A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 orHT-2 toxin tracers were carried out and their binding response with sevenmonoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2- specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high crossreactivity for T-2 toxin (CR%=100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 ?g kg-1) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 ?g kg-1 for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinityclean-up showed a good correlation (r=0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.

A value chain approach is needed for an efficient mycotoxin reduction in the food and feed chain, starting from measures to prevent contamination in the field, to control measures to avoid mycotoxin production during storage and over the process line, until final consumption. Grain cleaning is the most effective post-harvest mitigation strategy to reduce high levels of mycotoxins due to the efficient removal of mold-infected grains and grain fractions with high mycotoxin content. Several studies have been performed during the last years to investigate the reduction of deoxynivalenol in wheat and barley, ergot in rye, and total aflatoxins in peanuts and maize. Typical cleaning steps include (i) mechanical size separation and dust removal by aspiration, (ii) separation based on density differences, and finally (iii) optical sorting. Within grain milling, often a fourth cleaning step is included, (iv) separation of bran, and for wet processing as in malting, (v) washing can be introduced. Recently a completely new technology was developed for grain cleaning and monitoring based on the spectral properties of fluorescence. These well proven and recent innovations for mycotoxin reduction will be described further in the talk, taking a whole value chain approach.

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.

Grain cleaning is the most effective post-harvest measure to reduce elevated mycotoxin levels. The focus lies on the efficient removal of mould-infested grains and grain fractions on the basis of features such as size, density and optical properties. In this study, the reduction of total aflatoxins (AFB1, AFB2, AFG1, AFG2) in naturally contaminated maize was tested in three cleaning steps using industrial-scale cleaning Bühler machines. The first step included (i) the Grain Plus for mechanical size separation and dust removal by aspiration, the second (ii) separation based on density differences with a Concentrator, and the third (iii) optical sorting with SORTEX. Four batches of maize (about 3 tons each) with different levels of aflatoxin contamination were used for the trials. Furthermore, different process settings and cleaning intensities were tested. Sampling (3 replicates/batch) was performed according to the Commission Regulation N. 401/2006 and the collected samples were analyzed by HPLC/FLD with photochemical derivatization. In addition, the incoming material was analyzed by the Eurofins' Rapidust® system for on-site sampling and analysis of mycotoxins in grains. First of all, trials once again highlighted the difficulties of sampling for aflatoxins. Samples showed a large variability with respect to aflatoxin level. However, high levels of aflatoxin contamination were observed in the removed product streams, with values up to 250 µg/kg. Consistent results were achieved by calculating the aflatoxin level of the incoming material from the removed products taking into account the mass balance. These values compared well with the analyzed levels from the Rapidust®. Aflatoxin levels were reduced from about 10 and 20 µg/kg to 3-4 µg/kg and 2-3 µg/kg for the low and high contaminated material, respectively. The results showed that i) with the Grain Plus an aflatoxin removal of 10-15% was achieved by size separation and aspiration; ii) with the Concentrator, another 10-70% aflatoxins was removed by density separation and finally, iii) another 20-90% aflatoxins was removed by optical sorting with SORTEX. In conclusion, the combination of the tested cleaning machines could allowed a total aflatoxin removal of 60-90% in maize.

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.

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36 h in YES medium, and 72 h in hazelnut agar medium. Significantly, the amount of anatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease. potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular. ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts. (C) 2009 Elsevier B.V. All rights reserved.

Fungal biodiversity is one of the most important contributors to the occurrence and severity of mycotoxin contamination of crop plants. Phenotypic and metabolic plasticity has enabled mycotoxigenic fungi (MF) to colonize a broad range of agriculturally important crops and to adapt to a range of environmental conditions. New mycotoxin-commodity combinations provide evidence for the ability of fungi to adapt to changing conditions and the emergence of genotypes that confer enhanced aggressiveness toward plants and/or altered mycotoxin production profiles.Among diseases caused by MF, the most important are the result of attacks carried out by species complexes. Examples of these diseases are the Fusarium ear rot of maize, caused by species of the Fusarium fujikuroi species complex; Fusarium head blight of small-grain cereals (e.g. wheat, barley, and oat) caused by Fusarium graminearum species complex and a number of other Fusarium species; black point of wheat kernels caused by Alternaria alternata species complex and related species; grape rot caused by the black Aspergilli. Thus, the ability of various toxigenic species within the complexes to produce different classes of secondary metabolites, combined with their ability to coexist in the same host or/and occur in quick succession have allowed these complexes to become "invincible armadas" against many plants. Plant infections by MF can occur at all developmental stages, from germinating seeds to mature vegetative tissues, depending on the host plant and MF species involved. Therefore, since most toxigenic fungi have specific mycotoxin profiles, early and accurate identification of the species occurring in the plants at every step of their growth is critical to predict the potential toxicological risk to which plants are exposed and to prevent toxins entering the food chain. Moreover, the great biodiversity of MF species/strains is clearly the main factor responsible for the multi-toxin contamination risk in food commodities due to the co-occurrence of groups of toxigenic fungi genetically closely related or distant on the same crop. In addition, a great contribution to qualitative differences in mycotoxin production among fungi is variation in mycotoxin biosynthetic genes. We will report in the presentation main genetic, biochemical and chemical investigations carried out at the ISPA in order to: i) establish phylogenetic relatedness among the main species belonging to Alternaria, Aspergillus and Fusarium genera occurring on agro-food host plant products by using a multi-locus approach; ii) elucidate some specific differences in biosynthetic genes that are responsible for intra- and inter-specific differences in fumonisin and trichothecene production in Aspergillus and Fusarium, respectively; iii) develop rapid, easy and robust multi-mycotoxin detection methods for an accurate and reliable assessment of the risks related to the mycotoxin contamination of food products in the field.

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 significance of laboratory sample preparation for the determination of two important mycotoxins, ochratoxin A (OTA) and deoxynivalenol (DON), in wheat was investigated by comparing water-slurry mixing and dry-milling procedures. The distribution of OTA and DON in 10 kg samples of naturally contaminated wheat was established by analyzing one hundred 100 g subsamples of each sample. A normal distribution and a good repeatability of DON measurements was observed for both water-slurry mixing (mean 2290 microg/kg, CV 4.6%, median 2290 microg/kg) and dry milling (mean 2310 microg/kg, CV 6.4%, median 2290 microg/kg) procedures. For OTA determinations, reliable results could be obtained only by slurry mixing sample preparation (mean 2.62 microg/kg, CV 4.0%, median 2.62 microg/kg), whereas dry-milling comminution resulted in an inhomogeneous distribution with a high variability (mean 0.83 microg/kg, CV 75.2%, median 0.60 microg/kg) and a positive skewness (2.12). Ad hoc experiments were performed on different size portions of the same sample (10 kg) to assess accuracy and precision of the comminution/homogenization procedures (slurry mixing and dry milling). Very good results were obtained for DON determination with both procedures in terms of accuracy (>98.7% of the "weighted value") and precision (CV <3%). For OTA determination good results were only obtained by slurry mixing (99.4% of the "weighted value," CV 10%), whereas dry milling provided results with low accuracy (43.2% of the "weighted value") and high variability (CV 110%). This study clearly demonstrated that sample preparation by slurry mixing is strictly necessary to obtain reliable laboratory samples for OTA determination in wheat to minimize misclassification of acceptable/rejectable lots, mainly within official control.

Safety of milk may be affected by toxic contamination. Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Milk is the only food of animal origin where a significant aflatoxin feed-food carry over may occur. The main AFB1-related compound present in milk is aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, classified by the IARC as "group 1 human carcinogen". Regulation No. 1881/2006/EC established the maximum level for AFM1 in raw milk, heat-treated milk and milk for the manufacture of dairy products at 50 ng/kg. Scientific and technological research is called to develop cost- and time-effective field methods that can be transferred to producers for self-control purposes. When validated according to official guidelines,such as those defined in the Commission Regulation 519/2014/EU, rapid methods are expected to complement the consolidated European system for official control that is actually based on sophisticated and expensive laboratory instruments and techniques requiring extensive sample pre-treatment and personnel training.Aim of this work was to evaluate and compare analytical performances of widely applied commercial immunoassays for the detection of AFM1 in raw cow milk, namely lateral flow immunoassay (LFD) and ELISA. Analytical performances such as precision profile, cuf-off, false positive and false negative rates were evaluated for each assay by single laboratory validation performed at AFM1 levels of 0, 25, 50 and 75 ng/kg. Fifty ng/kg was the screening target concentration (STC), that was the concentration of interest for the detection of the mycotoxin in the milk sample. The two assays showed similar performances in terms of cut-off (37.7 ng/kg and 39.4 ng/kg for LFD and ELISA respectively), false suspects rate for blanks (< 0.1% for both assays) and false negative rate for samples containing AFM1 at levels higher than STC (0.3% for both assays). False suspect rate for samples contaminated at 25 ng/kg (50% STC) were 3% and 23% for LFD and ELISA respectively. Similar values were also obtained for the precision at all tested validation levels which was < 19% for the LFD and < 24 %for the ELISA.Furthermore, a satisfactory correlation of the results obtained with the rapid immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated at AFM1 levels in the range n.d. - 50 ng/kg. Finally, the extension of the scope of the LFD method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation. The obtained data showed the applicability of the LFD immunoassay to goat and sheep milk provided that a specific calibration curve was used.

Relazione tenuta durante il primo incontro dell'evento "Future for Food and Food for Future" presso il CNR - Area della ricerca I di Milano sul contributo della ricerca e dell'innovazione per garantire alimenti sicuri in uno scenario futuro.

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (?=220 nm) in less than 3min. Mean recoveries from blank wheat samples spiked with DONand NIV at levels of 100-2,000 ?g/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student-Newman-Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P< 0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). Nosignificant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 ?g/kg for DON and 20 ?g/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 ?g/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 ?g/kg. NIV was detected in two samples at negligible toxin levels (up to 46 ?g/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat.

A rapid and accurate fluorescence polarization (FP) immunoassay has been optimized for the determination of deoxynivalenol (DON) in wheat bran and whole-wheat flour. A preliminary treatment with activated charcoal was used to eliminate the strong matrix effect due to highly colored interfering compounds present in raw wheat bran extracts. In particular, matrix effect was removed by adding activated charcoal to the wheat bran extract (3.5 mg/mL) and mixing for 3 min of incubation time prior to the FP immunoassay analysis. No preliminary treatment was necessary for whole-wheat flour. Average recoveries from samples spiked with DON at levels of 500, 1,000, and 1,500 mu g/kg were 95 % for wheat bran and 94 % for whole-wheat flour, with relative standard deviation generally lower than 13 %. Limits of quantification of the optimized FP immunoassay were 120 mu g/kg for both matrices. The overall time of analysis was lower than 15 min for wheat bran and 10 min for whole-wheat flour. Good correlations (r > 0.971) were observed between DON contents obtained by both FP immunoassay and high-performance liquid chromatography with immunoaffinity cleanup for 37 and 23 samples of naturally contaminated wheat bran and whole-wheat flour, respectively. These results show that the FP immunoassay is suitable for high-throughput screening as well as for quantitative determination of DON in wheat bran and whole-wheat flour.

European intake estimates indicate that the presence of HT-2 and T-2 toxins in cereals, mainly in oats, can be of concern for human health. Therefore, the development of sensitive, rapid and reliable methods for determining these mycotoxins in cereals, in particular oats, has high priority. A rapid ultra-performance liquid chromatographic (UPLC) method has been developed for the simultaneous determination of HT-2 and T-2 toxins in oats and wheat at ?g kg(-1) level. Ground samples were extracted with methanol/water (90:10, v/v) and the diluted extracts were cleaned up through immunoaffinity columns. HT-2 and T-2 toxins were separated and quantified by UPLC with photodiode array (PDA) detector (?=202 nm) in less than 5 min. Mean recoveries from blank oats samples spiked with HT-2 and T-2 toxins at levels of 50-1000 ?g kg(-1) ranged from 87 to 96%, with relative standard deviations (RSDs) lower than 7%; mean recoveries from wheat spiked with HT-2 and T-2 toxins at levels of 25-100 ?g kg(-1) ranged from 91 to 103%, with RSDs lower than 5%. The limit of detection of the method was 8 ?g kg(-1) for both toxins (signal-to-noise ratio 3:1). The method was successfully applied to the analysis of HT-2 and T-2 toxins in naturally contaminated oats and wheat samples. A good correlation was found by comparative analysis of naturally contaminated samples of oats (r=0.9985) and wheat (r=0.9058) using the proposed method or a reliable HPLC method with fluorescence detection after pre-column derivatization with 1-anthroylnitrile.

A rapid fluorescence polarization immunoassay (FPIA) was optimized and validated for the determination of ochratoxin A (OTA) in rye and rye crispbread. Samples were extracted with a mixture of acetonitrile/water (60:40, v/v) and purified by SPE-aminopropyl column clean-up before performing the FPIA. Overall mean recoveries were 86 and 95% for spiked rye and rye crispbread with relative standard deviations lower than 6%. Limits of detection (LOD) of the optimized FPIA was 0.6 mug/kg for rye and rye crispbread, respectively. Good correlations (r > 0.977) were observed between OTA contents in contaminated samples obtained by FPIA and high-performance liquid chromatography (HPLC) with immunoaffinity cleanup used as reference method. Furthermore, single laboratory validation and small-scale collaborative trials were carried out for the determination of OTA in rye according to Regulation 519/2014/EU laying down procedures for the validation of screening methods. The precision profile of the method, cut-off level and rate of false suspect results confirm the satisfactory analytical performances of assay as a screening method. These findings show that the optimized FPIA is suitable for high-throughput screening, and permits reliable quantitative determination of OTA in rye and rye crispbread at levels that fall below the EU regulatory limits.

A new analytical method for the determination of ochratoxin A (OTA) in red wine has been developed by using adouble-extract cleanup and a fluorometric measurement after spectral deconvolution. Wine samples were diluted with a solutioncontaining 1% polyethylene glycol and 5% sodium hydrogencarbonate, filtered, and purified by immunoaffinity and aminopropylsolid-phase column. OTA contents in the purified extract were determined by a spectrofluorometer (excitation wavelength, 330nm; emission wavelength, 470 nm) after deconvolution of fluorescence spectra. Average recoveries from wine samples spikedwith OTA at levels ranging from 0.5 to 3.0 ng/mL were 94.5-105.4% with relative standard deviations (RSD) of <15% (n = 4).The limit of detection (LOD) was 0.2 ng/mL, and the total time of analysis was 30 min. The developed method was tested on 18red wine samples (naturally contaminated and spiked with OTA at levels ranging from 0.4 to 3.0 ng/mL) and compared withAOAC Official Method 2001.01, based on immunoaffinity column cleanup and HPLC with fluorescence detector. A goodcorrelation (r2 = 0.9765) was observed between OTA levels obtained with the two methods, highlighting the reliability of theproposed method, the main advantage of which is the simple OTA determination by a benchtop fluorometer with evidentreductions of cost and time of analysis.

European intake estimates indicate that the presence of T-2 and HT-2 toxins in cereals can be of concern for human health. Oats, barley and rye have been shown to be frequently contaminated by T-2 and HT-2 toxins, with high incidence and levels of contamination. Furthermore cereal-based products have also been shown to be contaminated. Therefore, the European Commission has recommended to collect more monitoring data on the presence of T-2 and HT-2 toxins in cereals and cereal-based products in order to assess changes and trends in human and animal exposure to these mycotoxins. The development of rapid, sensitive and reliable methods for monitoring T-2 and HT-2 toxins in cereals and cereal-based products has high priority in order to protect consumer health from the risk of exposure to these toxins. Fluorescence Polarization (FP) immunoassay is a homogeneous technique that is getting attention as screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. Fluorescence polarization immunoassays have been optimized for rapid quantification of T-2 and HT-2 toxins in oats, barley, rye and cereal-based products. Samples were extracted with 90% methanol and the extract was filtered and diluted with sodium chloride solution prior to the FP immunoassay. Overall mean recoveries from spiked oats, rye, barley, pasta, oat flakes and oats crispbread ranged from 101% to 107% with relative standard deviations lower than 6%. Limits of detection (LODs) of the FP immunoassay were 70 µg/kg for oats, 40 µg/kg for oat flakes and barley, 25 µg/kg for pasta and 20 µg/kg for rye and oats crispbread. Good correlations (r > 0.953) were observed between T-2 and HT-2 toxins contents in naturally and artificially contaminated barley, oats, oat flakes and pasta samples determined by both FP immunoassay and UHPLC with immunoaffinity column clean-up. These results, combined with rapidity (less than 10 min) and simplicity of the assay, show that the optimized assay is suitable for high-throughput screening as well as for reliable quantitative determination of T-2 and HT-2 toxins in oats, barley, rye and cereal-based products.

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.

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, aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains. In this framework, a multiplex dipstick immunoassay based method for the simultaneous quantitative determination of major Fusarium toxins, namely deoxynivalenol (DON), zearalenone (ZEA), and fumonisins (FUM) in cereals (wheat, barley and maize) is under development. The dipstick format is based on an indirect competitive approach. The DON/ZEA/FUM prototype is based on a nitrocellulose lateral flow device using a reader to enable quantitative determination of mycotoxin contamination in cereal extracts. Three test lines (mycotoxin-BSA conjugates) and one control line are located on the strip membrane, whereas labeled antibodies were freeze-dried within a microwell. The prototype strip test development included the following steps: definition of coating process and detection reagent formulation parameters; definition of assay parameters like incubation time, migration time, volume sampling to reach minimum performance requested in term of LoD (limit of detection), LoQ (limit of quantification); development of a sample preparation protocol allowing satisfactory mycotoxin recoveries. The total time of analysis is 25 minutes including pre analytical treatment.Preliminary results showed the DON/ZEA/FUM prototype to be compatible with the minimum acceptable performances, and to be suitable for its application to the analysis of real samples containing the target mycotoxins at levels close to EU regulatory levels.

Metabolomics is a powerful method for comprehensive investigation of metabolite variations in biological systems. Currently liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) represent the most powerful metabolomic platform. Provided that a proper sample preparation is performed, this technology may allow for the detection of thousands of metabolites and therefore may provide a comprehensive view of the metabolome.Untargeted metabolite mass profiles can be used for biological interpretations, however approaches that do not require the identification of the metabolic features should be used with extreme caution because they may lead to false interpretations. The identification of metabolites with a high level of confidence is required in order to improve the meaning of metabolomics in biological systems, such as plant-pathogen interaction, and possible applications.Well-established computational tools and workflows are important for highly reproducible and repeatable metabolomic studies. In addition, knowledge-based workflows for metabolite annotations should be built, integrating information relevant to MS peaks relationships (adducts and neutral losses), MS/MS data, retention time modeling, with biochemical knowledge. Sharing workflows (research data and software) helps to validate the findings reported in publications and, more importantly, lets researchers freely reuse the data as they are or as a reliable basis to move forward. For these reasons, the aim of this study was to develop an integrated and open source platform for LC-HRMS metabolomic studies. The platform enables processing of data from targeted and untargeted LC-HRMS analysis (profiling and compound annotation). The applicability of the developed approach is here demonstrated through a preliminary investigation of the metabolic response of maize induced by Fusarium verticillioides infection in maize kernels.

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, non-targeted 1H-NMR fingerprinting, in combination with different multivariate statistical analysis techniques, was used to classify lentils according to their geographical origin. In particular, 85 lentil samples from two different countries, i.e. Italy and Canada, were collected from retail markets and analysed by using an optimized 1H-NMR protocol. Principal component analysis showed partial grouping of samples on the basis of origin with overlapping zones. Therefore, two class-modeling techniques such as Soft Independent Modelling of Class Analogy (SIMCA) and UNEQual dispersed classes (UNEQ) and three discriminant techniques, such as k - Nearest Neighbor (k-NN), Linear Discriminant Analysis (LDA), Partial Least Squares - Discriminant Analysis (PLS-DA), were used and the performances of the resulting models were compared. The best average recognition and cross-validation prediction abilities, 100% and 93.7% respectively, were obtained by the LDA model, performed on a set of 20 principal components previously selected by a stepwise decorrelation procedure. The other models, except the SIMCA one, also showed good performances (above 90%). All tested statistical models were validated by evaluating the prediction abilities on an external set of lentil samples. LDA model showed the best results with an external prediction ability of 100%, but also the other models showed remarkable performances (above or near 90%).These findings demonstrated the suitability of the methods developed to discriminate geographical origin of lentils and confirmed the applicability of the NMR data, in combination with chemometrics, to solve geographic origin issues of foodstuffs.

An untargeted method using headspace solid-phase microextraction coupled to electronic nose based on mass spectrometry (HS-SPME/MS-eNose) in combination with chemometrics was developed for the discrimination of oranges of three geographical origins (Italy, South Africa and Spain). Three multivariate statistical models, i.e. PCA/LDA, SELECT/LDA and PLS-DA, were built and relevant performances were compared. Among the tested models, SELECT/LDA provided the highest prediction abilities in cross-validation and external validation with mean values of 97.8% and 95.7%, respectively. Moreover, HS-SPME/GC-MS analysis was used to identify potential markers to distinguish the geographical origin of oranges. Although 28 out of 65 identified VOCs showed a different content in samples belonging to different classes, a pattern of analytes able to discriminate simultaneously samples of three origins was not found. These results indicate that the proposed MS-eNose method in combination with multivariate statistical analysis provided an effective and rapid tool for authentication of the orange's geographical origin.

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.

The effect of processing on mycotoxin content in milling fractions has been investigated in 10 samples of durum wheat contaminated with T-2 and HT-2 toxins at levels ranging from 97 to 5,954 ?g/kg (sum of T-2 and HT-2 toxins). Either naturally contaminated samples or samples artificially inoculated with Fusarium sporotrichioides under field conditions were used. A method based on liquid chromatography-tandem mass spectrometry coupled with immunoaffinity column cleanup was validated in-house for the simultaneous analysis of both toxins in a variety of matrices, including uncleaned wheat, cleaned wheat, screenings, bran, red dog, fine middlings, and semolina. Mean recoveries from samples spiked with T-2 and HT-2 toxins at levels of 100 ?g/kg ranged from 85 to 107%, with relative standard deviations (RSDs) lower than 14%. The milling process led to an increase of T-2 and HT-2 toxin contents up to 13-and 5-fold in screenings and bran, respectively, compared with occurrence in the uncleaned wheat; however, an overall reduction of T-2 and HT-2 toxins by 54% (RSD, 20%) and 89% (RSD, 3%) was observed in cleaned wheat and in semolina, respectively. Copyright © International Association for Food Protection.

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.

Grains and grain-based products provide the largest contribution to the sum of HT-2 and T-2 toxin exposure for humans and their monitoring is of high priority. The European Commission has recently published a recommendation on the presence of T-2 and HT-2 toxins in cereals and cereal products. An indicative level of 50 ?g/kg has been fixed for the sum of T-2 and HT-2 toxins in durum wheat (Triticum turgidum ssp. durum Desf.) and other cereals for direct human consumption. Wheat debranning, a process by which outer kernel layers are removed through both friction and abrasive scouring actions, positively affects several quality features of semolina such as the improvement of milling yield and the reduction of heat damage. Moreover, the same process provides a powerful tool to improve hygienic-sanitary quality of derived products. Aim of the present study was to evaluate the effectiveness of debranning process to reduce the levels of T-2 and HT-2 toxins in treated kernels and milling fractions. A durum wheat sample contaminated with T-2 and HT-2 toxins at level of 122 ?g/kg (sum of T-2 and HT-2 toxins) was subjected to two consecutive debranning steps at laboratory scale, each one of the duration of 30s. Debranned and not-debranned kernels were processed by a pilot milling plant. All samples, including debranned kernels, debranning wastes and milling fractions (semolina, coarse bran, fine bran and fine middlings) were analysed for the sum of T-2 and HT-2 toxins by enzyme-linked immunoassay (Veratox® for T-2/HT-2, Neogen) and by LC-MS/MS after immunoaffinity column clean up. Results showed that the debranning process significantly reduced levels of T-2 and HT-2 toxins in the kernels with a reduction rate of 72% after the first 30s step. No significant toxin reduction was observed after the second 30s debranning step. High mycotoxin levels were found in the debranning wastes. The semolina fractions, after 0.4 mm sieving, obtained from both debranned and unprocessed kernels showed not significant differences in toxin content, suggesting an accumulation of these toxins in the most peripheral layers. Interestingly, coarse bran obtained from debranned kernels at first step (30s) resulted to be less contaminated (201 µg/kg) than that obtained from unprocessed kernels (433 µg/kg). Preliminary results obtained in the present study indicate that the debranning process is useful to reduce T-2 and HT-2 toxin contamination present on the surface of the wheat kernel and could play an important role to obtain whole-wheat products or less refined products with high nutritional value and hygienic-sanitary quality.

The effect of triazole-based treatments on Fusarium head blight (FHB), grain yields and the accumulation of deoxynivalenol (DON) in harvested wheat kernels was evaluated by means of twenty multi-site field experiments performed during five consecutive growing seasons (from 2004-2005 to 2008-2009) in Italy. Fungicide treatments were carried out on different cultivars of common wheat (cv. Serio, Blasco, Genio and Savio) and durum wheat (cv. Orobel, Saragolla, San Carlo, Levante, Duilio, Karur and Derrik) after artificial inoculation with a mixture of toxigenic Fusarium graminearum and F. culmorum strains. The application of fungicides containing prothioconazole (Proline® or Prosaro®) at the beginning of anthesis (BBCH 61) resulted in a consistent reduction of FHB disease severity (by between 39 and 93%) and DON levels in wheat kernels (by between 40 and 91%) and increased wheat yields (from 0.4 to 5.6 t ha-1, average 2.2 t ha-1), as compared to the untreated/inoculated control. Fungicides containing tebuconazole (Folicur® SE) and cyproconazole plus prochloraz (Tiptor® Xcell) showed a reduced effectiveness compared with prothioconazole-based treatments. All fungicide treatments were more effective in reducing DON and increasing grain yields of common wheat than durum wheat. Results showed that the application of fungicides containing prothioconazole at the beginning of anthesis provided a strong reduction of FHB disease, allowing both an increase in grain yields and a considerable reduction of DON content in wheat kernels.

The efficacy of agrochemical treatments, based on three different fungicides combined with an insecticide,was tested in southern Italy for two years on three maize hybrids to control Fusarium ear rotof maize and the accumulation in the maize kernels of the carcinogenic mycotoxins fumonisins. Insectdamage incidence and severity, disease incidence and severity, identification of Fusarium species andlevels of fumonisin contamination in kernels were determined. Field trials showed in both years thatnatural colonization of maize kernels by the fumonisin producing species Fusarium proliferatum and F.verticillioides (up to 81.5 and 26.5%, respectively) and total fumonisin contamination (up to 68.2gg-1)were highly severe. For all hybrids and in both years, the treatment with the insecticide applied alonereduced the insect damage severity consistently and the content of fumonisins in the kernel only in halfof the cases, whereas fungicide treatments applied in combination with the insecticide showed a furthersignificant reduction of fumonisin contamination in the three hybrids and in both years.

L'interesse per l'ozono quale agente sanitizzante nell'industria alimentare è aumentato negli ultimi anni in risposta ad una sempre crescente richiesta di una 'chimica verde'. L'ozono è infatti un composto rispettoso dell'ambiente in quanto si decompone rapidamente in ossigeno e non lascia residui negli alimenti. Tale gas è considerato un additivo alimentare GRAS (Generally Recognised As Safe) ed il suo utilizzo come additivo antimicrobico per il contatto diretto con gli alimenti è stato recentemente approvato dalla Food and Drug Administration (FDA). Recenti studi hanno mostrato come l'ozono sia efficace nel controllo di insetti, batteri e funghi e nel degradare pesticidi e micotossine che possono contaminare i cereali. Scopo del presente studio è stato quello di valutare l'effetto dei trattamenti con ozono gassoso a diverse concentrazioni (9,0-15,4-26,1 g/m3) e tempi di contatto (2-8-12-24 ore) sulla contaminazione da funghi filamentosi, lieviti e micotossine (in particolare deossinivalenolo e tossine T-2 e HT-2) in campioni di frumento duro utilizzando un prototipo di generatore di ozono progettato ad hoc per il trattamento delle cariossidi. E' stato inoltre valutato l'effetto dei trattamenti su alcuni parametri di qualità del frumento, in particolare sul contenuto in ceneri, proteine, amido, fibra, glutine e indice di giallo. I trattamenti con ozono alle concentrazioni di 9,0 e 15,4 g/m3 non hanno evidenziato effetti significativi sulla contaminazione da funghi filamentosi e lieviti per tutti i tempi di contatto, rispetto al controllo non trattato. In tali condizioni operative è stata osservata una riduzione del contenuto di DON (fino al 19%), rispetto al controllo non trattato, già a partire dalle 8 ore di contatto. I trattamenti con ozono a concentrazioni maggiori (26,1 g/m3) hanno determinato sia una riduzione significativa della carica microbica già a partire dalle 2 ore, sia una maggiore riduzione del contenuto di DON, fino al 32%, a partire dalle 12 ore di trattamento. Non è stata invece osservata alcuna variazione significativa del contenuto di tossine T-2 e HT-2 per tutti i trattamenti. Nelle diverse condizioni sperimentali non sono state osservate variazioni significative dei parametri qualitativi del frumento.

I cereali e i prodotti a base di cereali costituiscono la maggiore fonte di esposizione dell'uomo alle tossine T-2 e HT-2, metaboliti tossici prodotti da funghi del genere Fusarium. Lo scopo del presente lavoro è stato quello di valutare l'effetto del processo di decorticazione sul contenuto delle tossine T-2 e HT-2 nella granella e nelle frazioni di macinazione del frumento duro. Le analisichimiche hanno evidenziato un'efficace azione del processo di decorticazione nel ridurre i livelli di tossine T-2 e HT-2 nella granella e nelle principali frazioni di scarto di macinazione ad elevato valore nutritivo (crusca e farinetta), suggerendo una distribuzione delle due micotossine limitata ai tegumenti esterni delle cariossidi. I livelli di contaminazione nelle semole setacciate ottenute sia dai campioni decorticati, sia tal quali, sono risultati trascurabili.

La contaminazione da micotossine rappresenta una delle maggiori problematiche nella gestione del rischiodella sicurezza e della salubrità dei prodotti alimentari. I cereali -come frumento, orzo e mais -e i prodotti derivati, risultano frequentemente contaminati dai tricoteceni quali deossinivalenolo (DON) e dalle tossine T-2 e HT-2 come risultato dell'accrescimento di specie fungine Fusarium su piante o granaglie. È stato ampiamente dimostrato che il processo di trasformazione degli alimenti può notevolmente influire sulla stabilità delle micotossine, alterandone i contenuti nei prodotti finali. Nel presente lavoro è stato valutato l'effetto della panificazione sulla stabilità dei tricoteceni DON, T-2 e HT-2 in panetti prodotti a partire da farine naturalmentecontaminate dalle tre micotossine. Insieme alle forme native, sono state analizzate anche alcune forme glicosilate ovvero DON-3 Glucoside, T-2 Glucoside e HT-2 Glucoside che, al pari delle micotossine native,potrebbero costituire un rischio per la salute umana. I risultati mostrano che i livelli finali di DON aumentanoin seguito alla panificazione, mentre quelli del DON-3 Glucoside appaiono dimezzati. Per le tossine T-2e HT-2 si osserva in generale una variabile suscettibilità alla cottura.

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, T-2 and HT-2 toxins are mycotoxins frequently occurring in cereals and cereal-based products along with their conjugated forms. In this paper, we provide insights into the fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside derivatives during bread making, using naturally contaminated wheat flour. High-resolution mass spectrometry was used to assess the extent of degradation of the three mycotoxins during bread baking and to identify some glucoside conjugates, namely deoxynivalenol, T-2 and HT-2 mono-glucosides, detected both in the flour and in the respective breads. Our findings show deoxynivalenol's levels markedly increased upon baking, whereas those of HT-2 and T-2 toxins were decreased in the final bread with special regard to the T-2 toxin.

Mycotoxins are naturally occurring toxic metabolites produced by filamentous fungi under a wide range of climatic conditions on different agricultural crops during growth, drying and subsequent storage. Monitoring, control, risk assessment and prevention of mycotoxins in foods are important issues worldwide associated with public health, agricultural production, food processing and trade. For these reasons the European Commission has set recommended levels or maximum permitted levels for mycotoxins of major concern in a wide range of foodstuffs.Analytical methods for the determination of mycotoxins in foods are commonly based on chromatographic techniques (GC, HPLC or LC-MS). Although these methods permit a sensitive and accurate determination of the analyte, they require skilled personnel and are time-consuming, expensive, and unsuitable for screening purposes. Simple, rapid, and more effective screening methods for mycotoxins determination are highly demanded.Fluorescence polarization immunoassay (FPIA) is a homogenous assay that measures competition between a fluorescently labelled antigen (tracer) and unlabelled antigen in solution for binding a specific antibody. The FP signal is inversely related to the antigen content that competes with the tracer, and it increases when the binding of specific antibody to the tracer increases. Unlike most immunoassays (e.g., ELISA), the main advantage of this format is that additional manipulation steps, as multiple washing steps or separation of free from antibody-bound analyte, are not necessary. The selection of the appropriate antibody-tracer combination determines the speed, accuracy, precision and sensitivity of a FPIA. Incubation times, cross-reactivity, compatibility with organic solvents and matrix effects are analytical parameters to be evaluated and optimized in the development of a FPIA. We have recently developed several FPIAs for the determination of mycotoxins in cereals and processed products, including deoxynivalenol in wheat and derived products, ochratoxin A in wheat, T-2 and HT-2 toxins in wheat, barley, oats and oatflakes [1-3]. An accurate validation of these assays has been performed on each tested matrix using either artificially and naturally contaminated samples, and reference materials. These FPIAs are rapid, easy-to-use, readily automated, and suitable for high-throughput screening as well as for the quantitative determination of mycotoxins in foodstuffs at levels below regulatory levels.

T-2 toxin (T-2) and HT-2 toxin (HT-2) are type A trichothecene mycotoxins produced by several Fusarium species, mainly Fusarium sporotrichioides, Fusarium langsethiae and Fusarium poae. Generally, these Fusarium species can grow on cereals and produce T-2 and HT-2 under moist cool conditions already prior to harvesting [1]. Among cereals, oats, wheat, rye and derived products are key dietary sources of T-2 and HT-2 exposure [1]. Fluorescence polarization (FP) immunoassay is a homogeneous technique that is getting attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. A rapid, sensitive and reliable FP immunoassay has been recently reported for the determination of the sum of T-2 and HT- 2 toxins in wheat [2]. The aim of present work was to evaluate the applicability of the above-mentioned FP immunoassay to other unprocessed cereals, such as rye, and cereal-based products, such as oats crispbread, for the quantitative determination of the total content of T-2 and HT-2. The concept of determining the total content of T-2 and HT-2 in cereal samples for both official control purposes and risk assessment studies results in line with the EC Recommendation [3]. No purification step of extracts was required, although in order to reduce the matrix effect a dilution step with NaCl solution (4 % for rye and 1% for oats crispbread), in a ratio 1:5 (v/v), was necessary to let precipitation of proteins and matrix interfering compounds. For the optimized FP immunoassay, LOD of 0.20 ng/mL (equivalent to 20 ?g/kg in rye and oats crispbread) was calculated. Overall mean recoveries of the optimized FP immunoassay were 105 and 107% for rye and oats crispbread, respectively, with relative standard deviations lower than 4%. The analytical performances of the optimized FP immunoassay in terms of accuracy and precision fulfill the criteria established by the European Commission [4]. In addition, a comparative analysis of the levels of contamination in spiked and blank samples was performed by both FP immunoassay and UHPLC method. In particular, a total of 30 rye and oats crispbread samples, of which 20 spiked samples at levels from 50 to 700 ?g/kg and 10 uncontaminated samples, were analyzed. The proposed method coupled performances in terms of sensitivity, accuracy and precision comparable to those of a chromatographic technique with rapidity (20 min), costs and simplicity typical of a high-throughput screening method and can be used as a valid alternative to more expensive and time-consuming LC methods for quantitative determination in rye and oats crispbread [5].ACKNOWLEDGMENTSThis work has been supported by the Italian Ministry of Education, University and Research (MIUR) project no. CTN01_00230 CL.A.N. Cluster Tecnologici Nazionali - SAFE&SMART project "New enabling technologies for food safety and food chain integrity within a global scenario". REFERENCES[1] EFSA Panel on Contaminants in the Food Chai

A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC50 value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 mu g/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 mu g/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r = 0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable quantitative determination of OTA in wheat at level far below the EU regulatory limits.

Fusarium toxins, a group of mycotoxins, can be produced by Fusarium fungi under temperate climatic conditions on agricultural commodities, mainly cereals, in field as well as during storage. As a defensive response of the host plant, Fusarium toxins can be metabolized by forming modified mycotoxins, often called "masked" mycotoxins. It has been shown that many modified forms are hydrolysed into the parent mycotoxin during digestion. In order to protect consumer health from the risk of exposure to modified and parent forms of Fusarium toxins, the development of rapid, sensitive and reliable methods for their simultaneous determination in cereals is highly demanded. Currently, fluorescence polarization immunoassay (FPIA) is getting the attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. The focus of our work is to develop and validate quantitative FPIAs for simultaneous determination of DON and its acetylated (3-acetyl-DON, 15-acetyl-DON) and glycosylated forms (DON-3-glucoside) and T-2/HT-2 toxins and their glycosylated forms (T2-glucoside, HT2-glucoside) in wheat. A fluorescein-label (tracer) of DON (DON-FL) and four T2- and HT2-fluerescein tracers (T2-FL, HT2-FL1a, HT2-FL1b and HT2-FL2) were synthesized and purified. The assessment of the antibody-tracer binding was performed using four DON monoclonal antibodies (MAbs), and ten T2-glucoside MAbs, one HT-2 MAb and two T-2 MAbs, at different concentrations. Concerning the FPIA for the determination of DON, its acetylated and glycosylated forms, the highest antibody-tracer binding was observed for the clone 22/DON-FL combination, while in the FPIA for the determination of T-2/HT-2 toxins and their glycosylated forms, the highest bindings were observed for thirteen T2-glucoside MAbs with T2-FL and HT2-FL1b combinations, as well as for HT-2 MAb/HT2-FL1a combination and for T-2 MAb/T2-FL and HT2-FL1a combinations. Competitive FPIAs were performed with the selected antibody combinations. In particular, FPIA for the determination of DON, its acetylated and glycosylated forms showed IC50=23.5 ng/mL for DON and exhibited 204%, 45% and 11% as cross-reactivity, respectively, for 3-acetyl-DON, DON-3-glucoside and 15-acetyl-DON. While, among the selected combinations for T-2 and HT-2, the HT-2/HT2-FL1a combination exhibited 80% as cross-reactivity for T-2 and its glycosylated form and the highest sensitivity, with IC50 = 2.0, 2.6 and 2.7 ng/mL for HT2, T-2 and T2-glucoside, respectively. These findings showed the applicability of the developed FPIAs to the determination of parent and modified mycotoxins, expressed as sum, in solution. This work was supported by the MYCOKEY project which has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 678781.

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.

Trichothecene mycotoxins occur in cereal grains and are produced by different Fusarium species, which may develop in the field, principally in wheat, maize, barley and oats. Trichothecene glucosylation can be included among naturally occurring mechanisms in cereals that lead to a reduction of native mycotoxin accumulation in grains. Although toxicological data are scarce, these modified forms may represent a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing [1]. With respect to type-A trichothecenes, the recently issued Recommendation No. 165/2013/EC of the European Commission, setting indicative maximum permitted levels for T-2 toxin (T-2) and HT-2 toxin (HT-2), also takes into account the increasing knowledge made available on T-2/HT-2 glucosides stating that 'attention should be paid to the occurrence of masked forms, in particular their glycosylated-conjugates'.The increasing instrumental availability and technological advances in LC-MS, often allowing compound identification without the need for reference standards, have had a strong impact on the know how about formation and characterization of modified mycotoxins. Detection of known or "novel" trichothecene-related compounds in fungal cultures, plant material or processed foods can provide insights in detoxification and metabolisms of trichothecenes. Furthermore, the direct determination of free and conjugated forms might enable a more accurate evaluation of the risk of exposure.Recent advances in applications of LC-MS techniques for chemical characterization and quantitative determination of type-A trichothecene glucosyl derivatives will be reviewed and discussed. High-resolution mass spectrometry (HRMS), based on Orbitrap technology, has been applied to investigate on the presence of T-2 and HT-2 glucoside derivatives in naturally contaminated cereals (including wheat, oats and barley) and Fusarium fungal cultures. Molecular structure details obtained by measuring exact masses of main characteristic fragments, with high mass accuracy led to the identification of a monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin [2].Two mono-glucosyl derivatives of neosolaniol (NEO) and one mono-glucoside derivative of diacetoxyscirpenol (DAS) have been identified and characterized by liquid chromatography tandem mass spectrometry (LC-MS/MS). These compounds were detected either in fungal cultures or in cereal samples naturally contaminated with the parent toxins [3]. Furthermore, a preliminary screening for the presence of these modified forms of mycotoxins, carried out on naturally contaminated cereal samples (i.e. wheat, oats and barley) showed a widespread occurrence of type-A trich

The presence of glucoside derivatives of T-2 and HT-2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high-resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T-2 and HT-2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk.

Fra i cereali maggiormente soggetti a contaminazione da tossine T-2 e HT-2 ritroviamo avena, frumento, orzo e segale. A causa della limitate informazioni disponibili in merito alla presenza delle due tossine in cereali e prodotti derivati in Europa, recentemente la Commissione Europea ha evidenziato la necessità di raccogliere dati supplementari sull'incidenza della contaminazione per una completa valutazione del rischio, ai fini della salvaguardia della salute umana ed animale. Per tale ragione, assume notevole rilevanza la necessità di sviluppare nuovi metodi rapidi, sensibili ed accurati per la loro determinazione sia in cereali non processati che nei relativi prodotti finiti. Nell'ambito di tali metodi risulta crescente l'attenzione verso gli immunosaggi in fase omogenea basati sulla Polarizzazione di Fluorescenza (FP), vista la loro semplicità di utilizzo, rapidità, affidabilità ed economicità. A tal proposito, sono stati sviluppati e validati immunosaggi FP per la determinazione rapida e quantitativa di tossine T-2 e HT-2 (espressa come somma delle due tossine) in avena, segale, orzo, pasta, fiocchi di avena e prodotti da forno a base di avena (oats crispbread). I campioni sono estratti con una miscela metanolo/acqua (90:10, v/v), successivamente filtrati, diluiti con una soluzione acquosa di cloruro di sodio ed infine analizzati mediante l'immunosaggio FP. I valori dei recuperi medi ottenuti per tutte le matrici analizzate rientrano nell'intervallo tra 101% e 107% e presentano deviazioni standard relative inferiori al 6%. I limiti di determinazione (LODs) degli immunosaggi FP sviluppati risultano pari a 70 µg/kg per avena, 40 µg/kg per fiocchi di avena e orzo, 25 µg/kg per pasta e 20 µg/kg per segale e prodotti da forno a base di avena. Infine, è stata osservata una buona correlazione (r > 0,953) tra il contenuto delle tossine T-2 e HT-2 in campioni di avena, orzo, fiocchi di avena e pasta naturalmente ed artificialmente contaminati ottenuto mediante gli immunosaggi FP sviluppati ed il metodo UHPLC con purificazione con colonnine ad immunoaffinità (metodo di riferimento). In conclusione, tali risultati combinati con la rapidità (10 - 15 minuti) e semplicità del protocollo messo a punto, dimostrano che i saggi sviluppati possono essere applicati sia per uno screening rapido, sia per una determinazione quantitativa delle tossine T- 2 e HT-2 in campioni di avena, orzo, segale e prodotti a base di cereali.

A series of field experiments has been conducted in North West Italy over a period of 3 years to evaluate the effect of fungicide applications on common wheat yield and safety, combined with different agronomic conditions (high: a susceptible variety to FHB planted in an untilled field; low: a medium tolerant variety to FHB planted in a ploughed field) for Fusarium head blight (FHB) infection risk. A azole mixture (prochloraz + epoxiconazole), applied at heading, was compared in each year and for each agronomic condition with a triazole with high activity against F. graminearum and F. culmorum (metconazole), a strobilurin-azole mixture with elevated action to control leaf diseases and delay leaf senescence (azoxystrobin + prochloraz) and an untreated control. The following parameters were analyzed: flag leaf greenness, grain yield, test weight, thousand kernel weight (TKW), FHB incidence and severity and deoxynivalenol (DON) contamination. The results of this research, conducted over three years with different meteorological conditions, underline the important link between fungicide efficacy and environmental and agronomic conditions that can influence fungal disease pressure. The fungicide effect on the control of FHB and the increase in flag leaf longevity and grain yield were greater with an increase in the disease pressure. On the other hand, the DON contamination was reduced by the fungicide to a greater extent in the low risk agronomic and environmental conditions compared to the high risk ones. Prochloraz + epoxiconazole showed a lower efficacy in reducing DON contamination compared to metconazole, particularly in the higher FHB pressure conditions. No significant differences were observed between the azole mixture and the strobilurin-azole mixture for flag leaf greenness, grain yield, test weight or TKW. This study provides useful information that can be used to evaluate, in naturally-infected field conditions, the effect of a fungicide application at heading on wheat yield and safety in distinct scenarios for FHB and foliar disease pressure. © M. Blandino et al., 2011 Licensee PAGEPress, Italy.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low g/kg range.

The management of good agricultural practices during pre-harvest is a key issue for minimising the risk of Fusarium mycotoxin accumulation in the crops. Such practices involve crop rotation, tillage, appropriate fertilisation and fungicide application, use of biological control agents, variety selection, timely planting and harvests as well as control of insects, which frequently facilitate Fusarium species infections. On the other hand, the reduction of Fusarium mycotoxins along the malting and brewing chain is also highly dependent on a correct post-harvest and malting management that must aim firstly at the separation of the infected crop products from the healthy material. Therefore, the use of different tools such as manual sorting or optical sensors is also a crucial aspect for reducing the level of mycotoxin contamination. Moreover, it is extremely important to prevent post-harvest contamination and develop practical and effective post-harvest procedures for mycotoxin reduction in the food supply chains and to provide alternative and safe use options for contaminated batches. An updated review will be given on integrated management of pre-and post-harvest practices aiming at minimising the risk of mycotoxin contamination along the malting and brewing chain and main effective solutions, including the development of a MycoKey app, proposed and to be achieved within the EU project MycoKey (http://www.mycokey.eu/) .

Fusarium head blight (FHB) disease and deoxynivalenol (DON) contamination of wheat grains depend on multiple factors, above all climatic conditions, but also agronomic factors such as crop rotation, debris management, variety susceptibility and fungicide applications. Although it is generally believed that multiple strategies are more successful than a single strategy, only a few studies have shown the quantitative effect of combining multiple strategies.Field experiments have been conducted over three growing seasons in three sites in Northern Italy to evaluate the effect of previous crop residue management through tillage, variety susceptibility and triazole fungicide application on common wheat, according to a full factorial scheme. The following parameters were analyzed: FHB severity, grain yield and DON contamination.The collected data have clearly shown a close interaction between the factors involved in FHB severity and DON content, while the interactions were less significant for grain yield. In all nine trials, the DON contamination was significantly affected by the interaction of at least two of the compared factors, while the interaction between all three factors involved was significant in four trials. The most favourable scenario to avoid DON contamination (ploughing, moderately resistant variety, triazole application at heading) reduced the DON content by 97% compared to the worst one (direct sowing, susceptible variety, no fungicide application).Since the interaction between the agricultural practices have shown a synergistic effect, integrated multiple strategies, in areas characterized by a high risk of FHB, can be considered the very effective management means of reducing FHB and DON contamination in wheat.

T-2 and HT-2 toxins are mycotoxins produced by several Fusarium species that are commonly found in various cereal grains, including oats, barley, wheat and maize. Intake estimates indicate that the presence of these mycotoxins in the diet can be of concern for public health. In this work, the inclusion processes occurring between fluorescent anthracene-derivatives of T-2 and HT-2 toxinsand different cyclodextrin (CD) molecules were investigated in aqueous solutions by means of UV-Vis absorption, fluorescence emission and dynamic light scattering. Binding constant values and chemico-physical parameters were calculated. It was found that b-CDs give stronger inclusion reactions with both T-2 and HT-2 derivatives, as stated by important emission intensity increments. Suchinteractions were found to be fundamentally enthalpy-driven. Among b-CDs, the effect of the methylation at hydroxyl groups was tested: as a result, the di-methyl form of b-CD was found to induce the best fluorescence intensity enhancements.

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.

In the present investigation, an LC-MS method for sensitive multiplex detection of five allergenic ingredients in a processed food matrix is presented. Cookie was chosen as complex food model and was incurred with egg, milk, soy, hazelnuts and peanuts before baking. Extraction, purification and pre-concentration protocols were applied to ground cookie basing on protocols described elsewhere. Specific instrumental features of a dual cell linear ion trap MS instrument were exploited to identify suitable peptide markers for each allergen and to deliver a sensitive multiplex SRM-based method for the simultaneous detection of common allergenic ingredients which might contaminate such a commodity.

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.

Fusarium head blight (FHB) is one of the major diseases of cereals mainly caused by phytopatogenic Fusarium species. Most of the Fusarium species associated with FHB, under favourable environmental conditions, can produce various toxic secondary metabolites (mycotoxins) that can contaminate grains. Mycotoxin contamination of crops may cause economic losses at all levels of food and feed production, therefore, mycotoxin prevention and control is a global concern and contaminated commodities impair trade and threaten consumer safety. Deoxynivalenol (DON) is a type B trichothecene mycotoxin mainly produced by F. graminearum and F. culmorum and is a common natural contaminant of wheat. T-2 and HT-2 toxins are type A trichothecenes mainly produced by F. sporotrichiodes, F. langsethiae and F. poae that, in favorable environmental conditions, may grow on a variety of cereals, including wheat, oats, barley and maize. Analytical methods for the determination of DON, T-2 and HT-2 toxins in cereals are commonly based on chromatographic techniques. Although these analytical methods permit sensitive and accurate determination of the toxins, they require a preliminary extract clean-up and are time-consuming, expensive, and unsuitable for screening purposes. The aim of this study, carried out within the AGER project "From Seed to Pasta", was to develop rapid and reliable methods for the determination of DON, T-2 and HT-2 toxins in durum wheat.Fourier-Transform-Near Infrared (FT-NIR) spectroscopy has been used to develop quantitative and classification models for DON prediction in durum wheat samples in the range of <50-16000 ?g/kg. Partial least-squares (PLS) regression analysis displayed a large root mean square error of prediction value (1977 ?g/kg) as compared to the EU maximum limit for DON in unprocessed durum wheat, thus making the PLS approach unsuitable for quantitative predictionof DON in durum wheat. On the other hand, Linear Discriminant Analysis (LDA) was successfully used to classify contaminated wheat samples based on their DON content with an overall classification and false compliant rates of 90% and 5%, respectively, when a cut-off of 1400 ?g/kg was used. This result fulfils the requirement of the European official guidelines for screening methods, and confirms the suitability of FT-NIR to screen a large number of wheat samples for DON contamination.A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. A limit of detection of 8 ?g/kg for the combined toxins was determined.Analytical performances in terms of accuracy (96%) and precision (8%) values of FP immunoassay fulfill the EU criteria established for acceptance of an analytical method for the determination of T-2 and HT-2 toxins. Comparative analyses of 45 naturally contaminated and spiked durum wheat samples by both the FP immunoassay and high-performance liquid chro

Fusarium langsethiae, formally described as a new species over a decade ago, has been identified as the main producer of HT-2(HT2) and T-2 (T2) toxins in Europe in small cereal grains. Mycotoxin contamination caused by this Fusarium species can representa food safety hazard that deserves further attention. In the present work, the mycotoxin profile in wheat cultures of F.langsethiae is presented with particular reference to the production of major type-A trichothecenes and their glucosyl derivatives.F. langsethiae isolates, representative of the major Italian wheat cultivation areas, were tested for the production of T2,HT2, diacetoxyscirpenol (DAS) and neosolaniol (NEO), and relevant glucosyl derivatives. Liquid chromatography-tandem massspectrometry (LC-MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiaeisolates under investigation resulted to be potent producers of T2, HT2 and NEO. Furthermore, a well-defined set of isolates,all originating from Central Italy, produced also DAS. All isolates were found to be able to produce HT2 glucosyl derivatives,whereas only traces of T2 glucoside were detected in one sample. Furthermore, two mono-glucosyl derivatives of NEO and onemono-glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylatedtrichothecenes in analyzed fungal extracts revealed a general co-occurrence of these derivatives with the parent toxin at levelsthat could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the productionof glucosylated trichothecenes by F. langsethiae cultured on small grains.

Grain cleaning is the most effective post-harvest measure to reduce high levels of mycotoxins due to the efficient removal of mould-infected grains and contaminated grain fractions on the basis of features such as size, density and optical properties. Several studies were performed during the last years to investigate the reduction of e.g. deoxynivalenol in wheat, ergot in rye, and total aflatoxins in peanuts. In this study, the reduction of total aflatoxins (AFB1, AFB2, AFG1, AFG2) in naturally contaminated maize was tested in three cleaning steps using industrial-scale cleaning Bühler machines. The first step included (i) mechanical size separation and dust removal by aspiration with the "Grain Plus", the second (ii) separation based on density differences with a "Concentrator", and the third (iii) optical sorting with "SORTEX". Four batches of maize (about 3 tons each) with different levels of aflatoxin contamination were used for the trials. Furthermore, different process settings and cleaning intensities were tested. Sampling (3 replicates/batch) was performed according to the Commission Regulation N. 401/2006 and the collected samples were analyzed by HPLC/FLD with photochemical derivatization. In addition, the incoming material was analyzed by the Eurofins' rapidust® system for on-site sampling and analysis of mycotoxins in grains. Firstly, trials once again highlighted the difficulties of sampling for aflatoxins. Samples showed a large variability with respect to aflatoxin level. However, high levels of aflatoxin contamination were observed in the removed product streams, with values up to 250 µg/kg. Consistent results were achieved by calculating the aflatoxin level of the incoming material from the removed products taking into account the mass balance. These values compared well with the analyzed levels from the rapidust® system. Aflatoxin levels were reduced from about 10 and 20 µg/kg to 3-4 µg/kg and 2-3 µg/kg for the low and high contaminated material, respectively. In conclusion, the combination of the tested cleaning machines could allow a total aflatoxin removal of 60-90% in maize.

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.

As determined by the Intergovernmental Panel on Climate Change, warming of the climate system is unequivocal and has been associated with rising sea levels, diminished amounts of ice and snow and increasing oceanic and atmospheric temperatures. Such climate changes have a significant impact on stages and rates of toxigenic fungi development and can modify host-resistance and host-pathogen interactions, influencing deeply also the conditions for mycotoxin production that vary for each individual pathogen. Moreover, the new combinations mycotoxins/host plants/geographical areas are arising to the attention of the scientific community and require new diagnostic tools and deeper knowledge of both biology and genetics of toxigenic fungi. In this review, it is underlined that an extension of the aflatoxin contamination risk in maize in South and Central-Europe is highly likely in the next 30 years, due to favorable climatic conditions to the growth of Aspergillus flavus. Moreover, the mycotoxigenic Fusarium species profile on wheat in Europe is in continuous change in Northern, Central and Southern-Europe with, in particular, a worrisome growing contamination of F. graminearum in the Central and Northern Europe.

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.

T-2 and HT-2 toxins are two of the most toxic members of type-A trichothecenes, produced by a number of Fusarium species. The occurrence of these mycotoxins was studied in barley samples during a survey carried out in the 2011-2014 growing seasons in climatically different regions in Italy. The percentage of samples found positive ranges from 22% to 53%, with values included between 26 and 787 µg/kg. The percentage of samples with a T-2 and HT-2 content above the EU indicative levels for barley of 200 µg/kg ranges from 2% to 19.6% in the 2011-2014 period. The fungal species responsible for the production of these toxins in 100% of positive samples has been identified as Fusarium langsethiae, a well-known producer of T-2 and HT-2 toxins. A positive correlation between the amount of F. langsethiae DNA and of the sum of T-2 and HT-2 toxins was found. This is the first report on the occurrence of F. langsethiae--and of its toxic metabolites T-2 and HT-2--in malting barley grown in Italy.

Surveys on the occurrence of type A trichothecenes in wheat, and particularly for the T-2 and HT-2 toxins, and information on the biology and epidemiology of the causative Fusarium species (i.e. F. langsethiae, F. sporotrichioides) are scarce in Italy, as compared to the more common type B trichothecene, deoxynivalenol and its producers. This 4-year monitoring of phytopathogenic Fusarium species on 183 seed lots of durum wheat shows wide distribution of F. langsethiae in Italy and the potential of several isolates of this fungus to produce high amounts of T-2 and HT-2 in wheat. Fusarium langsethiae was observed for approximately 48% of the analysed samples, with a maximum incidence for a single lot of 10.5%. Fusarium sporotrichioides was observed only in 2011, with an average incidence of 2% (range, 0-3%). A collection of F. langsethiae isolates representative of the main cultivation areas in Italy was established. These isolates showed great variability for their toxin production in vitro. Of 28 strains, all except one isolate can produce the T-2 and HT-2 toxins. HT-2 was generally in greater amounts than T-2, with an average concentration ratio for HT-2 to T-2 of 2.1 (range, 0.7-5.4). The artificial inoculation of wheat with three isolates of F. langsethiae produced no Fusarium head blight symptoms under field conditions. However, significantly higher incidence of F. langsethiae was seen on the kernels of inoculated plants, compared to the uninoculated controls.

A fluorescence polarization (FP) immunoassay has been optimized and validated for rapid quantification of T-2 and HT-2 toxins in both unprocessed cereals, including oats, barley and rye, and cereal-based products for direct human consumption, such as oat flakes, oats crispbread and pasta. Samples were extracted with 90 % methanol, and the extract was filtered and diluted with water or sodium chloride solution prior to the FP immunoassay. Overall mean recoveries from spiked oats, rye, barley, oat flakes, oats crispbread and pasta ranged from 101 to 107 %, with relative standard deviations lower than 7 %. Limits of detection (LODs) of the FP immunoassay were 70 mu g/kg for oats, 40 mu g/kg for oat flakes and barley, 25 mu g/kg for pasta and 20 mu g/kg for rye and oats crispbread. The trueness of the immunoassay was assessed by using two oat and oat flake reference materials for T-2 and HT-2 toxins, showing good accuracy and precision. Good correlations (r > 0.953) were observed between T-2 and HT-2 toxin contents in naturally and artificially contaminated samples determined by both FP immunoassay and ultra-high-performance liquid chromatography (UHPLC) with immunoaffinity column cleanup used as reference method. These results, combined with rapidity and simplicity of the assay, show that the optimized assay is suitable for high-throughput screening, as well as for reliable quantitative determination of T-2 and HT-2 toxins in cereals and cereal-based products.

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.

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.

Rapid test methods for measuring Aflatoxin M1 (AFM1) in milk are available either as commercial kits or research methods. Enzyme-lynked immunosorbent assays (ELISA), lateral flow tests, immunoaffinity columns coupled with fluorimetric assay are common formats in the current market. Based on recent research developments it is expected that innovative technologies such as electrochemical affinity biosensors, aptamer based biosensors, dynamic light scattering, might be available in the next future for new kits development.The main purpose of screening methods is to detect the presence of a contaminant at level of interest allowing rapid decision making. European Union has set a maximum permitted limit of 0.050 µg/kg for AFM1 in milk. Besides the high sensitivity required for AFM1 detection in milk, the major analytical challenge when developing screening tests is to make them reliable and robust for laboratory and in-field use. This means to cope with differences from matrix to matrix, environmental conditions, operator skills, lot-to-lot reproducibility. A commonly recognized evaluation system needs to be in place so that commercial kits can be evaluated against the same standard. Evaluation programs for commercial testing kits have been established by the United State Department of Agricultural - Grain Inspection Packers and Stockyards Administration (USDA-GIPSA) and AOAC Research Institute. Recent efforts of the European Union, for establishing practical guidelines for the generation of fit-for-purposes performance parameters for screening methods for mycotoxins in foods, resulted in the Regulation 519/2014/EC. The EC validation scheme and its practical application to evaluate performances of a commercial kit for AFM1 detection in milk will be presented and discussed.

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.

The availability of rapid diagnostic methods for monitoring ochratoxigenic species during the seasoning processes for dry-cured meats is crucial and constitutes a key stage in order to prevent the risk of ochratoxin A (OTA) contamination. A rapid, easy-to-perform and noninvasive method using an electronic nose (e-nose) based on metal oxide semiconductors (MOS) was developed to discriminate dry-cured meat samples in two classes based on the fungal contamination: class P (samples contaminated by OTA-producing Penicillium strains) and class NP (samples contaminated by OTA non-producing Penicillium strains). Two OTA-producing strains of P. nordicum and two OTA non-producing strains of P. nalgiovense and P. salamii, were tested. The feasibility of this approach was initially evaluated by e-nose analysis of 480 samples of both Yeast Extract Sucrose (YES) and meat-based agar media inoculated with the tested Penicillium strains and incubated up to 14 days. The high recognition percentages (higher than 82%) obtained by Discriminant Function Analysis (DFA), either in calibration and cross-validation (leave-more-out approach), for both YES and meat-based samples demonstrated the validity of the used approach. The e-nose method was subsequently developed and validated for the analysis of dry-cured meat samples. A total of 240 e-nose analyses were carried out using inoculated sausages, seasoned by a laboratory-scale process and sampled at 5, 7, 10 and 14 days. DFA provided calibration models that permitted discrimination of dry-cured meat samples after only 5 days of seasoning with mean recognition percentages in calibration and cross-validation of 98 and 88%, respectively. A further validation of the developed enose method was performed using 60 dry-cured meat samples produced by an industrialscale seasoning process showing a total recognition percentage of 73%. The pattern of volatile compounds of dry-cured meat samples was identified and characterized by a developed HS-SPME/GC-MS method. Seven volatile compounds (2-methyl-1-butanol, octane, 1R-?-pinene, D-limonene, undecane, tetradecanal, 9-(Z)-octadecenoic acid methyl ester) allowed discrimination between dry-cured meat samples of classes P and NP. These results demonstrate that MOS-based electronic nose can be a useful tool for a rapid screening in preventing OTA contamination in the cured meat supply chain.

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.

Mycotoxins are natural contaminants of several agricultural products and derivatives produced as secondarymetabolites by several filamentous fungal species. Their presence in food and feed can cause serious risks to humanand animal health due to adverse toxic effects; therefore maximum permitted levels have been fixed for the majoroccurring mycotoxins in several commodities at European and international level. The mycotoxins of major concernare: the aflatoxins (aflatoxin B1 is the most potent naturally occurring carcinogen known), the trichothecenes(a structurally related family of cytotoxic compounds) including deoxynivalenol (DON), T-2 and HT-2 toxins, thezearalenone (a potent estrogenic mycotoxin), the fumonisins (predominantly fumonisin B1, a possible carcinogen tohumans) and the ochratoxins (predominantly ochratoxin A, a potent nephrotoxin). Sensitive, reliable and accuratemethods of analysis are hence required to gather adequate information on the levels of exposure to these mycotoxinsand to fulfill regulatory requirements.

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.

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 recent Commission Recommendation 2013/165/EU, asked for collection of more data on T-2 and HT-2 toxins occurrence in cereals and cereal products and emphasized that "in case the used method of analysis enables it, it would be appropriate to analyze also the masked mycotoxins, in particular the mono- and di-glycosylated conjugates of -T2 and HT-2 toxin". Addressing these issues, the aim of this work was to obtain more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area, were analyzed by LC-HRMS for the presence of T-2, HT-2, and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. A widespread co-occurrence of the glucosyl derivative of HT-2 with the unconjugated toxin was observed in unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low µg/kg levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2 to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their mono-glucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.

This paper reports a new method for the determination of T-2 and HT-2 toxins and their glucosylated derivatives in cereals, and some survey data aimed at obtaining more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area were analysed by LC-HRMS for the presence of T-2, HT-2 and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. The glucosyl derivative of HT-2 was detected at levels up to 163 mu gkg(-1) in 17 of the 18 analysed unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low mu gkg(-1) levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2% to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full-scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their monoglucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.

Soybean and soy meal samples collected during the harvest season 2008-2009 in the soybean-growing area of Córdoba Province in Argentina were analysed for T-2 and HT-2 toxins occurrence. These mycotoxins were detected using HPLC analysis with fluorescence detection after derivatisation with 1-anthronylnitrile and immunoaffinity column clean-up. Characteristics of in-house validated method such as accuracy, precision, detection and quantification limits were defined by means of recovery test with spiked soybean and soy meal samples. Mean recoveries for T-2 within the spiking range 125-500 ?g/kg, were 90.9 and 81.3% for soybean and soy meal, respectively with a within-laboratory relative standard deviation <10%. Analysis of samples spiked with HT-2 in the same range gave a mean recovery of 70.2 and 77.5% for soybean and soy meal, respectively, with relative standard deviations <12%. The limit of detection for the method was 25 ?g/kg for T-2 and HT-2, based on a signal-to-noise ratio 3:1 and the limit of quantification was established as three times the detection limit. Out of 64 samples, only two soybean samples showed contamination with A-type trichothecenes evaluated. Confirmatory analyses of the contaminated samples were performed by LC-MS/MS. This study demonstrated low incidences and levels of T-2 and HT-2 in soybean harvested among the areas in the Cordoba Province.

Fusarium equiseti and Fusarium semitectum represent the most abundant species in the Fusarium complex isolated from flowers, soybean pods and seeds in Argentina. The aim of the present study was to assess the production of major type A and type B trichothecenes (diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin, nivalenol, deoxynivalenol) and zearalenone by 40 F. equiseti and 22 F. semitectum isolates on rice culture. Mycotoxins were determined by HPLC with fluorescence detection after derivatisation with 1-anthronylnitrile for type A trichothecenes (i.e. diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin), by HPLC with UV detection for type B trichothecenes (i.e. nivalenol and deoxynivalenol), and by TLC for zearalenone. A total of 22 of 40 F. equiseti isolates produced diacetoxyscirpenol, nivalenol and ZEA alone or in combination, whereas only two of 20 F. semitectum isolates were nivalenol and ZEA producers. Both Fusarium species did not produce any deoxynivalenol, neosolaniol, T-2 toxin and HT-2 toxin. The variable retention in toxigenicity displayed by both fungal species suggests that these species have a saprophytic lifestyle in the soybean agroecosystem in Argentina.

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.

The analytical performances of a lateral flow immunoassay (AFLA-V AQUA(TM), Vicam a Waters Business) forthe determination of aflatoxins in maize were evaluated according to Commission Regulation (EU) No. 519/2014. This case study showed how a critical evaluation of validation results can provide a quite informativepicture of kit performances for its use for different purposes. The validation study, performed at aflatoxin B1levels encompassing EU maximum permitted levels (up to 8 ug kg-1), provided information on the methodprecision profile, cut off values, and false suspect and false negative rates. The total precision expressed asrelative standard deviation varied from 14 to 29% for contaminated samples. Cut off values calculatedconsidering 2 ug kg-1 or 4 mg kg-1 as the screening target concentration were 1.24 and 2.18 ug kg-1,respectively, whereas the resultant false suspect rate for blanks was 42% and 8%, respectively. The falsenegative rate for samples containing the analyte at higher concentrations was found to be lower than1%. Analysis of naturally contaminated maize samples performed by the lateral flow immunoassay andthe AOAC official method revealed a good correlation (parameters of the linear regression: r = 0.97,slope = 0.96) between the obtained results. A satisfactory agreement between reference values and testresults was also obtained in the analysis of reference materials. Finally, a QC protocol based onguidelines suggested in the EU regulation was designed to verify kit performances over time and in newproduction lots.

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.

A method is described for the total phosphine detection in cereal caryopses, comprising the steps of inserting a sample of cereal caryopses inside a container (1) equipped with hermetic closure (2); adding to the sample an aqueous solution of H 2 SO 4 with a v/v concentration in the range of 5-20%, with obtainment of an aqueous dispersion, and hermetically closing the first container (1); subjecting the aqueous dispersion contained in the first container (1) to the action of microwaves for a time not greater than 3 minutes; drawing a predetermined volume of gas overlying the aqueous dispersion and detecting any phosphine that may be present by means of colorimetric and/or spectrophotometric methods, preferably by bringing it into contact with a predetermined volume of an aqueous solution of AgNO 3 of known molarity, inside a second container (3) with hermetic closure and visually analysing the obtained colour and/or spectrophotometrically measuring the absorbance at 400 nm of the aqueous solution. Equipment for the execution of the aforesaid method is described