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

A real-time PCR method for the detection of pine nuts in food.

Pine nuts, worldwide consumed as such, or as ingredient in desserts, pastries, sauces (Italian pesto) or salads, is also known to be a source of food allergens. Several allergic reactions (in most cases anaphylaxis) referred to pine nuts have been reported, after ingestion or by simple inhalation [1,2]. In order to protect allergic consumers, methods to unequivocally detect the presence of pine nuts in complex food matrices need to be developed. A Taqman-based real-time PCR method for the detection of Pinus spp. DNA was set up. Genomic DNA was isolated from pine nuts purchased in local markets, from pine needles belonging to nine different species of pine tree producing edible nuts, and from other sources commonly employed in the preparation of pine nut-containing foods. Moreover, model foods spiked at known concentration of pine nut powder were prepared in lab. Finally, DNA was purified from commercial foods declaring (or not) the presence of pine nuts. Selected primer pairs and probe displayed an efficiency near to 100% when applied to DNA isolated from pine nut seeds. Eight out of nine species of Pinus employed in the specificity assay were positively amplified, while none of the 18 negative controls displayed amplification. Both the intrinsic detection limit and the practical one evaluated on model spiked foods was similar or higher than that of other commercially available detection kit. Finally, the presence/absence of pine nut DNA in commercial foods was confirmed by our method

Pru du 2S albumin or Pru du vicilin?

A short partial sequence of 28 amino acids is all the information we have so far about the putative allergen 2S albumin from almond. The aim of this work was to analyze this information using mainly bioinformatics tools, in order to verify its rightness. Based on the results reported in the paper describing this allergen from almond, we analyzed the original data of amino acids sequencing through available software. The degree of homology of the almond 12kDa protein with any other known 2S albumin appears to be much lower than the one reported in the paper that firstly described it. In a publicly available cDNA library we discovered an expressed sequence tag which translation generates a protein that perfectly matches both of the sequencing outputs described in the same paper. A further analysis indicated that the latter protein seems to belong to the vicilin superfamily rather than to the prolamin one. The fact that also vicilins are seed storage proteins known to be highly allergenic would explain the IgE reactivity originally observed. Based on our observations we suggest that the IgE reactive 12kDa protein from almond currently known as Pru du 2S albumin is in reality the cleaved N-terminal region of a 7S vicilin like protein

Insects in food and their relevance regarding allergenicity assessment

Abstract Within the European Green Deal, the ‘Farm‐to‐Fork’ strategy aims to accelerate the transition to a sustainable food system and to make food systems fair, healthy and environmentally friendly. Insects contribute to the circularity of agriculture, and are ideal candidates to complement traditional sources of protein. In this context, a working programme within the European Food Risk Assessment (EU‐FORA) Fellowship Programme framework was developed at the German Federal Institute for Risk Assessment in collaboration with the Spanish National Research Council. The purpose of this technical report is to describe the activities in which the fellow was involved. As part of the training, the fellow performed a literature search regarding insects as food and allergenicity resulting in 493 hits. Out of the literature search a comprehensive scientific database with 200 publications has been built using the application ‘EndNote’. Furthermore, an extensive scientific review with the title ‘Sustainable food systems: EU regulatory framework and contribution of insects to the Farm‐to‐Fork strategy’ approaching several important issues regarding insects (Regulatory frame, Market situation, Labelling and Control, Application as food/feed, Consumer acceptance and Allergenicity risk assessment) has been drafted and sent for publication in a peer reviewed journal. In order to analyse the impact of food processing on the allergenicity of insects, different food samples were prepared and artificially digested using a protocol simulating the gastrointestinal tract. Further laboratory work to analyse the readouts, including enzyme‐linked immunosorbent assay (ELISA), has been discussed and proposed, scheduled for the end of July. In conclusion, the present working programme, together with additional activities and training provided by different institutions, enabled the fellow to gain a broader perspective in food safety, particularly concerning insects as novel foods and their safety assessment

Isolation and full characterisation of a potentially allergenic lipid transfer protein (LTP) in almond

Non-specific lipid transfer proteins (nsLTP) were shown to be among the most significant allergens, in particular in several fruits belonging to the Rosaceae family. The molecular features of LTPs, such as the presence of eight cysteine residues forming four disulfide bridges, confer a compact structure, decreasing the probability of degradation due to cooking or digestion, thereby increasing the chance of systemic absorption and severe allergic reactions. Few studies on LTP-induced allergies regarding almond (Prunus dulcis L) are available in the literature. In the present work, we describe for the first time the extraction and purification of an almond LTP, achieving its full characterisation by using liquid chromatography and exact mass spectrometry; the full sequence was identified by means of LC-ESI-Orbitrap-MS applying a bottom-up approach. The characterised protein consists of 92 amino acids and has a calculated exact MW of 9579.0. The presence of four disulfide bridges was confirmed after reduction, as shown by a mass increment of 8 Da. Finally, its potential allergenicity was confirmed via an in silico approach. The results presented here demonstrate the enormous potential of advanced MS techniques for obtaining high-quality structural and functional data of allergenic proteins in a short time

Non-Targeted NMR Method to Assess the Authenticity of Saffron and Trace the Agronomic Practices Applied for Its Production

The development of analytical methods aimed at tracing agri-food products and assessing their authenticity is essential to protect food commercial value and human health. An NMR-based non-targeted method is applied here to establish the authenticity of saffron samples. Specifically, 40 authentic saffron samples were compared with 18 samples intentionally adulterated by using turmeric and safflower at three different concentration levels, i.e., 5, 10, and 20 wt%. Statistical processing of NMR data furnished useful information about the main biomarkers contained in aqueous and dimethyl sulfoxide extracts, which are indicative of the presence of adulterants within the analyzed matrix. Furthermore, a discrimination model was developed capable of revealing the type of agronomic practice adopted during the production of this precious spice, distinguishing between organic and conventional cultivation. The main objective of this work was to provide the scientific community involved in the quality control of agri-food products with an analytical methodology able to extract useful information quickly and reliably for traceability and authenticity purposes. The proposed methodology turned out to be sensitive to minor variations in the metabolic composition of saffron that occur in the presence of the two adulterants studied. Both adulterants can be detected in aqueous extracts at a concentration of 5 wt%. A lower limit of detection was observed for safflower contained in organic extracts in which case the lowest detectable concentration was 20%