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.

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 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.

Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus.Temperature and water activity are the two key determinants in pre and post-harvest environmentsinfluencing both the rate of fungal spoilage and aflatoxin production. Varying the combination ofthese parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it isfundamental to know which combinations can control or be conducive to aflatoxin contamination.Little information is available about the influence of these parameters on aflatoxin production onalmonds. The objective of this study was to determine the influence of different combinations oftemperature (20°C, 28°C, and 37°C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth,aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and twostructural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on analmond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1production was obtained at 28°C and 0.96 aw; no fungal growth and AFB1 production wereobserved at 20°C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37°C AFB1production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptasequantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed atmaximum (28°C) and minimum (20°C and 37°C) AFB1 production. Conversely the two structuralgenes (aflD and aflO) were highly expressed only at maximum AFB1 production (28°C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which isstrictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO),but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are mostlikely subordinated to other regulatory processes acting at post translational level.The results of this study are useful to select conditions that could be used in the almond processingchain to suppress aflatoxin production in this important product

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.

Ochratoxin A (OTA) is a renal mycotoxin and transplacental genotoxic carcinogen. The aim of this study was to evaluate the natural occurrence of OTA in equine blood samples and its placental transfer. For the assessment of OTA levels, serum samples were collected from 12 stallions, 7 cycling mares and 17 pregnant mares. OTA was found in 83% of serum samples (median value = 121.4 pg/mL). For the assessment of placental transfer, serum samples were collected from the 17 mares after delivery and from the umbilical cords of their foals, after foaling. Fourteen serum samples from pregnant mares contained OTA (median value = 106.5 pg/mL), but only 50% of their foals were exposed (median values = 96.6 pg/mL). HPLC analysis carried out on four serum samples (collected from two mares and their respective foals) supported the ELISA results on OTA placental transfer. This is the first report on the natural occurrence of OTA in horse serum samples and placental transfer in horses. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

L'ocratossina A (OTA) è una nefrotossina prodotta da funghi del genere Aspergillus e Penicillium con nota attività cancerogena, genotossica e teratogena. Riduzione della funzionalità riproduttiva è stata riportata nel suino (Birò et al 2003), ma non ci sono informazioni sui livelli di esposizione e sulla tossicità riproduttiva nell'equino. Scopo del lavoro è stato di valutare l'esposizione naturale degli equini all'OTA e il passaggio placentale, quantificando OTA nel siero raccolto da stalloni, cavalle cicliche e gravide e dal cordone ombelicale di puledri alla nascita. Inoltre, dopo esposizione in vitro di spermatozoi equini a livelli di OTA compresi tra 750 nM a 24 µM, si è valutata la vitalità (con il SYBR14/ioduro di propidio), la stabilità cromatinica utilizzando il test SCSA (con l'arancio di acridina), la produzione di ROS (utilizzando il 2'7'diclorofluorescin-diacetato) tramite analisi citofluorimetriche e la motilità spermatica tramite il sistema CASA (Giannoccaro et al 2010; Minervini et al 2010). Sono stati esaminati campioni di seme prelevati nella stagione riproduttiva (Aprile-Luglio) e non riproduttiva (Novembre-Gennaio). Le analisi citofluorimetriche sono state condotte dopo 2 ore (vitalità e SCSA) e dopo 30 min (ROS) di esposizione all'OTA. La motilità è stata analizzata dopo 30 min. L'OTA è stata ritrovata nell'83% dei campioni di siero di stalloni (valore mediano=121.4 pg/ml). In 14 campioni di siero su 17 raccolti da cavalle gravide, l'OTA è stata riscontrata con un livello simile (livello mediano=106.6 pg/ml), mentre è stata riscontrata solo nel 50% dei campioni serici prelevati dal cordone ombelicale, con un livello mediano pari a 96.6 pg/ml. La tossicità dell'OTA, dopo esposizione in vitro, è stata ritrovata solo in spermatozoi raccolti durante la stagione non riproduttiva. In entrambe le stagioni esaminate, la vitalità spermatica e la stabilità cromatinica non sono state influenzate da tutte le concentrazioni testate. Al contrario l'OTA ha indotto un significativo effetto pro-ossidante (evidenziato come aumento dei livelli di ROS) dopo esposizione in vitro a concentrazioni da 12 a 24 µM. Inoltre, un significativo aumento della motilità totale è stato registrato dopo esposizione per 30 minuti alle stesse concentrazioni. Gli equini sono risultati esposti a bassi livelli di OTA la cui presenza potrebbe rappresentare un rischio per l'efficienza riproduttiva di tale specie, dovuto ad un possibile accumulo nel plasma seminale e tossicità sui parametri spermatici, come riscontrato anche nel suino (Birò et al 2003). L'esposizione in vitro per brevi tempi all'OTA ha determinato un aumento dei livelli di ROS e di motilità solo negli spermatozoi equini raccolti nella stagione non riproduttiva. Questa maggiore sensibilità degli spermatozoi prelevati nella stagione non riproduttiva potrebbe essere dovuta a modificazioni morfologiche e funzionali presenti nelle due stagioni del cavallo conseguenti a fluttuazioni ormonali stagional

Ochratoxin A contamination of red wines might be quite severe in certain high-risk regions and vintages, thus requiring corrective measures to fulfill acceptable standards for human consumption. This work proposes an innovative and environmentally friendly corrective measure to reduce ochratoxin A levels by repassage of contaminated musts or wines over grape pomaces having no or little ochratoxin A contamination. Grape pomaces have a high affinity for ochratoxin A and have been shown to remove ochratoxin A from must and wine during vinification. Time course experiments showed that ochratoxin A adsorption by pomaces is a rapid process, reaching equilibrium in less than 10 h, and is not affected by the tested toxin concentrations. Repassage of wine from Primitivo grapes spiked with 2-10 mu g/kg ochratoxin A over pomaces obtained from the same grapes removed up to 65% ochratoxin A within 24 h. Similar results (50-65% ochratoxin A reduction) were obtained with Primitivo or Negroamaro wines repassed over pomaces from different grape varieties including white grapes (Malvasia, Greco di Tufo) and red grapes (Sangiovese, Aglianico). Grape pomaces maintained a good efficacy in removing ochratoxin A after being reused four times. Unlike other enological fining agents, the use of grape pomaces to adsorb ochratoxin A from red wines of the same grape variety (Primitivo) did not affect relevant wine quality parameters, including color intensity and health-promoting phenolic content (trans-resveratrol, quercetin, total polyphenols). These quality parameters were instead positively or negatively affected when contaminated wines were repassed over grape pomaces from other grape varieties, the effect being related to the intrinsic characteristics of the pomace variety. The proposed decontamination procedure can be applied in a modern winery provided that contaminated grapes are identified early and processed separately from uncontaminated grapes.

Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock. © 2014 by the authors; licensee MDPI, Basel, Switzerland.