The analytical performances of a novel DNA-ligand system using the time-resolved fluorescence (TRF) response of ochratoxin A (OTA)-terbium-DNA aptamer interaction were tested for the quantitative determination of OTA in wheat. Wheat was extracted with acetonitrile/water (60:40, v/v) followed by clean-up through affinity columns containing a DNA-aptamer-based oligosorbent. Then, OTA was detected by TRF spectroscopy after reaction with a terbium fluorescent solution containing the DNA-aptamer probe. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. The average recovery from samples spiked with 2.5-25 ?g kg(-1) OTA was 77%, with a relative standard deviation lower than 6% and a quantification limit of 0.5 ?g kg(-1). Comparative analyses of 29 naturally contaminated (up to 14 ?g kg(-1)) wheat samples using the aptamer-affinity column/TRF method or the immunoaffinity column/high-performance liquid chromatography method showed good correlation (r = 0.985) in the range tested. The trueness of the aptamer-based method was additionally assessed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.

Aptamers are single-stranded oligonucleotides that are mainly selected using SELEX (Systematic Evolution of Ligands by EXponential) enrichment and are able to discriminate target molecules with high affinity and specificity, even in the case of very closely related structures. Because of their in vitro selection and production, the technology of aptamers is emerging as a viable alternative for use in a broad range of applications including affinity chromatography, lateral flow devices and biosensors. Aptamers have been produced for several targets, including peptides, proteins, drugs, whole cells, and, recently, for mycotoxins, e.g. ochratoxin A (OTA) and fumonisin B1 (FB1). The DNA aptamer with high affinity and specificity to OTA was used as oligosorbent for the preparation of aptamer-based solid phase extraction (SPE) columns. The procedure for the preparation of SPE columns was standardised after evaluating the effect of different parameters, such as oligosorbent volume, column size and breakthrough volume. SPE columns packed with 300 µl oligosorbent (24 nmol aptamer) were successfully used for the clean-up of durum wheat extracts prior to OTA determination by high performance liquid chromatography (HPLC) and fluorescence detection (FLD) in unprocessed durum wheat. The SPE-columns showed a linearity of the dose-response curve in the range of 0.4-500 ng OTA. Average recoveries from wheat samples spiked at levels of 0.5-50 ng/g ranged from 74% to 88% (relative standard deviation <6%). The limit of quantification was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 ng/g). Comparison of HPLC-FLD analyses of 33 naturally contaminated durum wheat samples after clean-up by aptamer-SPE or immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance. These results provide evidence that this DNA aptamer is a promising material for the analysis of OTA in food and can be used to replace antibodies as binding reagents in diagnostic assays. Recently, six DNA sequences binding to FB1 have been selected in our laboratories after 18 SELEX rounds. In particular, a sequence (FB1 39) showed the highest binding efficiency towards FB1 with a dissociation constant (KD) in the nanomolar range. Further processing of the selected sequence are in progress to shorten the length and improve the binding affinity and to evaluate cross-reactivity towards other similar compounds. The final aptamer for FB1 will be evaluated for potential use in fumonisin detection systems.

Aptamers are synthetic oligonucleotides that are mainly produced using a procedure known as SELEX (Systematic Evolution of Ligands by EXponential enrichment) which does not require the use of animals and offers a greater degree of control with respect to binding conditions. Their ability to fold into distinct tertiary structures forms the basis for target recognition, even in the case of very closely related structures. Based on their characteristics, aptamer-based technologies are becoming a promising and convenient alternative to antibodies for the detection of contaminants in foods. Aptamers have been produced for several targets, including mycotoxins, e.g. ochratoxin A (OTA), fumonisin B1 and aflatoxin B1. One of the reported DNA aptamers with high affinity and specificity to OTA was used as oligosorbent for the preparation of aptamer-based solid phase extraction (SPE) columns. Once standardising the procedure for their preparations, SPE columns were successfully used in combination with high performance liquid chromatography and fluorescence detection (HPLC-FLD) for the analysis of OTA in wheat. Columns showed equivalent performance to relevant immunoaffinity columns (IACs). Average recoveries from wheat samples spiked at levels of 0.5-50 µg/kg ranged from 74% to 88% (relative standard deviation <6%). The quantification limit was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 µg/kg). These findings were potentially useful to develop optimized DNA-aptamer SPE columns and bring the technology to commercial readiness. As a follow-up, aptamer-SPE columns were used for the first time in combination with a novel DNA-ligand system for high throughput OTA analysis in wheat. The technology was based on the measurement of Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) response of OTA-terbium-aptamer interaction. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. Average recoveries from wheat samples spiked at 2.5-25 µg/kg OTA ranged from 72% to 81% (relative standard deviation <6%) with a quantification limit of 0.5 µg/kg. Comparative analyses of 29 naturally contaminated (up to 14 µg/kg) wheat samples by aptamer-SPE columns/TR-FRET or IACs/HPLC-FLD showed a good correlation (r = 0.985) in the tested range. The trueness of the aptamer-based method was additionally confirmed by analysis of two quality control wheat materials for OTA.The DNA-ligand system is innovative, simple, and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.

Fumonisins (FBs), Fusarium mycotoxins common food contaminant, are a potent inducer of oxidative stress and lipid peroxidation in intestinal cells. In order to verify this toxic effect in intestine tract, the aim was to assess lipid peroxidation (as malondialdehyde MDA increased levels) on intestine rat samples exposed to chyme samples from in vitro digestion of FBs contaminated corn samples. Naturally (9.61 ± 3.2 ?g/gr), artificially (726 ± 94 ?g/gr) and spiked corn samples at EU permitted FBs levels were digested and added to luminal side of Ussing chamber for 120 min. Fumonisins-free corn sample was used as control. The MDA increase was observed just in 83% of intestine samples exposed at EU FBs levels and the digestion process seems to reduce this incidence (50% of samples). Malondialdehyde levels were FBs dose- and subject-related and ranged from 0.07 ± 0.01 to 3.59 ± 0.6 nmol/mg. Highest incidence and MDA % increment (I) were found when intestine tracts were exposed to chymes from artificially corn sample. The induction of lipid peroxidation induced by FBs could be due to interactions between FBs and intestinal membranes, with consequent modifications in membrane permeability and oxygen diffusion-concentration, as suggested by other authors.

The effect of nixtamalization on the content of fumonisins (FBs), hydrolysed (HFBs) and partially hydrolysed (PHFBs) fumonisins in maize was investigated at laboratory-scale. Maize naturally contaminated with FBs and PHFBs was cooked with lime. Starting raw maize, steeping and washing waters and final masa fractions were analysed for toxin content. Control-cooking experiments without lime were also carried out. The nixtamalization reduced the amount of FBs and PHFBs in masa and converted them to HFBs. However, the three forms of fumonisins collected in all fractions amounted to 183%, indicating that nixtamalization made available forms of matrix-associated fumonisins that were then converted to their hydrolysed forms. Control-cooking enhanced FBs and PHFBs reduction, due to the solubility of fumonisins in water during the steeping process, but did not form HFBs. These findings indicate that benefits associated with enhancing the nutritional value of nixtamalized maize are also associated with a safer product in terms of fumonisin contamination.

The gut is a possible target toward mycotoxin fumonisins (FBs) exposure. The study aims to investigatethe effects induced by FBs contaminated-corn chyme samples on functional parameters of human and ratintestine by using Ussing chamber. Fumonisins-contaminated corn and processed corn samples wereundergone to in vitro digestion process and then added to luminal side. A reduction (about 90%) of shortcircuit current (Isc lA/cm2) during exposure of human colon tissues to fumonisins-free corn chyme sampleswas observed, probably related to increased chyme osmolality. This hyperosmotic stress could drainwater towards the luminal compartment, modifying Na+ and Cl transports. The presence of FBs in cornchyme samples, independently to their concentration, did not affect significantly the Isc, probably relatedto their interference towards epithelial Na+ transport, as assessed by using a specific inhibitor (Amiloride).The rat colon tract represents a more accessible model to study FBs toxicity showing a similar functionalresponse to human. In the rat small intestine a significant reduction (about 15%) of Isc parameterduring exposure to uncontaminated or FBs contaminated corn chyme samples was observed; thereforesuch model was not suitable to assess the FBs toxicity, probably because the prevalent glucose and aminoacids electrogenic absorption overwhelmed the FBs influence on ionic transport.

The natural co-occurrence of aflatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) in dried split ginger purchased from different local markets in Lagos, South West Nigeria has been investigated. A total of 120 ginger samples, 31 collected during the rainy season and 89 during the dry season, were analyzed. Mycotoxins were determined according to the AOAC Official Method 2008.02 based on multi-toxin immunoaffinity column clean up and liquid chromatography quantification. The incidence of contamination with aflatoxins (AFs) and OTA was significantly higher during the rainy season (81% and 77%, respectively) than the dry season (46% and 37%, respectively). Average levels of AFs and OTA in positive samples were 3.13 and 5.10 ?g/kg in the rainy season (range 0.11-9.52 ?g/kg and 0.20-9.90 ?g/kg) and 1.18 and 2.76 ?g/kg (range 0.20-3.57 ?g/kg and 0.17-12.02 ?g/kg) in the dry season, respectively. Furthermore, the levels of AFB1 detected in 7 out of 31 samples (23%) collected during the rainy season were above the European Union (EU) maximum permitted level (i.e. 5 ?g/kg). No samples were found above the EU regulatory limits established for OTA in ginger (i.e. 15 ?g/kg). Moreover, a higher co-occurrence of AFs and OTA was observed in samples collected during the rainy season (65%) than the dry season (21%). Data showed that high humidity and temperature occurring during storage, which are prevalent in the rainy season, offer favorable conditions for AFs and OTA fungal production. This is the first report on the co-occurrence of AFs and OTA in ginger samples from Nigeria. Our results demonstrate that, in order to minimize the risks for consumers, the monitoring of the co-occurrence of these mycotoxins in ginger is highly recommended.

The use of synthetic materials as alternative to antibodies are increasingly being investigated and proposed for use in mycotoxin analysis. Aptamers are single-stranded oligonucleotides that are mainly selected using SELEX (Systematic Evolution of Ligands by EXponential) enrichment for their ability to bind targets with high affinity and specificity. A DNA aptamer with high affinity and specificity towards OTA was produced and preliminarily used as an oligosorbent for the preparation of affinity columns. Recently, an optimised procedure for the preparation of aptamer-SPE (solid phase extraction) columns has been described and successfully applied to the quantitative determination of OTA in unprocessed wheat at levels below the EU maximum permitted level. The effect of different parameters, such as oligosorbent volume, column size and breakthrough volume (maximum eluting solvent) has been tested. The SPE columns, packed with 300 µl oligosorbent (24 nmol aptamer), showed a linearity of the dose-response curve in the range of 0.4-500 ng OTA and were used for the clean-up of durum wheat extracts prior to OTA determination by HPLC-FLD (high performance liquid chromatography and fluorescence detection). OTA mean recoveries (from 74 to 88%) from wheat samples spiked at levels from 0.5 to 50 ng/g fulfilled EU requirements for acceptability of the analytical method. Limit of quantification of the method was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 ng/g). The comparative HPLC-FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and IMA (immunoaffinity) columns showed a good correlation. Furthermore, aptamer-SPE columns could be re-used up to five times without any loss of performance.Recently, the selection of aptamer sequences binding for fumonisin B1 (FB1) has been also described. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to FB1. Six unique sequences were obtained, each showing improved binding to FB1 compared to controls. Between them, the sequence containing the lowest guanosine content (only 8%) yielded the highest binding to FB1 with a dissociation constant of 100 ± 30 nM. Experiments to identify the minimal target-binding sequence to shorten the aptamer and improve binding are in progress.The successful outcome of this research provides evidence that DNA aptamers can be used to replace antibodies as binding reagents in diagnostic assays with commercial application for an economically important mycotoxin analysis

Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 ?M with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust andinnovative biosensor platforms for the detection of tyramine in food and beverages.

The synthesis of partially hydrolyzed fumonisins (PHFB1 and PHFB2) and hydrolyzed fumonisins (HFB1 and HFB2) by chemicalhydrolysis of pure fumonisins (FB1 and FB2) is reported together with the isolation and characterization by liquidchromatography-high-resolution mass spectrometry (LC-HRMS). Two structural isomers of partially hydrolyzed forms ofFB1 and FB2 were identified, namely PHFB1a and PHFB1b and PHFB2a and PHFB2b. Reaction yields were 21% for PHFB1(sum of the two isomers), 52% for HFB1, 31% for PHFB2 (sum of the two isomers) and 30% for HFB2. Purity of each isolatedcompound was >98%.An LC-HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzedderivatives was applied to 24 naturally contaminated samples of maize and maize-based products. The majority of samples(18 out of 24) were contaminated with fumonisins B1 and B2. Fumonisins co-occurred with both partially hydrolyzed andhydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co-occurrence of fumonisins withpartially hydrolyzed fumonisins was also recorded in one sample ofmaize kernels and four samples ofmaize-based products (i.e.maize meal, cous-cous, corn-cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 ?g/kg for fumonisins (sum of FB1and FB2), from 10 to 210 ?g/kg for partially hydrolyzed fumonisins (sum of PHFB1 and PHFB2) and from 30 to 200 ?g/kg forhydrolyzed fumonisins (sum of HFB1 and HFB2). This is the first report of the isolation of PHFB2 and the co-occurrence of FB1,FB2, PHFB1, PHFB2, HFB1 and HFB2 in maize products. Considering the growing use of nixtamalized and maize-based products,the monitoring of fumonisins and their partially and totally hydrolyzed forms in these products may represent an importantcontributing factor in evaluating the relevant human risk exposure.

Le fumonisine B1 (FB1) e B2 (FB2) sono micotossine prodotte principalmente dai funghi del genere Fusarium, frequenti contaminanti del mais. Per il suo valore nutritivo e per le svariate utilizzazioni dei suoi prodotti e sottoprodotti, il mais rappresenta una componente importante dell'alimentazione umana. In alcuni paesi del Sud America e nel Messico il mais viene consumato previa cottura in presenza di una soluzione di idrossido di calcio. Questo processo, noto come nixtamalizzazione, produce un impasto chiamato "masa" che possiede proprietà nutrizionali superiori a quelle del mais di partenza. La "masa" viene poi impiegata per la produzione di svariati prodotti derivati come tortillas, tamales e arepas. Durante il processo di nixtamalizzazione si verifica la rimozione di uno o di entrambi i residui di acido tricarballilico delle fumonisine dando origine rispettivamente alle fumonisine parzialmente idrolizzate (PHFBs) o alle fumonisine idrolizzate (HFBs). Recentemente presso i laboratori ISPA-CNR sono stati prodotti, isolati e caratterizzati per la prima volta standard puri (purezza >98%) di PHFB1, PHFB2, HFB1 e HFB2. La disponibilità di tali standard ha permesso di valutare l'effetto del processo di nixtamalizzazione (condotta su scala da laboratorio) sul contenuto delle fumonisine e delle relative forme idrolizzate. Il mais di partenza, le frazioni intermedie (acque di steeping e di lavaggio) e la "masa" finale sono state analizzate mediante LC-HRMS per determinarne il contenuto di FBs, PHFBs e HFBs. Sono stati condotti anche esperimenti di controllo che prevedevano la cottura del mais in assenza di idrossido di calcio. I risultati hanno evidenziato che la nixtamalizzazione riduceva il contenuto di FBs e PHFBs nella masa finale, mentre favoriva la produzione di HFBs. Tuttavia, il contenuto complessivo delle tre forme di fumonisine nelle frazioni raccolte risultava ben superiore al 100% (fino al 180%) indicando che la nixtamalizzazione rendeva disponibili forme di fumonisine complessate con i componenti della matrice che venivano a loro volta convertite nelle relative forme idrolizzate. Al contrario, la cottura del mais in assenza di idrossido di calcio non rilasciava HFBs e quasi il 100% di fumonisine iniziali veniva raccolto nelle frazioni intermedie e nella mais cotto. Considerata la minore tossicità delle forme idrolizzate delle fumonisine rispetto alle forme native si può affermare che la nixtamalizzazione produce alimenti più salubri rispetto al mais di partenza.