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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

Towards honey authentication: assessing European honey entomological origin by a molecular identification approach

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

Following the European Union (EU) legislation, honey should be produced by the western honey bee, Apis mellifera. Across Europe, 10 different A. mellifera subspecies can be found, comprising 3 different lineages (A, M and C) based on mtDNA [1]. In general, honey bees occupy allopatric geographical ranges according to their evolutionary lineages, allowing to establish an entomological origin for honey produced in different EU countries. Additionally, several honeys with protected designation of origin (PDO) detail the subspecies traditionally used in their production [2]. While numerous works focused on the botanical and/or geographical authenticity of honey, only a few have attempted its entomological authentication. For that purpose, DNA-based methods have been considered as the most suitable tools since they allow the unequivocal species identification. So far, only few works described the use of DNAbased methods to establish the entomological origin of honey [3,4] and those were focused on different species of honey bees, including Meliponini and/or Trigonini stingless bees. To our knowledge, this is the first attempt to distinguish among different European honey bee subspecies commonly used in honey production, with further application to honey authentication. In this work, DNA markers were developed for the differentiation of A. mellifera subspecies DNA in honey. For this purpose, individuals of A. m. iberiensis lineage A (n=22) from Portugal and Spain (n=5), A. m. iberiensis lineage M from Spain (n=7), A. m. mellifera lineage M from France, Netherlands, Scotland and Norway (n=7), A. m. ligustica lineage C from Italy (n=4), A. m. carnica lineage C from Croacia and Serbia (n=4) and commercial Buckfast lineage C bees (n=10) were tested. Different sets of primers were designed targeting the cytochrome oxidase I gene. The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). Species-specific primers successfully allowed the identification of A. m. iberiensis lineage A by end-point PCR. The use of real-time PCR coupled with High Resolution Melting analysis allowed the separation of A. mellifera honey bee subspecies in different clusters according to their lineages. The developed methodologies were applied to the analysis of authentic honey samples from Portugal (produced by A. m. iberiensis lineage A), Spain (produced by A. m. iberiensis lineage M), and Italy (produced by A. m. ligustica lineage C), allowing its successful entomological origin identification.

2017Conference object

Open access

Development of molecular markers for honey entomological origin identification

Publication . 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.

2017Conference object

Open access

Assessing the variability of the fatty acid profile and cholesterol content of meat sausages

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

Eighteen different brands of meat sausages including pork, poultry and the mixture of both meats (pork and poultry) in sausages, were analysed for their nutritional composition (total fat, moisture, crude protein and ash), cholesterol content and fatty acid composition. As expected, the pork Frankfurter sausages presented a higher fat content compared to sausages that include poultry meat in their composition. A multivariate statistical analysis was applied to the data showing the existence of significant differences among samples. Regarding fatty acid composition, significant differences were verified in canonical variate plots when the samples were grouped by sausage type, suggesting that the fatty acid profile is strongly influenced by the type of meats, as well as other ingredients such as vegetable oil and lard, used in its formulation. The group of poultry Frankfurter sausages presented lower levels of SFA and higher levels of PUFA, which can point to a healthier profile compared to the pork and meat mixture sausages. Nevertheless, some poultry sausages showed a higher cholesterol content compared to the pork Frankfurters. The lowest mean cholesterol content was obtained for the group of pork Frankfurters, which somehow contradicts the consumers' idea that pork meat products should be avoided due to its high cholesterol levels.

2014Journal article

Open access

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