Safety of Plant Food Supplements: advanced genomic and chemical approaches to assess botanical origin, illegal drugs and contaminants

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Publications

Botanical authentication of lavender (Lavandula spp.) honey by a novel DNA-barcoding approach coupled to high resolution melting analysis

Publication . Soares, Sónia; Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Oliveira, Beatriz; Mafra, Isabel

Monofloral honeys (such as, lavender honey) are highly appreciated by the consumers due to their flavour, taste and properties. However, since they are considered prime products, they are often targets of adulteration. This work exploits DNA barcoding combined with high resolution melting (HRM) analysis to establish the botanical origin of honey, using lavender honey as a case study. The plastidial matK gene was targeted as a candidate barcode for Lavandula species identification. The method allowed differentiating the species in three clusters with confidence levels >99%, being the results well correlated with the sequencing analysis. It was successfully applied to identify the botanical origin of several lavender honeys, which were grouped in the cluster of the most common species in Portugal (L. stoechas subsp., L. penduculata and L. viridis). The proposed method represents a simple, specific and cost-effective tool to authenticate the botanical origin of honey.

2018Journal article

Open access

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

Publication . Amaral, Joana S.; Raja, Fatima Zahra; Costa, Joana; Grazina, Liliana; Villa, Caterina; Charrouf, Zoubida; Mafra, Isabel

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.

2022Journal article

Open access

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

Publication . Grazina, Liliana; Amaral, Joana S.; Costa, Joana; Mafra, Isabel

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.

2020Journal article

Open access

High-resolution melting analysis as a tool for plant species authentication

Publication . Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Mafra, Isabel

High-resolution melting (HRM) analysis is a cost-effective, specific, and rapid tool that allows distinguishing genetically related plants and other organisms based on the detection of small nucleotide variations, which are recognized from melting properties of the double-stranded DNA. It has been widely applied in several areas of research and diagnostics, including botanical authentication of several food commodities and herbal products. Generally, it consists of the main steps: (1) in silico sequence analysis and primer design; (2) DNA extraction from plant material; (3) amplification by real-time PCR with an enhanced fluorescent dye targeting a specific DNA barcode or other regions of taxonomic interest (100–200 bp); (4) melting curve analysis; and (5) statistical data analysis using a specific HRM software. This chapter presents an overview of HRM analysis and application, followed by the detailed description of all the required reagents, instruments, and protocols for the successful and easy implementation of a HRM method to differentiate closely related plant species.

2021Book part

Open access

Evaluation of fatty acids of salmon from different origins: comparison of extraction and derivatization methodologies

Publication . Grazina, Liliana; Nunes, Maria A.; Mafra, Isabel; Oliveira, Beatriz; Amaral, Joana S.

Global demand for fish and fish products has increased significantly over the last decades, which led to a simultaneous increase of aquaculture production around the world, currently corresponding to almost 50% of the global fish market [1]. Among different concerns regarding the fish that consumers are eating, nowadays, there is a demand for correct information about the species, production method (farmed vs. wild) and the catch origin/provenience of fish. Salmon, one of the most popular fish in Europe, can have different geographical origins and generally command higher prices when caught in the wild. Moreover, the commercially important species of salmon belong to different genus, namely Salmo and Oncorhynchus. Therefore, this work intended to compare the fatty acid composition of salmon from diverse origins, testing different extraction and derivatization methodologies. Farmed salmon specimens were obtaining from Chile, Canada and Norway. Two lipid extraction methods, namely conventional Soxhlet extraction using n-hexane added with butylated hydroxytoluene (BHT) and an adaptation of the Bligh and Dyer extraction using ultra-turrax homogenisation with 1% NaCl, followed by extraction with chloroform and methanol, were tested. Additionally, fatty acid methyl esters (FAME) were prepared by two methodologies, namely by alkaline transmethylation using KOH and by acidcatalysed transmethylation using boron trifluoride-methanol solution. FAME were analysed in a Shimadzu GC-2010 Plus gas chromatograph equipped with a Shimadzu AOC-20i auto-injector and a flame ionisation detector (Shimadzu, Japan). A CP-Sil 88 silica capillary column (50 x 0.25 mm i.d, 0.20 μm) from Varian (Middelburg, Netherlands) was used for FAME separation. Injector and detector temperatures were 250 and 270 °C, respectively. The compounds were identified by comparison with standards (FAME 37, Supelco, Bellefonte, PA, USA). Based on the obtained results, the ultra-turrax 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. Nonadecanoic acid (C19:0) was submitted to BF3/MeOH derivatization resulting in a high transmethylation yield (90.3%). In general, salmon samples showed high contents of polyunsaturated fatty acids, including ω-3 fatty acids, which supports its consumption as part of a healthy diet.

2017Conference object

Open access