Structural features and immunoreactivity of plant food allergens: impact of technological food processing and in vitro digestibility

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DNA mini-barcodes coupled to high resolution melting (hrm) analysis for the botanical authentication of rosemary honey

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

Honey is a natural product highly consumed for its taste, nutritional value and health benefits. Monofloral honeys are the most appreciated by consumers and frequently attain high market values, thus being prone to fraudulent practices. Therefore, the development of methodologies to assess and authenticate the botanical origin of honey is of utmost importance. For this purpose, traditional methods based on pollen identification by microscopic analysis are still being used, but they are time‐consuming and greatly dependent on the experience/skill of trained analysts. As an alternative, the use of DNA markers represents promising approach for the identification of botanical species in honey. Currently, DNA barcoding has been regarded with increasing interest for the taxonomic identification of plants, with two plastidial genes (matK and rbcL) being proposed for their differentiation (Bruni et al., 2012). Thus, the objective of this work was to identify the botanical species in rosemary honey using mini‐barcode regions coupled to high resolution melting (HRM) analysis. For this purpose, different plant species (Lavandula spp.) and ten mono‐ and multifloral honeys were used. Three DNA barcoding loci, namely the plastidial coding genes rbcL and matK and the noncoding intergenic trnH‐psbA region, were used to design primers targeting Lavandula spp. (GenBank Z37408.1, KJ196360.1 and HQ902822.1). DNA from plants and honeys was extracted with NucleoSpin Plant II kit (method A), according to Soares et al. (2015). The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR) and real‐time PCR. Prior to the specific amplifications, DNA extracts were positively tested targeting a universal eukaryotic sequence (18S rRNA gene). Results from specific PCR assays were further confirmed by real‐time PCR amplification using EvaGreen fluore scence dye. The application of HRM analysis allowed discriminating Lavandula spp. into distinct clusters with high level of confidence. When applying the developed methodology to rosemary honey, samples were classified on the same cluster of Lavandula stoechas (endemic species in Portugal), therefore confirming its botanical origin. To our knowledge, this is the first study using HRM analysis for the rapid discrimination of plant species in honey.

2016Conference object

Open access

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

Publication . Batista, Andreia; Costa, Joana; Fernandes, Telmo J.R.; Amaral, Joana S.; Oliveira, Beatriz; Mafra, Isabel

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.

2016Conference object

Open access

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

Publication . Batista, Andreia; Costa, Joana; Fernandes, Telmo J.R.; Amaral, Joana S.; Oliveira, Beatriz; Mafra, Isabel

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.

2015Conference object

Open access

Novel diagnostic tools for Asian (Apis cerana) and European (Apis mellifera) honey authentication

Publication . Soares, Sónia; Grazina, Liliana; Mafra, Isabel; Costa, Joana; Pinto, M. Alice; Duc, Hanh Pham; Oliveira, Beatriz; Amaral, Joana S.

Honey can be produced by different species of honeybees, with two being of economic importance due to their use in apiculture, namely Apis mellifera (known as European honeybee) and Apis cerana (known as Asian honeybee). Due to the decline of the wild populations of the Asian honeybee, this honey generally attains much higher market value, being prone to adulteration. This work aims at proposing new tools, based on the use of molecular markers, for the entomological authentication of honey. To this end, new species-specific primers were designed targeting the tRNA leu -cox2 intergenic region and allowing the detection of A. cerana DNA by qualitative polymerase chain reaction (PCR). Additionally, a novel real-time PCR method with high resolution melting analysis was developed to target the 16S rRNA gene of both bee species, allowing their discrimination in different clusters. The proposed methodologies were further applied with success in the authentication of Asian and European honey samples by the identification of honeybee DNA, demonstrating the usefulness of these simple and cost-effective new approaches.

2018Journal article

Open access

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

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

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.

2021Journal article

Open access