Mycotoxins are toxic fungal metabolites that may contaminate several agricultural products, both in the field and during storage. Foodstuffs and feedstuffs contaminated by these natural contaminants may cause serious risks to human and animal health due to their toxic effects.For reliable surveillance programs is mandatory the availability of rapid and cost-effective methods for mycotoxin monitoring that should be validated according to harmonized guidelines. Performance evaluation according to internationally recognized validation programs ensures compliance with regulations and facilitates trade, giving the stakeholders more confidence to choose the proper assay with respect to the application. Development, validation and one-site testing of immunoassays for rapid mycotoxin detection is one of the priority tasks of the MycoKey project (http://www.mycokey.eu/), an EU multidisciplinary project coordinated by the Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains.A brief overview of recent activities carried out at ISPA-CNR on the development and validation of immunoassays for the rapid determination of mycotoxins in cereals and cereal-based products will be presented, with a focus on multi-mycotoxin assays for on-site application. In particular, performances, advantages and limitations of newly developed fluorescence polarization immunoassays and immunochromatographic assays (i.e. lateral flow devices or dipsticks) for the determination of the major mycotoxins occurring in cereals and cereal-based products, as well as the use of DNA aptamers as alternative novel biorecognition agents in biosensor applications for mycotoxins detection, will be presented.

Conventional methods used for the determination of mycotoxins are sensitive and give both qualitative and quantitative information, although they are greatly restricted by long assay time, high cost and limited portability. As a consequence, more rapid, low cost, highly specific and portable methods for detecting these analytes are the focus of a great deal of research. In this perspective, this work describes a label free, simple and reliable method using a specific sequence of ssDNA aptamer for detecting OTA, a toxic fungal metabolite frequently occurring in a variety of foodstuffs and feeds. A piezoelectric (QCM) based biosensor was used for real time monitoring of four ssDNA aptamers-OTA interactions to select the most efficient one. Based on these results, a lab-made plasmonic sensing platform based on sinusoidal gratings was fabricated and functionalized with the most efficient selected aptamer. The sensitivity of the biosensor was found to be dependent on the aptamer immobilization strategy. In the optimized experimental conditions the biosensor was demonstrated to detect down to 0.2 ng/ml of OTA with a LOD of 0.005 ng/ml. These findings sounds very promising to produce high sensitivity, fast and potentially portable biosensors for the detection of OTA in food commodities.

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.

In previous trials the flavonoid quercetin proved to be effective in reducing Penicillium expansum infections and patulin accumulation in apples. Since quercetin resulted more effective in in vivo than in in vitro trials, a possible role of this substance in enhancing host resistance was hypothesized. To verify this hypothesis, a cDNA library of genes differentially expressed in response to quercetin application was constructed by using the suppression subtractive hybridization (SSH) approach. A total of 89 unique sequences were obtained. By homology search and functional analysis the identified sequences were putatively categorized as belonging to "metabolism", "subcellular localization" and "protein with binding functions or cofactor requirement" classes. Similarity was also found with genes coding proteins whose role in defence mechanisms is still unknown.

Penicillium expansum causes blue mould, a serious postharvest disease of apples, and is the main producer of the mycotoxin patulin. Since control by synthetic fungicides is less accepted by consumers, the demand for alternative means is pressing. In a recent study, the flavonoid quercetin, although scarcely effective in in vitro assays against P. expansum growth, significantly reduced blue mould rots on 'Golden Delicious' apples, suggesting an enhancement of host disease resistance. To confirm or reject this hypothesis, genes differentially expressed in quercetin-treated 'Golden Delicious' apples were identified by suppression subtractive hybridization (SSH). A pool of 88 unique gene transcripts was obtained. Several sequences revealed high similarities with different classes of pathogenesis-related proteins (RNase-like PR10 and PR8), or with proteins expressed under stress conditions. Other transcripts had high similarity to genes of unknown function or genes coding for proteins having a role in pathogen recognition and in signalling pathways. SSH data were validated by analysing the expression of 14 genes by quantitative real-time PCR (qPCR). Eleven genes proved to be up-regulated at a medium-high level in freshly harvested apples. Among these, 5 genes selected for temporal expression profiling revealed the existence of a combined effect, particularly at 24 or 48 h, between wounding and phenolic treatment. These results provide evidence that quercetin induces resistance to P. expansum in apples. by acting on the transcription level of genes involved in several distinct metabolic processes.

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications.

A comparison study of different extraction and clean-up procedures for the liquid chromatographic analysis offumonisins B1 (FB1) and B2 (FB2) in corn masa flour was performed. The procedures included extraction (heat orroom temperature) with acidic conditions or EDTA-containing solvents, and clean-up by immunoaffinity or C18solid-phase extraction columns. Thereafter an analytical method was optimised using extraction with an acidicmixture of methanol-acetonitrile-citrate/phosphate buffer, clean-up through the immunoaffinity column anddetermination of fumonisins by liquid chromatography with automated pre-column derivatisation witho-phthaldialdehyde reagent. Recovery experiments performed on yellow, white and blue masa flours at spikinglevels of 400, 800 and 1200 mgkg1 FB1 and of 100, 200 and 300 mgkg1 FB2 gave overall mean recoveries of 99%(6%) for FB1 and 88% (6%) for FB2. Good recoveries (higher than 90% for both FB1 and FB2) were alsoobtained with corn tortilla chips. The limits of quantification of the method (signal-to-noise ratio of 10) were25 mgkg1 for FB1 and 17 mgkg1 for FB2. The method was tested on different commercial corn masa flours aswell as on white and yellow corn tortilla chips, showing fumonisin contamination levels (FB1þFB2) up to1800 mgkg1 (FB1þFB2) in masa flour and 960 mgkg1 in tortilla chips. Over 30% of masa flours originatingfrom Mexico exceeded the European Union maximum permitted level.

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B-1, B-2, G(1) and G(2), fumonisins B-1 and B-2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analysed crude extracts. External (54%), matrix-matched (22%) or stable isotope-labelled internal standards calibration (24%) were used for toxin quantification. In general, extraction mixtures of water with acetonitrile, methanol or both provided good results for quantitative extraction of mycotoxins from maize. Laboratories using sample extract clean-up reported acceptable results for the majority of mycotoxins. Good results were also obtained by laboratories that analysed crude extracts although a high variability of results was observed for all tested mycotoxins. Matrix-matched calibration or isotope-labelled internal standards efficiently compensated matrix effects whereas external calibration gave reliable results by injecting <= 10 mg of matrix equivalent amounts. Unacceptable high recovery and high variability of fumonisin results were obtained by the majority of laboratories, which could not be explained and thus require further investigation. These findings provide the basis for the optimization and selection of methods to be used in future interlaboratory validation studies to derive their performance characteristics for simultaneous determination of mycotoxins in maize.

Alternaria species were reported to be the most commonly fungi affecting either tomato fruit and plantcausing the so called black mold of tomato. Rapid infection of Alternaria in tomato may occur on the crop orpost harvest yielding high economical losses due to spoilage of industrialized products such as tomato purée.Under optimal growth conditions, Alternaria spp. may also produce various mycotoxins. Alternariol (AOH),alternariol monomethyl ether (AME) and tenuazonic acid (TeA) are mycotoxins commonly found intomatoes and tomato products, representing a serious risk for human health related to the consumption ofthese products. This study aims at definying boundaries for growth and mycotoxin production in order tooptimize product formulation and shelf-life.

A liquid chromatographic method for thedetermination of fumonisins B1 (FB1) and B2(FB2) in corn-based foods for infants and youngchildren was subjected to an interlaboratoryvalidation study involving 11 laboratories. Fiveblind duplicate sample pairs of each matrixwere analyzed to establish the accuracy,repeatability, and reproducibility of the method.Mass fractions in the baby food samplesranged from 89.1 to 384.4 ?g/kg FB1 and from22.5 to 73.6 ?g/kg FB2. The method involved awarm extraction with citrate phosphate buffer-methanol-acetonitrile (50 + 25 + 25, v/v/v), acleanup through an immunoaffinity column,and an end-determination of fumonisins by LCafter automated precolumn derivatization witho-phthaldialdehyde reagent. RSDs for withinlaboratoryrepeatability (RSDr) ranged from 6.8to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDsfor between-laboratory reproducibility (RSDR)ranged from 15.4 to 26.2% for FB1 and 21.6 to36.3% for FB2. Mean FB1 recoveries from babyfoods spiked at 100.0 and 250.0 ?g/kg were 89and 96%, respectively; for FB2 spiked foods at25.0 and 62.5 ?g/kg recoveries were 90 and 85%,respectively. HorRat values ranged from 0.8 to1.2 for FB1, whereas for FB2 they ranged from0.9 to 1.4 when calculated according to Horwitz,and from 1.0 to 1.7 when calculated accordingto Thompson, indicating an acceptable amonglaboratoryprecision for all matrixes (HorRatvalues <2).

A method for the determination of fumonisin B1 (FB1) and B2 (FB2) in different commercial maize-based products for infants and young children was developed and tested in a limited validation study involving 3 laboratories. The method used extraction at 55 °C with an acidic mixture of methanol-acetonitrile-phosphate/citrate buffer, clean-up through immunoaffinity column and fumonisin determination by high performance liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde. Recovery experiments were performed at five spiking levels in the ranges of 80-800 ?g/kg FB1 and 20-200 ?g/kg FB2. Mean recoveries ranged from 83 to 97% for FB1 and from 61 to 78% for FB2. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 5 to 12% for FB1 and from 8 to 13% for FB2, whereas relative standard deviation for between-laboratory reproducibility (RSDR) ranged from 6 to 10% for FB1 and from 9 to 16% for FB2. The limit of quantification of the method (signal to noise ratio of 6) was 2.8 ?g/kg for FB1 and 2.2 ?g/kg for FB2. Fumonisins were found in 6 out of 19 maize-based baby foods obtained from the Italian retail market at levels up to 53 ?g/kg.

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling geland used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packedwith 300 ll oligosorbent (24 nmol DNA) showed a linear (r = 0.999) behaviour in the range of 0.4-500 ngOTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheatprior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveriesfrom wheat samples spiked at levels of 0.5-50 ng/g ranged from 74% to 88% (relative standard deviation<6%) with limits of detection and of quantification of 23 and 77 pg/g, respectively. The comparativeHPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columnscould be re-used up to five times without any loss of performance.

Results obtained within a project undertheEuropean Commission standardization mandateM520/EN (item 4) are herein presented. Aim of theproject was to develop and validate an analyticalmethod for the simultaneous determination ofnivalenol, deoxynivalenol and its acetyl derivatives,T-2 and HT-2 toxins, and zearalenone in cereals andcereal products by liquid chromatography - tandemmass spectrometry. The standardized method shallbe applied to perform official control of the abovefood products, and to determine whether aproduction batch can be put on the market.

For enforcement purposes and forreliable surveillance programs, the availability ofvalidated methods for determining foodcontaminants with performance characteristics thatmeet certain minimum criteria is mandatory. A briefoverview of recent activities carried out at theInstitute of Sciences of Food Production, NationalResearch Council of Italy (ISPA-CNR) on thedevelopment and validation of analytical methodsfor the determination of mycotoxins in food ispresented.

Deoxynivalenol (DON) is a Fusarium toxin which frequently occurs in grains. Because of the toxic effectsinduced by DON, many regulations worldwide have established safety levels in food and feed. For instance,the EC maximum limit for DON in unprocessed wheat bran has been set at 750 ?g/kg. New devices areenvisaged for the rapid detection of DON in grain stocks in order to verify the compliance with EUregulation and to perform a quick assessment of contamination without using chemicals and benchanalytical instruments. Optical spectroscopy is currently emerging as a modern and "green" analyticaltechnique for intact food analyses, thanks to the non-destructive nature of light measurements whichenable rapid checks without making use of reagents or chemical treatments, thus avoiding the problem ofwaste disposal.The objective of this study was to assess the use of Raman spectroscopy, excited at 1064 nm by using adispersive detection scheme, for rapid screening of DON in wheat bran. Twelve wheat bran samplescontaminated with DON in the range <=100-1600 ?g/kg were considered. Four replica measurements werecarried out for each sample, thus taking into account unavoidable inhomogeneity of contamination. Ramanspectra were processed using Standard Normal Variate (SNV) and Orthogonal Signal Correction (OSC) forcompensation of scattering influence, and removal of DON-independent effects. Then, Partial Least Squareregression was applied as a predictive model for DON quantification. A coefficient of determinationR2=0.72 was obtained, together with a root means square error of calibration RMSEC=313 ?g/kg, thusindicating that Raman spectroscopy has good potential as a rapid tool for DON detection.

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.

Rapid methods are nowadays recognized as a strategic tool for mycotoxin issues management. Specific guidelines for validation and verification of mycotoxin screening methods are set in Commission Regulation (EU) No 2014/519. This regulation establishes that the "aim of the validation is to demonstrate the fitness-for-purpose of the screening method" and focuses the entire validation procedure on the determination of specific cut-off levels ensuring a maximum rate of false-negative results of 5%, and the assessment of the rate of false-suspect (positive) results. With regards to rapid test-kits, 'fitness for purpose' includes not only the criteria more commonly considered when discussing laboratory-based methods (specificity, accuracy and precision), but also more "practical" parameters such as speed and ease to implementation in a new operational environment. The latter means demonstrating under local conditions that performance parameters as established during the validation, can be achieved by first time (unskilled) users. This goal can be achieved through "method verification".The aim of the present study was the verification of fitness for purpose of mycotoxin screening methods when applied by first time users. This was done in one laboratory facility, with multiple technicians attending, through results of a training course. The verification study was organized similar to a collaborative exercise, involving 10 groups comprised of two technicians that used the methods for the first time. Four different screening methods were applied, namely fast-ELISA, lateral flow device (LFD), fluorescence polarization immunoassay (FPIA), liquid chromatography - high resolution mass spectrometry (LC-HRMS), for deoxynivalenol screening in wheat and two screening methods (LFD and LC-HRMS) for aflatoxin determination in maize. The results of analyses were used to calculate precision, cut-off values and rate of false suspect results. The obtained performance characteristics were interpreted in terms of correct classification of samples as negative and suspect-positive and by comparing the results of the statistical evaluation with the results from previously performed validation studies. Finally, the statistical analysis of the results (ANOVA) allowed to draw some consideration on major factors affecting method precision.

Deoxynivalenol (DON) is the most commonFusariummycotoxin occurring in wheat and wheat-derived products,with several adverse and toxic effects in animals and humans. Although bran fractions produced by milling wheat havenumerous health benefits, cereal bran is the part of the grain with the highest concentration ofDON, thus representing a risk forconsumers. Increased efforts have been made to develop analyticalmethods suitable for rapid DON screening.RESULTS: The applicability of Fourier transform near-infrared (FTNIR), or mid-infrared (FTMIR) spectroscopy, and their combinationfor rapid analysis of DON in wheat bran, was investigated for the classification of samples into compliant andnon-compliant groups regarding the EU legal limit of 750 ?gkg-1. Partial least squares-discriminant analysis (PLS-DA) andprincipal component-linear discriminant analysis (PC-LDA) were employed as classification techniques using a cutoff value of400 ?gkg-1 DON to distinguish the two classes. Depending on the classification model, overall discrimination rates were from87% to 91% for FTNIR and from86% to 87% for the FTMIR spectral range. The FTNIR spectroscopy gave the highest overall classificationrate of wheat bran samples, with no false compliant samples and 18% false noncompliant samples when the PC-LDAclassification model was applied. The combination of the two spectral ranges did not provide a substantial improvement inclassification results in comparison with FTNIR.CONCLUSIONS: Fourier transform near-infrared spectroscopy in combination with classification models was an efficient toolto screen many DON-contaminated wheat bran samples and assess their compliance with EU regulations.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin mainly produced by several Fusarium species occurring in cereals. Chromatographic methods are the most widely used for quantitative determination of DON in foodstuffs and feedstuffs. However, these methods are destructive, time-consuming, expensive, unsuitable for screening purposes, and require a preliminary cleanup of the extracts. A range of alternative methods have been published, including infrared spectroscopy. Some studies on the use of near infrared spectroscopy and mid-infrared spectroscopy to predict DON contamination in whole grain and flour of wheat, maize and other grain cereals have been reported. The feasibility of using Fourier-transform near infrared (FT-NIR) spectroscopy for rapid and non-invasive analysis of DON in unprocessed durum wheat at levels close to the EU regulatory level (1750 µg/kg) has been recently reported. A partial least-squares (PLS) regression model was developed using correlation data between FT-NIR and HPLC/FLD (confirming method). We have further implemented the PLS model in a larger study involving more calibration (n = 230) and validation (n = 230) samples from different cultivars of wheat naturally contaminated with DON at levels up to about 16000 µg/kg DON. Slope, coefficients of correlation (r) and root mean square errors (RMSE) were close to 0.73, 0.85 and 300 µg/kg, respectively, in both calibration and validation PLS models. Similar results were obtained when the PLS model was developed by using the cross validation approach on the entire set of data.The reliability of FT-NIR spectroscopy for qualitative discrimination of wheat samples based on DON content was also investigated. Linear discriminant analysis (LDA) was performed on the same calibration and validation sets of durum wheat samples. When a cut-off limit of 1500 µg/kg was used to distinguish the samples classes, the LDA analysis was able to correctly classify more than 85% of wheat samples. Performances of LDA and of PLS regression models suggest that FT-NIR analysis might be a promising screening tool to rapidly analyse durum wheat samples for DON content. Further activities will be carried out to improve the predictive ability of the FT-NIR calibration models in the tested range

Deoxynivalenol (DON) is a mycotoxin mainly produced by several Fusarium species occurring in cereals andderived products. Rapid, robust and inexpensive methods using Fourier-Transform-Near Infrared (FT-NIR)spectroscopy have been recently developed at ISPA-CNR to predict DON levels in durum wheat. LinearDiscriminant Analysis (LDA) models were developed based on different cut-off limits (i.e. 1000, 1200 and1400 ?g/kg DON) that were set at levels lower than the EC maximum limit for DON in unprocessed durumwheat (i.e. 1750 ?g/kg). The overall classification rates of models were 89-91% with false compliant valuesof 3-7%. Model using a cut-off of 1400 ?g/kg fulfilled the requirement of the European official guidelinesfor screening methods. Partial Least-Squares (PLS) regression analysis was also used to determine DONcontent in wheat samples in the range of <50-6000 ?g/kg (as determined by a reference HPLC method). Themodel displayed good regression quality with a root mean square error (RMSE) of prediction of 868 ?g/kg.The feasibility of using FT-NIR spectroscopy was also investigated to rapidly predict DON in durum wheatbran at levels up to 1600 ?g/kg by both LDA and PLS analysis. The LDA model used a cut-off value of 400?g/kg that was lower than the EC maximum limit for DON in bran (i.e. 750 ?g/kg) and displayed aclassification rate of 80% with 5% of false compliant samples. Good performance results were also obtainedby applying the PLS statistical model, confirming a good fit between HPLC and FT-NIR data in the testedrange with an RMSE of cross-validation of 191 ?g/kg.These findings confirmed the suitability of FT-NIR to rapidly screen a large number of wheat samples forDON contamination and to verify the compliance with EU regulation.

Ochratoxin A (OTA) is a mycotoxin produced by several species of the genera Aspergillus and Penicillium, and can be frequently found in a variety of foods and beverages, including cereals, coffee, cocoa, spices, beer, wine, grape juice, and dried fruits. Effective monitoring of OTA should be undertaken and achieved through reliable and rapid analysis. Therefore, increased efforts have been made to develop analytical methods suitable for rapid OTA screening. In the present work the potential of using infrared spectroscopic for the screening of 229 wheat samples naturally contaminated with OTA in the range of < 0.15-54 µg/kg was investigated. Samples were analysed by both Fourier transform near- and mid-infrared spectroscopy (FT-NIR, FT-MIR). After a suitable pretreatment of the raw spectral data (baseline in combination with standard normal variate), Partial-Least Squares-Discriminant Analysis (PLS-DA) and Linear Discriminant Analysis (LDA) classification models were used to differentiate highly contaminated durum wheat samples from low contaminated ones and the performances of the resulting models were compared. Models were developed using a cut-off limit set at 2 µg/kg OTA that is lower than the EC maximum limit for OTA in unprocessed durum wheat (i.e. 5 µg/kg). The spectral ranges considered were between 7500-4000 cm-1 for FT-NIR and 4000-400 cm-1 for FT-MIR. For each spectral range, the classification results of the external validation (70 samples) were expressed in terms of average prediction abilities and false compliant rates. The average prediction were 94% for FT-NIR range and 96% for FT-MIR range, independently from the classification model used (i.e. PLS-DA or LDA) thus confirming the reliability of the two statistical approaches used. False compliant rates of 9% were obtained for both spectral ranges and both classification models.These findings indicates that FT-NIR, as well as FT-MIR analysis, might be a promising, inexpensive and easy-to-use screening tool to rapidly discriminate wheat samples for OTA content and verify the compliance with the EU regulatory level.This work has been supported by the Italian Ministry of Education, University and Research (P.O.N. 2007-2013), project S.I.Mi.S.A. "New Strategies for Improvement of Food Safety: Prevention, Control, Correction".

Deoxynivalenol (DON) is a type B trichothecene mycotoxin mainly produced by several Fusarium species occurring in cereals and derived products. Fourier-Transform-Near Infrared (FT-NIR) spectroscopy has been used to develop classification and quantitative models for the rapid analysis of DON in durum wheat and durum wheat bran.Linear Discriminant Analysis (LDA) was successfully used to differentiate highly contaminated durum wheat samples from low contaminated ones in the range of <50-16000 µg/kg. Three LDA models were developed based on different cut-off limits (i.e. 1000, 1200 and 1400 µg/kg DON) that were set at levels lower than the EC maximum limit for DON in unprocessed durum wheat (i.e. 1750 µg/kg). The overall classification and false compliant rates were 75-91% and 3-7%, respectively, with LDA model using a cut-off of 1400 µg/kg fulfilling the requirement of the European official guidelines for screening methods. On the other hands, the partial least-squares (PLS) regression analysis gave models with a large root mean square error (RMSE) of prediction value (1,977 ?g/kg) as compared to the EU maximum limit for DON, thus making the PLS approach unsuitable for quantitative prediction of DON in durum wheat.The feasibility of using FT-NIR spectroscopy to rapidly predict DON in durum wheat bran at levels up to 1600 µg/kg was also investigated by both LDA and PLS analysis. The LDA model used a cut-off value of 400 µg/kg that was lower than the EC maximum limit for DON in bran (i.e. 750 µg/kg) and displayed a classification rate of 80% with 5% of false compliant samples. Good performance results were also obtained by applying the PLS statistical model in the tested range with an RMSE of cross-validation of 191 µg/kg. These findings confirmed the suitability of FT-NIR spectroscopy to rapidly screen a large number of wheat samples for DON contamination and to verify the compliance with EU regulation.

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.

Mycotoxin contamination of agricultural food commodities and beverages poses a risk to human and animal health due to their toxic effects. Over 100 mycotoxins have been identified, although only a few of them present a significant source of food-borne illnesses and are of major concern worldwide. They are: aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), ochratoxin A (OTA), fumonisin B1 (FB1) and B2 (FB2), deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins. In the European Union, harmonized maximum levels for mycotoxins in foodstuffs have been specified in the Commission Regulation EC 1881/2006, and further amendments. Effective and efficient analytical methods are therefore required to identify and determine mycotoxins at legislated levels and to enforce regulatory limits. Within this context the application of LC-MS(MS) techniques is being largely explored since it enables the simultaneous monitoring of different mycotoxins in one run. Even though LC-MS(MS) methodologies for single or multiple mycotoxin determination are routinely used in control laboratories, to date none of official or standard methods approved by AOAC International or CEN (European Standardization Committee) is based on LC-MS. Proficiency Testing (PT) is an effective procedure to determine the performance of individual laboratories for specific measurements, providing a clear and a straightforward way of evaluating the accuracy (trueness and precision) of results obtained by different laboratories.An international PT was organized in 2014 to check, next to the laboratory performance, the state-of-art of currently used multi-mycotoxin methods and their implementation in the respective laboratory. The PT was free of charge and was organized by ISPA-CNR in the framework of the Italian project S.I.Mi.S.A. (PON02_00186_3417512) and promoted by the MoniQA Association (www.moniqa.org). Within the EU Network of Excellence MoniQA several efforts have been made for method comparison and deeper understanding of performances of the available LC-MS(MS) methodologies for multiple-mycotoxin analysis [1,2]. Participants were asked to determine DON, FB1, FB2, ZEA, T-2, HT-2, OTA and aflatoxins (AFB1, AFB2, AFG1, AFG2) in maize, and DON, ZEA, T-2, HT-2 and OTA in wheat. The contaminated test materials were produced and characterized by ISPA-CNR. The use of LC-MS(MS) methods, although not strictly required, was highly recommended, while the use of multi-mycotoxin methods was mandatory. Participants were not obliged to determine all toxins in each material, and let free to report only on those mycotoxins that were simultaneously determined with their multi-mycotoxin methodology. Twenty-two participants from 10 countries registered for the exercise. Nineteen laboratories returned 22 sets of results for various combinations of analytes. Three laboratories returned two sets of results obtained by using two different methods for both contaminated maize and wheat. The assign

Mycotoxins are toxic secondary metabolites of filamentous fungi affecting human and animal health. In the European Union harmonized maximum levels for the major mycotoxins occurring in food commodities have been fixed. Reliable analytical methods are therefore required to identify and determine mycotoxins at legislated levels and to enforce regulatory limits. Liquid chromatography-mass spectrometry (LC-MS) methods are being largely used since they enable the simultaneous detection of different mycotoxins. Moreover, they offer several advantages in terms of selectivity, sensitivity, substantial reduction of sample treatment, simultaneous quantification and confirmation of analyte identity. Participation in laboratory proficiency testing (PT) is an essential elements of laboratory quality assurance programmes in relation to mycotoxins analysis. In addition PT can be a useful tool to evaluate the state-of-art of methods for mycotoxin analysis by comparison of laboratory's performances. A number of PT schemes for individual or group of structurally related mycotoxins exist at international level. Only recently the interest is moving toward the organization of PTs for multi-mycotoxin determination by LC-MS. In 2014 the Institute of Sciences of Food Production of the National Research Council of Italy organized an international PT to check, next to the laboratory performance, the state-of-art of currently used multi-mycotoxin methods and their implementation in the respective laboratory. Test materials were maize contaminated with aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), ochratoxin A (OTA), fumonisin B1 (FB1) and B2 (FB2), deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins, and wheat contaminated with DON, ZEA, T-2, HT-2 and OTA. Eighteen laboratories from 10 Countries using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies participated in the study and returned results for various combinations of mycotoxins. The overall performance of participating laboratories and the assessment trend in multi-mycotoxin determination by LC-MS will be presented and discussed.

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008. ?g/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9. ?g/ml and 1.6-5.9. ?g/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices. © 2011 Elsevier B.V.

Negli ultimi anni il consumo di alimenti a base di crusca ha avuto un notevole incremento grazie al loro apporto di fibre, acidi grassi essenziali, amido, proteine, vitamine e minerali. Tuttavia diversi studi hanno anche dimostrato che la crusca di frumento duro e i prodotti derivati risultano essere frequentemente contaminati da deossinivalenolo (DON), una micotossina prodotta da funghi del genere Fusarium. Al fine di proteggere la salute del consumatore dall'esposizione al DON, la Commissione Europea ha fissato i limiti massimi ammissibili di DON in diversi prodotti, tra cui la crusca destinata al consumo umano diretto. I metaboliti fungini volatili sono stati utilizzati come indicatori della contaminazione da micotossine in cereali. A tal proposito, è stato sviluppato un metodo rapido, di facile realizzazione e non-distruttivo basato sull'impiego di un naso elettronico (e-nose) con sensori a tecnologia MOS (Metal Oxide Semiconductors) per distinguere campioni di crusca di frumento duro sulla base del contenuto di DON. In particolare i campioni, analizzati con metodo HPLC di riferimento, sono stati distinti in due classi: classe A ([DON] <= 400 µg/kg) e classe B ([DON] > 400 µg/kg). Lo sviluppo del metodo analitico è stato condotto su 410 campioni di crusca di frumento duro naturalmente contaminato da DON con livelli fino a 1600 µg/kg. L'analisi statistica multivariata, condotta mediante Discriminant Function Analysis (DFA), ha fornito un modello di calibrazione che permette di classificare i campioni di crusca con una percentuale di riconoscimento totale dell'89%. I campioni in classe A e classe B sono stati riconosciuti con percentuali rispettivamente dell'88% e 91%. La validazione del modello è stata condotta mediante procedura di cross-validazione (leave-more-out) escludendo in maniera random il 30% dei campioni dai dati in calibrazione e ponendo gli stessi in validazione. La percentuale di riconoscimento totale ottenuta in validazione è risultata pari all'87%, con valori percentuali simili per le classi A e B. E' stato inoltre ottimizzato un metodo SPME-GC-MS per caratterizzare la componente volatile di campioni di crusca di frumento duro in presenza ed in assenza di contaminazione da DON. La componente volatile ottenuta è risultata composta da idrocarburi alifatici e aromatici, acidi, esteri, alcoli, aldeidi, chetoni, terpeni e composti furanici. E' stato inoltre identificato un pattern di 8 molecole aventi correlazione positiva con il contenuto di DON, quali il 2-metil-1-propanolo, ?-caprolattone, 1-pentanolo, 1-otten-3-olo, esanale, 1-esanolo e tridecano o negativa come il 2-pentil-furano. Tali risultati confermano che il metodo e-nose sviluppato potrebbe essere un utile strumento per lo screening di DON in campioni di crusca di frumento duro.

Alternaria species are ubiquitous and includes both pathogens and saprophytes that may damage crops and cause postharvest decay. Alternaria spp. are able to grow at low temperature and may responsible for spoilage during refrigerated transport and storage. Certain species are also capable of producing mycotoxins which can contaminate plant products. Due to their thin skin, tomatoes are particularly at risk of being infected by Alternaria, and rapid growth may occur in soft tissues, causing the well-known black mold. In that context, predictive models of mycotoxin synthesis may be helpful in determining the levels of these mycotoxins for conditions supporting growth of Alternaria spp. The objective of this study was to evaluate the effects of temperature and pH on the toxin production of an A. alternata strain in a tomato-based mediumA full factorial design of six pH levels (ranging from 2 to 7) and 11 incubation temperatures (ranging from 3.2 to 37°C) was undertaken. Tomato-based agar plates at aw 0.99 were centrally inoculated with a standardized inoculum of 40 spores/5µL. Each experimental condition was tested in triplicate. For each of the 35 combinations supporting fungal development, mycotoxin accumulation was measured. Measurements of TeA, AOH and AME concentrations were performed on plates fully covered by the fungus, using HPLC. Quantification was performed by measuring peak areas at toxins retention time and comparing them with the relevant calibration curves. The experimental observations suggest that increasing pH induces a linear increase in the square root of mycotoxin synthesis, followed by a logistic decrease. The equation used to describe this relationship is based on the model of Bréand et al. (1997) and is defined by the following parameters: pHminTOX (minimum pH for toxin production), pHoptTOX (optimum pH for toxin production), TQoptTOX (toxin quantity at pHoptTOX) and TQminTOX (the toxin quantity at high pH values). Secondary models were developed to describe the relationship between temperature and the parameters (pHminTOX, pHoptTOX and TQoptTOX) while TQminTOX was assumed to be independent of temperatureTeA has been described as the major mycotoxin produced by Alternaria spp. on tomatoes. Perhaps not surprisingly, only TeA could be measured in this work while AOH and AME have not been detected. TeA measurements show an important contribution of acidic pH to mycotoxin synthesis, in particular near pH 4. At similar pH levels, incubation at low temperatures (from 6.5°C to 12°C) resulted in higher TeA production than at temperatures above 20°C. The developed model for TeA synthesis describes accurately the experimental measurements (R2=0.98). The predicted maximum toxin quantity TQoptTOX increases from of 180 µg/g at 6.5°C to 290 µg/g at 12°C. It then decreases until reaching 70 µg/g at 30°C. The parameters pHminTOX and pHoptTOX were also found to be temperature dependentThe model for toxin production can be used togethe

Le Fumonisine (FBs) sono micotossine prodotte da funghi del genere Fusarium che contaminano mais e suoi derivati. La Fumonisina B1 (FB1) nell'uomo è correlata a cancro esofageo e difetti del tubo neurale e classificata da IARC come possibile cancerogeno (IARC 2003). La FB1 inibisce il metabolismo degli sfingolipidi competendo con le basi sfingoidi libere (sfinganina e sfingosina) e inducendo l'inibizione dell'enzima ceramide sintetasi.Scopo dello studio è stato valutare la stabilità delle FBs dopo un processo di digestione in vitro e l'influenza del chimo prodotto sulla funzionalità intestinale, utilizzando modelli ex vivo (colon umano e intestino di ratto) mediante la camera di Ussing. Campioni di mais a diverso livello di contaminazione di FBs, erano digeriti in vitro, mimando composizione fisiologica e pH dei fluidi intestinali nelle fasi della digestione orale, gastrica e intestinale, e l'esposizione naturale dell'intestino alle micotossine(Versantvoort et al. 2005).I livelli di FBs nel chimo ottenuto con digestione in vitro, determinati mediante LC-HRMS, hanno mostrato un'elevata stabilità delle FBs (> 60%). Inoltre, durante l'esposizione dei campioni di colon umano e di ratto al chimo di mais, è stata osservata una differente efficienza dei trasporti ionici in relazione alla presenza/assenza di FBs nel chimo. L'analisi dei parametri elettrici, dopo esposizione per 120 min. a campioni di mais non contaminato, mostrava la riduzione (fino al 90%) della corrente di cortocircuito (indice del trasporto ionico di membrana), variazione probabilmente legata all'elevata osmolalità del chimo e conseguente attivazione dei trasporti ionici. Al contrario, la presenza delle FBs, indipendentemente dalla concentrazione, non ha modificato in ambedue i modelli intestinali tale parametro. Le micotossine, presumibilmente hanno interferito con il trasporto del Na+, inibendo la risposta allo stress osmotico indotta dalla matrice (Minervini et al. 2014a). L'influenza delle FBs sul trasporto ionico intestinale potrebbe essere correlata ad alterazioni strutturali della membrana plasmatica. E' stato descritto che l'esposizione alla FB1 può indurre l'alterazione del bilancio dei lipidi di membrana e delle interazioni proteine-lipidi cruciali per la funzione delle proteine di membrana nel loro ruolo di trasporto o recettoriale (Devi Paila et al. 2010). Tale effetto delle FBs sulla membrana plasmatica è stato dimostrato da nostri studi in vitro sulla linea intestinale HT29. Infatti la FB1, inibendo il metabolismo sfingolipidico, ha indotto un aumento della microviscosità, e quindi della permeabilità della membrana, e un aumento della perossidazione lipidica (Minervini et al 2014b) secondaria all'interazione della FB1 con il doppio strato lipidico e alla formazione di radicali liberi (Yin et al. 1998). In conclusione l'esposizione a chimo di mais contaminato da FBs ha indotto modificazioni del trasporto ionico sia nel colon umano che di ratto. Questo è il primo st

Mycotoxin analytical methods in food matrices include rapid screening and confirmation techniques. Development of both quantitative (ELISA, FLISA, FPIA, immunosensors) and qualitative (lateral flow, membrane-based flow-through) systems for (multi)mycotoxin detection is one of the fast-growing trends. Developments of various recognition elements as well as novel sensitive labels are currently ongoing in order to design a reliable and rapid test-system.The Mycokey project aims for the development of different (multi)mycotoxin rapid screening methods. The presentation will highlight the current developments: 1. a multiplex strip test for easy and rapid simultaneous determination of Fusarium toxins (fumonisins, deoxynivalenol and zearalenone) in cereals; 2. DNA aptamer based strip tests for simultaneous analysis of aflatoxin B1 and fumonisinB1 in maize making use of quantum dots as highly sensitive labels; 3. fluorescence polarization immunoassay for the quantitativesimultaneous determination of DON, 3-acetyl-DON, 15-acetyl-DON and DON-3-glucoside in wheat; 4. fluorescence polarization immunoassay for the quantitative simultaneous determination ofT-2, HT-2, T2-glucoside and HT2-glucoside in wheat; and 5. a new biosensor platform for the detection of AFB1 based on real-time electrochemical profiling (REP).Moreover, a MycoKey survey was performed for the collection and sharing of current knowledge and experience on rapid methods for mycotoxin screening. This, together with some lessons learned during the MycoKey round table discussion on mycotoxin detection methods will promote interactive discussions between scientist involved in rapid method development and stakeholders as to provide rapid methods that fit the customers' needs and expectations.

Maize is the most important food crop in the world, although it is largely susceptible to mycotoxin contamination causing a negative economic impact and serious health risks to humans and animals. It is well-known that moldy, colored/discolored, injured, broken and damaged grain kernels, as well as dust within a contaminated batch of maize, contain high levels of mycotoxins. A combination of cleaning technologies to efficiently remove contaminated fractions can therefore significantly reduce mycotoxin contamination in the outcome product.Different batches of biomass/feed quality maize contaminated by aflatoxins and Fusarium toxins (namely deoxynivalenol, fumonisins and zearalenone) have been processed on different industrial plants in Italy, Germany and Spain to evaluate the effect of different cleaning solutions on the reduction of mycotoxins. The investigated cleaning solutions included i) mechanical size separation of coarse, small and broken kernels, ii) dust/fine particles removal through an aspiration channel, iii) separation of kernels based on gravity and iv) optical sorting of spatial and spectral kernel defects. A dynamic sampling according to the Commission Regulation No. 401/2006 was performed along the entire process lines. The frequency of sampling was estimated according to the DG-SANCO guidance by taking in account the number of incremental samples, the weight of portion to be sampled (ton) and the unloading speed (ton/hr). A number of incremental samples ranging from 3 to 60 (about 100-300 g each) was collected, depending on the sampled fractions. Mycotoxin analyses of the water-slurry aggregate samples were performed by validated HPLC methods based on immunoaffinity column clean-up of extracts. In addition, in some trials the incoming materials were analyzed by the Eurofin's Rapidust® system for on-site sampling of dusts.A significant reduction of mycotoxin content in the cleaned products was observed for aflatoxins (up to 90%), zearalenone (up to 88%), deoxynivalenol (up to 82%) and fumonisins (up to 69%), with respect to the uncleaned products. High levels of mycotoxins were found in the rejected fractions, with the highest levels in dusts and in the rejected fractions from aspirator and optical sorting.This study shows that a cleaning line combining both mechanical and optical sorting technologies can provide a reliable solution for reducing mycotoxin contamination in maize. In addition, a completely new sorting technology was recently developed by Bühler for grain cleaning and monitoring based on the spectral properties of fluorescence to reduce the risk of aflatoxin contamination in maize.

Two Proficiency Testings (PTs) involving eighteen laboratory participants from 10 Countries have been conducted in 2014 for the simultaneous determination of deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, ochratoxin A and aflatoxins B1, B2, G1 and G2 in maize and of deoxynivalenol, zearalenone, T-2 and HT-2 toxins and ochratoxin A in wheat, respectively. Theaim of PTs was to check next to the laboratory performance the state-of-art of the LC-MS multimycotoxin methods used by participants. In addition, the trend of performances of LC-MS methods for multi-mycotoxin determination in maize together with method-related issues was assessed by comparing three PTs organized over the years 2011-2014. Data showed the improvement of laboratory performances with the overall acceptable z-scores that progressively increased from 59% in 2011 PT to 85% in 2014 PT, while the rate of unacceptable z-score decreased from 25% in 2011 PT to 11% in 2014 PT.

A proficiency test for simultaneous determination of up to 11 mycotoxins in maize by using LC-MS(MS) methodology was conducted. Purpose of the study was to evaluate the state of the art on the simultaneous determination of mycotoxins by LC-MS(MS) methodology and to obtain information on currently used methodologies and method-related performances. Laboratories were not obliged to determine all target mycotoxins and were free to report only on those mycotoxins that can be simultaneously determined with their LC-MS(MS) methodology.Participants received two test materials: one maize sample naturally contaminated with aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, HT-2 toxin, zearalenone, fumonisins B1 and B2 and one blank maize sample (for spiking). A common mixed mycotoxins calibrant solution and a spiking solution together with spiking protocol, instructions and a comprehensive questionnaire were also distributed. Forty laboratories from fourteen Countries reported results for some combinations of the analytes. In particular, 25 participants analyzed all target mycotoxins whereas the other participants analyzed between 2-10 mycotoxins. Electrospray ionization was used by all laboratories whereas triple quadrupole and ion trap detector was used by 36 and 4 laboratories, respectively. Sample extract cleanup (IMA, SPE, liquid/liquid extraction) was used by 22 participants whereas 17 laboratories analyzed crude extract without purification and 1 participant analyzed the extract with and without purification before LC-MS(MS) determination. For quantification, 10 participants prepared matrix-assisted calibration curves whereas the other 30 participants prepared calibration curves in pure solvents. To compensate matrix effect, 7 of 30 participants using calibration curves prepared in pure solvents added isotope labeled internal standards to the final extract whereas 1 participant used other metabolites (related to zearalenone or deoxynivalenol) as internal standards. Results on the performance of participating laboratories will be presented with particular reference to z-Scores and percent recovery of the 11 mycotoxins.

Fourier-transform-near infrared (FT-NIR) spectroscopy has been used to develop quantitative and classification models for the prediction of deoxynivalenol (DON) levels in durum wheat samples. Partial least-squares (PLS) regression analysis was used to determine DON in wheat samples in the range of <50-16,000 g/kg DON. The model displayed a large root mean square error of prediction value (1,977 g/kg) as compared to the EU maximum limit for DON in unprocessed durum wheat (i.e., 1,750 g/kg), thus making the PLS approach unsuitable for quantitative prediction of DON in durum wheat. Linear discriminant analysis (LDA) was successfully used to differentiate wheat samples based on their DON content. A first approach used LDA to group wheat samples into three classes: A (DON <= 1,000 g/kg), B (1,000 < DON <= 2,500 g/kg), and C (DON > 2,500 g/kg) (LDA I). A second approach was used to discriminate highly contaminated wheat samples based on three different cut-off limits, namely 1,000 (LDA II), 1,200 (LDA III) and 1,400 g/kg DON (LDA IV). The overall classification and false compliant rates for the three models were 75%-90% and 3%-7%, respectively, with model LDA IV using a cut-off of 1,400 g/kg fulfilling the requirement of the European official guidelines for screening methods. These findings confirmed the suitability of FT-NIR to screen a large number of wheat samples for DON contamination and to verify the compliance with EU regulation.

Deoxynivalenol (DON) is a mycotoxin, mainly produced by Fusarium sp., most frequently occurring in cereals and cereal-based products. Wheat bran refers to the outer layers of the kernel, which has a high risk of damage due to chemical hazards, including mycotoxins. Rapid methods for DON detection in wheat bran are required.RESULTSA rapid screening method using an electronic nose (e-nose), based on metal oxide semiconductor sensors, has been developed to distinguish wheat bran samples with different levels of DON contamination. A total of 470 naturally contaminated wheat bran samples were analyzed by e-nose analysis. Wheat bran samples were divided in two contamination classes: class A ([DON] 400 mu g kg(-1), 225 samples) and class B ([DON] > 400 mu g kg(-1), 245 samples). Discriminant function analysis (DFA) classified wheat bran samples with good mean recognizability in terms of both calibration (92%) and validation (89%). A pattern of 17 volatile compounds of wheat bran samples that were associated (positively or negatively) with DON content was also characterized by HS-SPME/GC-MS.

Deoxynivalenol (DON) is a mycotoxin frequently occurring in cereals and derived products, and regulated in many countries Raman spectroscopy performed using optical fibers, with excitation at 1064 nm and a dispersive detection scheme, was utilized to analyze wheat bran samples naturally contaminated with DON. A multivariate processing of the spectroscopic data allowed to distinguish two classes of contamination, with DON below and above 400 mu g/kg, respectively. Only one highly contaminated sample was misclassified. This preliminary result demonstrates the potential of Raman spectroscopy as a useful analytical tool for the non-destructive and rapid analysis of mycotoxins in food.

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

Le micotossine sono sostanze naturali con attività tossica prodotte da diverse specie fungine appartenenti principalmente ai generi Aspergillus, Penicillium e Fusarium. La loro presenza negli alimenti e nei mangimi può essere nociva per la salute umana ed animale. Al fine di garantire programmi di monitoraggio affidabili per una corretta valutazione del rischio associato alla loro esposizione e il rispetto dei livelli massimi ammissibili stabiliti in varie derrate alimentari, sono necessari metodi validati con caratteristiche che soddisfino determinati criteri di accettabilità. In questo articolo viene presentata una breve panoramica di recenti attività svolte presso l'Istituto di Scienze delle Produzioni Alimentari del Consiglio Nazionale delle Ricerche (ISPA-CNR) per lo sviluppo e validazione di metodi analitici per la determinazione di micotossine nei cereali e prodotti a base di cereali.

Introduction LC-MS/(MS) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by CEN and AOAC International. Objective Conduct a proficiency test (PT) for the simultaneous determination of up to 11 mycotoxins [aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B1 (FB1) and fumonisin B2 (FB2)] in maize using LC-MS/(MS) to benchmark laboratories currently using this technique and to obtain information on currently used methodologies and method-related performances. Methods Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB1, AFB2, AFG1 and AFG2, OTA, DON, T-2, HT-2, ZEA, FB1 and FB2); two test materials, namely a contaminated maize sample and a blank maize sample to be spiked with a spiking solution containing 11 mycotoxins. Laboratory results were rated with z-scores. Results Of the 64 laboratories enrolled in the PT, 41 laboratories from 14 countries returned 43 sets of results for various combinations of analytes. The majority of laboratories (61%) reported results for all 11 mycotoxins whereas the remaining laboratories reported results for a restricted combination (from 2 to 10 analytes). For contaminated maize and spiked maize the percentage of satisfactory z-score values (z < 2) were: DON 55% and 49%, FB1 50% and 30%, FB2 52% and 38%, ZEA 68% and 64%, T-2+HT-2 toxins 82% and 85%, OTA 58% and 60%, AFB1 56% and 62%, AFG1 73% and 84%, AFB2 40% and 78%, AFG2 64% and 78%. The poorest performance (z > 3) was obtained for FB1 (31%), FB2 (32%), AFB1 (32%) and AFB2 (32%) in contaminated maize and for DON (35%), FB1 (63%) and FB2 (52%) in spiked maize. Mean recovery results were acceptable for all mycotoxins (74% to 109%), except for fumonisins, where these were unacceptably high (159% for FB1 and 163% for FB2). Conclusion A robust and reliable method for simultaneous determination of 11 mycotoxins in maize could not be identified from the results of this PT. Additional experimental work is necessary to set up a method suitable for interlaboratory validation. The results of this PT and the relevant method's details can be useful to identify methodology strengths and weaknesses.

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.

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

Lentil (Lens culinaris Medik.) is the fourth most important pulse crop in the world after bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and chickpea (Cicer arietinum L.). Canada is the world's largest exporter of lentils, while in Italy lentils are a minor legume and can be found in restricted areas. However, Italian lentils present unique and characteristic qualities giving them a higher value, so that many of them have obtained international and national marks linked to their geographical origins, such as "protected geographical indication" (PGI), "traditional food products" (PAT) and Slow Food Presidium. For these reasons, there is a growing demand for analytical methods able to certify the declared geographical origin of lentils, in order to protect consumers and producers from fraud and unfair competition. In the present work, the potential of infrared spectroscopic fingerprinting technique for the geographical origin traceability of lentils was investigated. In particular, lentil samples from two different countries, i.e. Italy and Canada, were collected from retail markets and analysed by Fourier transform near- and mid-infrared spectroscopy (FT-NIR, FT-MIR). After a suitable pretreatment of the raw spectral data, Linear Discriminant Analysis (LDA) was used examining the FT-NIR and FT-MIR fingerprints separately and in combination in order to evaluate the spectral range mostly influenced by geographical origin. The LDA classification results were expressed in terms of recognition and prediction abilities (cross validation and external validation). Good classification results were obtained for both FT-NIR and FT-MIR ranges with FT-MIR one giving better prediction abilities, i.e. 95% and 92% for cross and external validation, respectively. The combination of the FT-MIR and F-NIR did not improve the model performances. These findings demonstrated the suitability of the methods developed to discriminate geographical origin of lentils and confirmed the applicability of the infrared spectroscopy, in combination with chemometrics, to solve geographic origin issues of foodstuffs.

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.