Browsing by Author "Grazina, Liliana"
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- Applicability of HRM analysis for carnaroli rice authentication based on polymorphisms of the waxy genePublication . Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Garino, Cristiano; Arlorio, Marco; Oliveira, Beatriz; Mafra, IsabelRice (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.
- Authentication of Argan (Argania spinosa L.) oil using novel DNA-based approaches: detection of olive and soybean oils as potential adulterantsPublication . Amaral, Joana S.; Raja, Fatima Zahra; Costa, Joana; Grazina, Liliana; Villa, Caterina; Charrouf, Zoubida; Mafra, IsabelArgan 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.
- Authentication of carnaroli rice by HRM analysis targeting nucleotide polymorphisms in the Alk and Waxy genesPublication . Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Garino, Cristiano; Arlorio, Marco; Mafra, IsabelCarnaroli 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.
- Authentication of ginkgo biloba herbal products by a novel quantitative real-time PCR approachPublication . Grazina, Liliana; Amaral, Joana S.; Costa, Joana; Mafra, IsabelGinkgo 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.
- Authentication of incense (Pittosporum undulatum Vent.) honey from the Azores (Mel dos Acores) by a novel real-time PCR approachPublication . Lopes, Ana; Moura, Monica B.M.V.; Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Pinto, M. Alice; Mafra, Isabel'Mel dos Acores' is a unique nectar honey produced from the exceptional and diverse flora of the Azores archipelago, categorised as incense honey ('mel de incenso') or multifloral honey ('mel multiflora'). Incense honey should contain over 30 % of pollen grains of Pittosporum undulatum Vent. In this work, a real-time PCR method targeting the ITS region was proposed for the first time to detect P. undulatum in the honey from the Azores. The approach exhibited high analytical performance, achieving a quantification limit of 0.01 pg of incense DNA. The method was successfully applied to 22 honey samples, from which incense was detected in all 9 monofloral incense honeys and in 5 out of 10 multifloral samples from the Azores. Generally, the quantitative results for incense DNA were in good agreement with the melissopalynological data. Therefore, a simple, cost-effective and reliable tool was herein proposed to authenticate and valorise the Azores honey.
- Botanical authentication of globe artichoke-containing foods: Differentiation of Cynara scolymus by a novel HRM approachPublication . Grazina, Liliana; Batista, Andreia; Amaral, Joana S.; Costa, Joana; Mafra, IsabelCynara 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.
- Botanical authentication of lavender (Lavandula spp.) honey by a novel DNA-barcoding approach coupled to high resolution melting analysisPublication . Soares, Sónia; Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Oliveira, Beatriz; Mafra, IsabelMonofloral 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.
- Comparative analysis of fatty acid composition of wild vs. farmed salmonPublication . Grazina, Liliana; Nunes, Maria; Mafra, Isabel; Oliveira, Beatriz; Amaral, Joana S.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.
- Detection of soybean oil as a potential adulterant of argan oil based on a novel DNA approachPublication . Raja, Fatima Zahra; Amaral, Joana S.; Charrouf, Zoubida; Costa, Joana; Grazina, Liliana; Villa, Caterina; Kartah, Badr Eddine; Oliveira, Beatriz; Mafra, IsabelArgan oil is a non-refined vegetable oil obtained from the fruits of the argan tree (Argania spinosa L.) and produced almost exclusively in the southwestern Morocco, where the argan forest is found. Different grades of argan oil are available, namely edible/food and cosmetic grades, depending on the use of roasted or raw kernels, respectively. Argan oil is considered one of the most prized oils in the world, with its demand growing worldwide mainly due to its success as an ingredient in cosmetic products. In Europe, the price of the edible grade oil is also very high as it is perceived as a luxury product [1]. Being a premium product, argan oil is highly prone to adulteration by admixing with cheaper vegetable oils or even its total substitution. Therefore, it is important to develop methodologies that can be used in the control of the authenticity of pure argan oil. Considering that several factors can affect the chemical composition of the oil, in this work novel approaches based on DNA markers are proposed to detect the presence of soybean oil as adulterant of argan oil.
- Development of molecular markers for honey entomological origin identificationPublication . Soares, Sónia; Grazina, Liliana; Mafra, Isabel; Costa, Joana; Pinto, M. Alice; Duc, Hanh Pham; Oliveira, Beatriz; Amaral, Joana S.Honey is the natural sweet substance produced by honey bees. According to the European Union legislation, it should be produced by the westem honey bee, Apis mellifera. However, in Ásia, honey is traditionally obtained from other bee species, mainly the eastera honey bee Apis cerana. So far, only a few protein-based methods have been proposed to assess honey entomological origin[ ], which in fact is related to its geographical origin since bee species generally occupy different geographical ranges according to their evolutionary lineages [ ]. In this work, DNA markers were developed for the specifíc identification ofA. mellifera and A. cerana in honey. For this purpose, bees of A. cerana from Thailand, China and Vietnam and honey bees of 4 different subspecies of A. mellifera (iberiensis, mellifera, ligustíca, carnica) from EU countries were used. Different sets ofprimers were designed targeting the 16S rRNA gene and the tRNAleu - COII intergenic region. The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). Primers targeting the intergenic region successfully differentiated A. cerana from A. mellifera. Positive amplifications were obtained for ali the bees with 16S rRNA primers. However, the use of real-time PCR coupled with High Resolution Melting analysis allowed the separation of the two honey bee species in different clusters. The developed methodologies were applied to the analysis ofauthentic honey samples from Vietnam (produced from A. cerana and A. mellifera bees) and from Portugal allowing its successful entomological origin identification.
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