatty acid composition combined with chemometrics was evaluated as a potential tool to authenticate salmon samples. In particular, the work aimed at identifying the geographical origin and production method (farmed vs. caught in the wild) of salmon.info:eu-repo/semantics/publishedVersio

Authentication of Argan (Argania spinosa L.) oil using novel DNA-based approaches: detection of olive and soybean oils as potential adulterants

Argan oil is a traditional product obtained from the fruits of the argan tree (Argania spinosa L.), which is endemic only to Morocco. It is commercialized worldwide as cosmetic and food-grade argan oil, attaining very high prices in the international market. Therefore, argan oil is very prone to adulteration with cheaper vegetable oils. The present work aims at developing novel real-time PCR approaches to detect olive and soybean oils as potential adulterants, as well as ascertain the presence of argan oil. The ITS region, matK and lectin genes were the targeted markers, allowing to detect argan, olive and soybean DNA down to 0.01 pg, 0.1 pg and 3.2 pg, respectively, with real-time PCR. Moreover, to propose practical quantitative methods, two calibrant models were developed using the normalized ΔCq method to estimate potential adulterations of argan oil with olive or soybean oils. The results allowed for the detection and quantification of olive and soybean oils within 50–1% and 25–1%, respectively, both in argan oil. Both approaches provided acceptable performance parameters and accurate determinations, as proven by their applicability to blind mixtures. Herein, new qualitative and quantitative PCR assays are proposed for the first time as reliable and high-throughput tools to authenticate and valorize argan oil.This work was supported by the FCT (Fundação para a Ciência e Tecnologia) through projects FCT/CNRST (Portugal/Morocco) (FCT/6460/6/6/2017/S) and the strategic funding of UIDB/50006/2020 | UIDP/50006/2020. This work was also funded by the European Union (EU) through the European Regional Development Fund (FEDER funds through NORTE-01-0145-FEDER- 000052) and the project SYSTEMIC (Knowledge Hub on Food and Nutrition Security, ERA-Net Cofund ERA-HDHL no. 696300). J. Costa and I. Mafra thank the FCT for funding through the Individual Call to Scientific Employment Stimulus (2021.03583.CEECIND/CP1662/CT0012 and 2021.03670.CEECIND/CP1662/CT0011, respectively). L. Grazina is grateful to the FCT for the grant (SFRH/BD/132462/2017) financed by POPH-QREN (subsidized by FSE and MCTES). The authors are grateful to the Groupement des Coopératives Targanine for supplying the argan oil sample. J.S. Amaral is grateful to the FCT for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 e UIDP/00690/2020) and SusTEC (LA/P/0007/2020).info:eu-repo/semantics/publishedVersio

Comparative analysis of fatty acid composition of wild vs. farmed salmon

To respond to the increasing global demand for fish, nowadays, almost 50% of the global fish market comes from aquaculture production [1]. Thus, there is the need to assure a correct information, not only about the species, but also about the production method (farmed vs. wild) and the catch origin of fish. Salmon, a hightrophic- level carnivorous species with high economic value due to its popularity, is among the fish species that is frequently produced in aquaculture. Although the feed given to farm-raised salmon is designed to meet its nutritional requirements, it can present differences compared to the diet of wild salmon that can be reflected on the muscle composition of farmed versus wild salmons. Therefore, this work aims at comparing the fatty acid composition of salmon from aquaculture and caught in the wild. Salmon specimens caught in the wild (n = 25) and farm-raised (n = 25) were obtained from West of Vancouver Island and Campbell River (Canada), respectively. Two lipid extraction methods (Soxhlet extraction with n-hexane and an adaptation of the Bligh and Dyer extraction method) and two derivatization procedures (alkaline transmethylation using KOH and acid-catalyzed transmethylation using BF3/MEOH solution) were tested. Fatty acid methyl esters (FAME) were analyzed in a Shimadzu GC-2010 Plus gas chromatograph equipped with a Shimadzu AOC-20i auto-injector, a flame ionization detector and a CP-Sil 88 silica capillary column (50 x 0.25 mm i.d., 0.20 μm). The injector and detector temperatures were 250 and 270 °C, respectively. The compounds were identified by comparison with standards (FAME 37, Supelco). Based on the obtained results, the modified Bligh and Dyer method was chosen for lipid extraction since it allowed obtaining higher amounts of long chain unsaturated fatty acids, particularly of docosahexaenoic acid (DHA). Similar results were obtained for both tested derivatization methodologies. In general, the two groups of salmon samples showed different profiles, with wild samples presenting significantly higher contents of omega-3 fatty acids, in particular docosahexaenoic and eicosapentaenoic acids, while farmed salmon had higher amounts of oleic and linoleic acids.To the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, No 613688), the project UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and by the project NORTE-01-0145-FEDER-000011. L. Grazina and M.A. Nunes acknowledge the PhD fellowship (SFRH/BD/132462/2017 and SFRH/BD/130131/2017) funded by FCT.info:eu-repo/semantics/publishedVersio