The food safety issue is among the cornerstones of the environmental protection schemesworldwide and beside hazard issues, there is great concern about monitoring of foodspoilage in a reliable way. Current practices of assessment of food spoilage still relies heavilyon regulatory inspection and sampling regimes. A portable photonic multisensor devicefor the detection of food contaminations, spoilage and fraud is the objective of the EUPhasmaFOOD project. It will integrate different capabilities: spectroscopic detection (VIS/NIR), imaging, smart signal processing, data analysis and comparison with updated modelson cloud platform hosting data set for training and calibration of food analysis algorithms,user friendly interfaces by smartphone/tablet/PC also through wireless connection.

Total mycotoxins exposure in humans could be estimated by the combined measures of urinary free mycotoxins, phase I metabolites and their glucuronides and/or sulphates derivatives. The predigestion of urine with ?-glucuronidase/sulfatase enzymes deconjugate the glucuronides and sulphates derivatives to form free mycotoxins and phase I metabolites, thus reducing the number of analytes (mycotoxin biomarkers) to be measured. It has been demonstrated that foods can be frequently contaminated by mixtures of mycotoxins and their modified forms. These last can be hydrolysed in the gut to form the parent mycotoxins. Therefore the analysis of urine for multi-biomarker is a powerful approach to identify the type and the number of mycotoxins ingested by each individual. Moreover, the urinary concentrations of mycotoxin biomarkers can be used to estimate the probable daily intake of those mycotoxins for which human excretion rate is available. We have developed an highly sensitive multi-biomarker LC-MS/MS method based on predigestion of urine with ?-glucuronidase/sulfatase enzymes and SPE/immunoaffinity concentration and cleanup for the determination of urinary biomarkers of deoxynivalenol (DON), zearalenone (ZEA), aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA). These are the main mycotoxins frequently co-occurring in food/beverage worldwide and are mainly produced by Fusarium graminearum (DON, ZEA), Aspergillus flavus and A. parasiticus (AFB1), F. verticillioides and F. proliferatum (FB1), Penicillium verrucosum and A. carbonarius (OTA). Our method was used to analyse 356 human urines collected in Italy, South Africa and Sweden and allowed the identification of mycotoxins mostly ingested in each country as well as the probable daily intake of each mycotoxin.

Human exposure assessment to deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) can be performed by measuring their urinary biomarkers. Suitable biomarkers of exposure for these mycotoxins are DON + de-epoxydeoxynivalenol (DOM-1), aflatoxin M1 (AFM1), FB1, ZEA + alpha-zearalenol (alpha-ZOL) + beta-zearalenol (beta-ZOL) and OTA, respectively. An UPLC-MS/MS multi-biomarker method was used to detect and measure incidence and levels of these biomarkers in urine samples of 52 volunteers resident in Apulia region in Southern Italy. The presence of ZEA + ZOLs, OTA, DON, FB1 and AFM1 were detected in 100%, 100%, 96%, 56% and 6%, of samples, respectively. All samples contained biomarkers of two or more mycotoxins. The mean concentrations of biomarkers ranged from 0.055 ng/mL (FB1) to 11.89 ng/mL (DON). Urinary biomarker concentrations were used to estimate human exposure to multiple mycotoxin. For OTA and DON, 94% and 40% of volunteers, respectively exceeded the tolerable daily intake (TDI) for these mycotoxins. The estimated human exposure to FB1 and ZEA was largely below the TDI for these mycotoxins for all volunteers.

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet.Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010-11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously.This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B<inf>1</inf> (FB<inf>1</inf>), fumonisin B<inf>2</inf> (FB<inf>2</inf>), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), ?-zearalenol (?-ZOL), ?-zearalenol (?-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.

It is well known that mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. Various foods may therefore be contaminated with a range of mycotoxins. Consequently, consumers are generally exposed to more than one mycotoxin at the same time. The aim of our studies was to determine the possible associations between mycotoxin exposure and diet as well as concurrent exposure to mycotoxins in randomly selected Swedish adults, aged 18-80 years. The participants took part in the Swedish national dietary survey "Riksmaten 2010-11" and urinary samples were collected on average 19 days after the 4-day web-based diet record. In the first study, total urinary deoxynivalenol (DON), i.e. free DON and the DON-glucuronide, was measured using immunoaffinity enrichment and LCMS quantification. In the second study, concurrent mycotoxin exposure was analysed using a multi-biomarker LC-MS/MS method. The first study showed that exposure of DON among the adult population was very common (>90%) with levels ranging from non-detectable (nd) to 65.8 ng DON/ml urine and a median level of 2.9 ng/ml. Furthermore, urinary DON (ng/mg creatinine) was associated with the intake (gram/day) of total cereal grain as well as with whole grain. DON was also significantly associated with breakfast cereals and porridge (p<0.05). Estimated intakes in this study ranged between 2.5 and 5443 ng/kg bw. In total 1% of the individuals had estimated intakes above the TDI whereas the mean and median intakes of 159 and 84 ng DON/kg bw respectively were considerably below the recommended TDI of 1000 ng/kg bw. To further explore the mycotoxin exposure among the Swedish population, the multi-biomarker assay was used including aflatoxin M1, DON, de-epoxy-deoxynivalenol (DOM-1), fumonisin B1 and B2, nivalenol, ochratoxin A, zearalenone, and ?- and ?-zearalenol. The purpose of the second study was to examine the extent of mycotoxin exposure and possible patterns of concurrent exposures. By utilizing consumption data from "Riksmaten 2010-11", we linked food patterns with the number of mycotoxins found in the urinary samples. Results revealed that concurrent exposure to more than one toxin was found in 69% of the study population. The most common combinations were DON+OTA and ZEA+FB2 which were found in 54 (21%) and 39 (15%) samples, respectively. Among the 67 samples (27%) with 3 different detectable mycotoxins DON and OTA and an additional toxin was found in 51 samples (76% of samples containing 3 mycotoxins). The most common combination was DON+OTA+NIV which was found in 38 samples (57% of samples containing 3 mycotoxins).However, when comparing the number of toxins detected with the reported diet data it was difficult to distinguish food patterns which would indicate an increased risk of being exposed to many mycotoxins simultaneously.

Deoxynivalenol (DON) exposure is estimated by the combined measures of urinary DON and DON-glucuronides. In this study, data from single-mycotoxin (SM) and a multimycotoxin (MM) methods were compared for 256 Swedish adult urine samples. Both methods included ?-glucuronidase predigestion, immunoaffinity enrichment, and LC-MS/MS. However, the specific reagents, apparatus, and conditions were not identical in part because the MM method measures additional mycotoxins. DON was detected in 88 and 63% of samples using the SM and MM methods, respectively, with the following mean and median concentrations: SM, mean = 5.0 ng/mL, SD = 7.4, range of positives = 0.5-60.2 ng/mL, median = 2.5 ng/mL, IQR = 1.0-5.5 ng/mL; MM, mean = 4.4 ng/mL, SD = 12.9, range of positives = 0.5-135.2 ng/mL, median = 0.8 ng/mL, IQR = 0.3-3.5. Linear regression showed a significant, albeit modest, correlation between the two measures (p = 0.0001, r = 0.591). The differences observed may reflect subtle handling differences in DON extraction and quantitation between the methods.

The performances of four LC-MS/MS methodologies for determination of up to eight mycotoxin biomarkers in human urines were compared by involving three laboratories that analysed common urine samples spiked at two levels of each biomarker. Each laboratory received a calibration solution, spiked urines and the corresponding unspiked urine. The two spiking levels for each biomarker were chosen by considering the levels naturally occurring in human urines and the limits of quantification of the LC-MS/MS methodologies used by participating laboratories. The results of each laboratory were evaluated for their z-score values. The percentage of satisfactory z-scores (z < 2) were: 100% for deoxynivalenol, de-epoxy-deoxynivalenol, aflatoxin M1, ?-zearalenol and zearalenone, 87% for ?-zearalenol, 50% for ochratoxin A and 42% for fumonisin B1. Good method performances were obtained for most biomarkers, at the levels tested in this study, as demonstrated by the overall percentage of satisfactory z-scores for all analytes (87%). Unsatisfactory/questionable z-scores (z > 2) were obtained for fumonisin B1 (7/12 results), ochratoxin A (4/8 results) and ?-zearalenol (1/8 results). The percentage of satisfactory z-scores for fumonisin B1 and ochratoxin A increased from 42% to 83% for fumonisin B1 and from 50% to 62% for ochratoxin A when laboratories 1 and 2 used own calibrants. Factors that could explain the different results obtained for fumonisin B1 and ochratoxin A with provided and own calibration solutions could not be identified in this study and should be carefully investigated in future studies.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) werescreened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were usedfor the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1and FB2), nivalenol (NIV), deoxynivalenol (DON), T-2 and HT-2 toxins and Alternaria mycotoxins altenuene (ALT),alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TTX) and tenuazonic acid (TeA). Frequencyof potential toxigenic fungal species occurrence was: 87% Aspergillus Sect. Nigri; 58% Aspergillus Sect. Flavi; 38%Aspergillus Sect. Circumdati; 42% Alternaria spp.; 33% Penicillium spp. and 20% Fusarium spp. Frequency ofmycotoxin occurrence and mean of positives were: 51% OTA, 29.5 µg/kg, 5 samples above the EU limit of 20 µg/kg;31% aflatoxins, 12.8 µg/kg, two samples above the EU limit of 5 µg/kg for aflatoxin B1; 91% ZEA, 1.4 µg/kg; 78%FB2, 7.6 µg/kg; 58% FB1, 22.8 µg/kg; 38% NIV, 39.5 µg/kg; 36% DON, 6.9 µg/kg; 20% T-2 toxin, 5.6 µg/kg and 22%HT-2 toxin, 13.8 µg/kg. For the Alternaria mycotoxins, 100% of samples contained TeA, 4817.9 µg/kg; 93% TTX,29.7 µg/kg; 56% AOH, 114.4 µg/kg; 33% AME, 13.0 µg/kg and 9% ALT, 61.7 µg/kg. Co-occurrence of mycotoxinsin each sample ranged from 2 to 16 mycotoxins (mean 7). No statistical correlation was found between mouldsand their mycotoxins occurrence. Within the four groups of peppers collected herein (fresh, dried, grounded andfried) higher percentages of contamination and mycotoxin levels were measured in grounded peppers, whereasmuch lower values were observed for fried peppers. The high percentages of positive samples and the high levelsof some mycotoxins observed in this study confirm the susceptibility of peppers to mycotoxin contamination andclaims for an improvement of the conditions used during production and drying process.

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

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

Le micotossine sono metaboliti secondari prodotti da funghi microscopici che infestano un gran numero di alimenti destinati al consumo umano e zootecnico. A causa dei loro molteplici ed importanti effetti tossici la loro presenza costituisce un serio pericolo per la salute degli uomini e degli animali. Al fine di tutelare la salute, l'Unione Europea, ha stabilito una serie di tenori massimi di tali sostanze ammessi negli alimenti e nei mangimi.La letteratura scientifica riporta non solo casi di contaminazione multipla da micotossine negli alimenti e mangimi, ma anche, effetti sinergici dovuti alla assunzione di più molecole contemporaneamente. Al fine di tutelare la salute pubblica è necessario mettere a punto metodiche analitiche che consentano la rilevazione simultanea di tutte le molecole per le quali il Legislatore Comunitario ha fissato dei limiti massimi in alimenti e mangimi e forniscano dati sulla loro copresenza per la valutazione del rischio complessivo. A tale scopo è stata messa a punto e validata una metodica analitica multiresiduo e multiclasse che consente, per mezzo della spettrometria di massa tandem accoppiata all'HPLC, la contemporanea rilevazione di aflatossine B1, G1, G2, B2, Ocratossina A, Zearalenone, Deossinivalenolo, Tossina T-2, HT-2, Fumonisine B1, B2 e B3 in cereali e alimenti ad uso zootecnico con purificazione mediante colonne ad immunoaffinità multianticorpo in tandem di tipo AOF (Afla/Ocra/Fum) e DZT (Don/Zon/T2HT2). Durante le fasi di messa a punto del metodo sono state valutate metodiche di estrazione e purificazione differenti quali il metodo dilute and shoot, la metodica QuEChERS (nella sua modalità originale e modificata), la SPE multiresiduo. È stato inoltre studiato il cosiddetto "effetto matrice" mediante la comparazione di curve di calibrazione in solvente ed in matrice con e senza l'ausilio di standard marcati con 13C in calibrazione interna. La separazione e rivelazione è stata ottenuta mediante un'unica corsa cromatografica con interfaccia di tipo ESI+ per tutte le molecole. Il metodo è stato validato su alimenti a base di cereali ed alimenti ad uso zootecnico verificando le performance in termini di linearità strumentale, effetto matrice, limite di quantificazione (LOQ), recupero, precisione in condizioni di ripetibilità e confrontando i risultati ottenuti con quanto prescritto dal Regolamento (CE) 401/2006 e s.m.i. e dalla norma UNI CEN/TR 16059. 74La metodica consente di rilevare tutte le molecole oggetto dello studio a livelli compatibili con i limiti di legge ed ha delle performance uguali o migliorative rispetto a quanto previsto dalla normativa vigente.

This PhD thesis deals with the development/optimization and validation of a sensitive, accurate, precise and robust LC-MS/MS analytical method for the simultaneous determination of biomarkers of DON, AFB1, FB1, ZEA and OTA in human and animal urine. This method was in-house validated and revalidated in a mini-intercomparison study for human urine. The applicability of the new method to human urine samples was demonstrated in a human pilot study conducted in South Africa and South Italy. The results of this study showed an high percentage of positive samples for biomarkers of DON, FB1, ZEA and OTA in both countries.

The fate of aflatoxins during processing of contaminated almonds into nougat, pastries, and almond syrup was evaluated by testing the effect of each processing step (blanching, peeling, roasting, caramelization, cooking, and water infusion) on the distribution and levels of aflatoxins. Blanching and peeling did not reduce total aflatoxins that were distributed between peeled almonds (90-93%) and skins (7-10%). Roasting of peeled almonds reduced up to 50% of aflatoxins. Up to 70% reduction of aflatoxins was observed during preparation and cooking of almond nougat in caramelized sugar. Aflatoxins were substantially stable during preparation and cooking of almond pastries. The whole process of almond syrup preparation produced a marked increase of total aflatoxins (up to 270%) that were distributed between syrup (18-25%) and spent almonds (75-82%). The increase of total aflatoxins was probably due to the activation of almond enzymes during the infusion step that released free aflatoxins from masked aflatoxins.

Deoxynivalenol (DON) is an important mycotoxin produced by several species of Fusarium. It occurs often in wheat grain and is frequently associated with significant levels of its modified form DON-3-glucoside (DON-3-Glc). Ozone (O3) is a powerful disinfectant and oxidant, classified as GRAS (Generally Recognised As Safe), that reacts easily with specific compounds including the mycotoxins aflatoxins, ochratoxin A, trichothecenes and zearalenone. It degrades DON in aqueous solution and can be effective for decontamination of grain. This study reports the efficacy of gaseous ozone treatments in reducing DON, DON-3-Glc, bacteria, fungi and yeasts in naturally contaminated durum wheat. A prototype was used to dispense ozone continuously and homogeneously at different concentrations and exposure time, in 2kg aliquots of durum wheat. The optimal conditions, which do not affect chemical and rheological parameters of durum wheat, semolina and pasta, were identified (55g O3h-1 for 6h). The measured mean reductions of DON and DON-3-Glc in ozonated wheat were 29% and 44%, respectively. Ozonation also produced a significant (p<0.05) reduction of total count (CFU/g) of bacteria, fungi and yeasts in wheat grains.

Il deossinivalenolo (DON), prodotto da diverse specie di Fusarium, si ritrova frequentemente come contaminante naturale nel frumento e in altri cereali. Spesso lo si ritrova associato a livelli significativi della sua forma modificata DON-3-glucoside (DON-3-Glc). L'ozono (O3) è un potente disinfettante e ossidante classificato come GRAS (Generalmente riconosciuto come sicuro). Esso reagisce facilmente con molti composti specifici compreso le micotossine, degradandole in soluzione acquosa, per cui ha il potenziale per essere efficace anche per la decontaminazione dei grani. In questo studio sono riportati i risultati sull'efficacia di trattamenti con ozono gassoso per la riduzione di DON, DON-3-Glc, batteri, funghi e lieviti in frumento duro contaminato naturalmente. Per le prove di ozonazione è stato usato un prototipo costituito da un cilindro rotante, contente il campione di cariossidi da trattare, in cui veniva insufflato ozono gassoso a diverse concentrazioni e tempi di esposizione. Sono state identificate le condizioni ottimali (55 gO3 h-1 per 6 h) che erano efficaci nel diminuire i livelli di contaminazione del frumento duro senza alterare i parametri chimici e reologici del frumento trattato, della semola e pasta da esso ottenuti. Le riduzioni medie di DON e DON-3-Glc nel grano ozonato erano rispettivamente del 29 e del 44%. L'ozonazione ha inoltre prodotto una riduzione significativa (p <0,05) del conteggio totale (CFU/g) di batteri, funghi e lieviti.

Contamination of vineyards from black Aspergilli is a well-known condition that cause the accumulation of ochratoxin A (OTA) in grapes and derived products. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, although, great variations may occur from one year to another. Among the black Aspergilli commonly found in infected grapes, Aspergillus carbonarius is considered the main responsible of OTA contamination, with A. niger at secondary extent. To minimise the black Aspergilli infection and limit OTA concentrations in grapes, several strategies are commonly adopted, including the implementations of good agricultural practices and the use of pesticides and fungicides. These strategies are essential to manage the problem, but since they are insufficient when extremely favourable condition occurs in the vineyard, new strategies, aimed to reduce OTA risk in vineyards, are necessary. In this respect, implementation of electrolysed oxidising water (EOW) in agriculture has arising during the last decade as an interesting alternative to replace or limit the use of chemicals. The efficacy of EOW was also demonstrated in post-harvest for reduction of gray mold and brown rot on surfaces of peaches and grapes. In this study, we screened for the first time the efficacy of EOW generated by EVA System® 100 apparatus (Industrie De Nora S.p.A., Milan, Italy) at different concentrations of free chlorine (ranging from 0.0125 to 0.4 g/L) on conidial germination and growth of A. carbonarius and A. niger. A good fungicidal activity was achieved after 2-10 minutes treatment with EOW containing 0.4-0.2 g/L of free chlorine, although A. carbonarius conidia were more resistant to EOW than A. niger conidia. Then EOW at 0.4 g/L free chlorine was tested on detached berries of Primitivo and Aglianico wine grape varieties that were singularly infected with A. niger and A. carbonarius. Treatments with Switch (cyprodinil and fludioxonil) at 0.8 g/L, a fungicide regularly used in vineyard against black Aspergilli, were used as positive controls. Percentage of infected berries, McKinney index, and OTA concentrations were used to evaluate the efficacy of the EOW treatment. On Aglianico grape berries EOW and Switch produced an almost complete reduction on percentage of infection, McKinney index and OTA concentration compared to the control. On Primitivo grape berries EOW treatment reduced more than half the percentage of infections and McKinney index for both A. carbonarius and A. niger, although Switch showed a better performance. A significant reduction of OTA concentration was observed for EOW and Switch treatments. These results evidence for the first time that EOW is effective to reduce black Aspergilli inoculum and OTA contamination on grape berries.

Biomarker-based methods are being more and more used to assess dietary exposure of mycotoxins in a population. The aim of the present study was to perform an extended analysis of urinary multiple mycotoxin levels and associations with background characteristics and food groups. Exposure assessment calculations were performed on three urine mycotoxins as described below and the probable daily intake (PDI) was compared with the established tolerable daily intake (TDI) to uncover potential exposure risks. The study population consisted of 250 adults and 50 school children in grade five from two surveys conducted by the Swedish National Food Agency. Six mycotoxins (deoxynivalenol (DON), zearalenone (ZEA), fumonisin B (FB ), fumonisin B (FB ), ochratoxin A (OTA), and nivalenol (NIV) and four metabolites (deepoxy-deoxynivalenol (DOM-1), aflatoxin M (AFM ), ?-zearalenol (?-ZOL) and ?-zearalenol (?-ZOL) were measured by an ultra-performance liquid chromatography tandem mass spectrometry based method (LC-MS/MS). OTA and DON were the most commonly occurring mycotoxins in urine of both adults and children, 51 and 63%, respectively in adults and 96 and 94%, respectively in children. A positive correlation was found between urinary NIV and total cereal consumption among adults. ZEA, ?-ZOL, ?-ZOL and FB were significantly higher in females than males (P<0.01 for all). Adjusted OTA levels were inversely correlated with income in men. In children, the percentage DOM-1 positive samples were much higher compared to adults, 76 and 8% respectively, indicating a higher capacity to detoxify DON. The small sample size among children made it difficult to study associations between urine mycotoxins levels and food group intake. All PDI estimates [DON (with and without DOM-1), ZEA (with and without ?-ZOL and ?-ZOL) and FB ] were below the TDI values except for DON exposure in adults, as reported previously, 1.3% of the volunteers were above the TDI.

Grape pomaces are increasingly being used as starting material in the industrial production of plant food supplements (PFS), food coloring, and tartrates, but they are at risk of ochratoxin A (OTA) contamination, a mycotoxin with nephrotoxic and carcinogenic effects. We analyzed 24 commercial PFS and 13 food coloring samples derived from Vitis vinifera, mainly pomaces, using a HPLC-FLD method for OTA determination. OTA was found in 75% of PFS samples and 69% of food coloring samples at levels of <1.16-20.23 ?g/kg and <1.16-32.00 ?g/kg, respectively. The four commercial leavening agents containing tartrates were found to be negative for OTA. All eight samples collected in two distilleries that use grape pomaces and wine lees to produce tartrates and other byproducts contained OTA at levels of <1.16-240.93 ?g/kg. The high incidence of OTA contamination in PFS and food coloring agents derived from V. vinifera suggests that maximum permitted level(s) should be established for this mycotoxin in these products.

The efficacy of four agricultural byproducts (ABPs) and two commercial binders (CBs) to reduce the gastrointestinal absorption of a mixture of mycotoxins was tested in piglets using urinary mycotoxin biomarkers as indicator of the absorbed mycotoxins. Twenty-eight piglets were administered a bolus contaminated with the mycotoxin mixture containing or not ABP or CB. Twenty-four hour urine was collected and analyzed for mycotoxin biomarkers by using a multiantibody immunoaffinity-based LC-MS/MS method. Each bolus contained 769 ?g of fumonisin B1 (FB1), 275 ?g of deoxynivalenol (DON), 29 ?g of zearalenone (ZEN), 6.5 ?g of aflatoxin B1 (AFB1) and 6.6 ?g of ochratoxin A (OTA) corresponding to 2.2,0.8, 0.08, 0.02, and 0.02 ?g/g in the daily diet, respectively. The percentage of ABP in each bolus was 50%, whereas for the two CBs the percentages were 5.2 and 17%, corresponding to 2.8, 0.3, and 0.9% in the daily diet, respectively. The reduction of mycotoxin absorption was up to 69 and 54% for ABPs and CBs, respectively. White grape pomace of Malvasia was the most effective material as it reduced significantly (p < 0.05) urinary mycotoxin biomarker of AFB1 (67%) and ZEN (69%), whereas reductions statistically not significant were observed for FB1 (57%), DON (40%), and OTA (27%). This study demonstrates that grape pomace reduces the gastrointestinal absorption of mycotoxins. This agricultural byproduct can be considered an alternative to commercial products and used in the feed industries as an effective, cheap, and natural binder for multiple mycotoxins.

Metabolic profile of urine from piglets administered with single boluses contaminated with mycotoxin mixture (deoxynivalenol, aflatoxin B1, fumonisin B1, zearalenone, and ochratoxin A) were studied by 1H NMR spectroscopy and chemometrics (PCA, PLS-DA, and OPLS-DA). The mycotoxin levels were close to the established maximum and guidance levels for animal feed (2003/100/EC and 2006/576/EC). Urine samples were obtained from four groups of four piglets before (control, C) or within 24h (treated, T) after receiving a contaminated boluses with increasing doses of mycotoxins (boluses 1-4). For the two highest dose groups, the urines were collected also after one week of wash out (W). For the two lowest doses groups no significant differences between the C and T samples were observed. By contrast, for the two highest doses groups the T urines separated from the controls for a higher relative content of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentration of betaine and TMAO. Interestingly, a similar profile was found for both W and T urines suggesting, at least for the highest doses used, serious alteration after a single bolus of mycotoxin mixture.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Todate, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonariushas allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previouslycharacterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotidesdownstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Itsproximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to theintroduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced proteinsequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTAcluster of A. niger. The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonariusand determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expressionprofile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlationof the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmedthat the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supportingour earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius.

Almonds are extremely nutrient-dense nuts. They provide generous amounts of calories, fats, complex carbohydrates, protein, vitamins, minerals and fiber. They are consumed as raw nuts or as ingredients of derived products. However, almonds can be susceptible to aflatoxins contamination, extremely harmful for the human health even at low concentrations. We evaluated the evolution of aflatoxins during processing of almonds into traditional Italian products, such as pastries, nougat and syrup. The mass balance approach was used to determine levels and distribution of aflatoxins in each fraction collected during processing steps. Experiments were conducted on almonds inoculated with a toxigenic strain of Aspergillus flavus. A robust and horizontal HPLC-FLD method was optimized and used for aflatoxins determination in all the matrices considered in this study. The two tested blanching processes (steaming and boiling in water) did not reduce aflatoxins levels in peeled almonds. Standard roasting conditions produced up to 50% reduction of aflatoxins. Higher aflatoxin reduction (82%) was observed by roasting at 150°C for 120 min, but almonds lost their organoleptic characteristics. The preparation and cooking of nougat produced a consistent reduction of 54-70% of aflatoxins due to the caramelisation of sugar on almond surface. Almond pastries were prepared by mixing almond paste, eggs and sugar and cooked at 140°C for 30 min, 160°C for 20 min or 180°C for 15 min. Aflatoxins were substantially stable during cooking of pastries. Almond syrup was prepared from ground peeled almond that was infused in water for 5 hours. After infusion spent almonds were discarded and the infuse was sugared and boiled until it reaches the consistency of a syrup. About 18-25% of total aflatoxins passed in the final syrup. The whole process of almond syrup preparation produced a marked increase of total aflatoxins. This increase was probably due to the activation of endogenous almond enzymes that release free aflatoxins from modified aflatoxins during the water infusion of ground peeled almonds.

Twelve different approaches commonly used forthe simultaneous LC tandem MS (MS/MS)determination of mycotoxins (deoxynivalenol,aflatoxins, ochratoxin A, T-2 and HT-2 toxins,fumonisins, and zearalenone) were tested in cerealsand feed materials. They comprised differentextraction solvents, types of cleanup [solid-phaseextraction, QuEChERS, and immunoaffinity (IMA)],and calibration approaches (external or matrixmatched).The percentage of mycotoxins withacceptable recovery, according to Regulation (EC)No. 401/2006, ranged from 9 to 100%. The approachgiving the highest percentage of acceptable resultswas selected and further tested for corn, rice, andfeed spiked at three different mycotoxin levels (low,medium, and high). The method is based onextraction with MeOH-water (70 + 30, v/v) andcleanup with two multiantibody IMA columns. Forcorn and rice spiked at low mycotoxin levels, asignificant matrix effect was observed and wascompensated by using 13C calibration. At highermycotoxin levels (medium and high), matrix effectswere negligible as no significant differences wereobserved for the majority of recovery resultscalculated by 13C calibration and external calibration.Although the proposed method still needsimprovement in terms of accuracy and, to a lesserextent, precision, it was successfully tested with fourproficiency tests in buckwheat, corn, rice, and feed,giving acceptable z-scores for 97% (34 out of 35) ofresults.

Aflatoxins and ochratoxin A are regulated in Europe for some spices (Capsicum spp., Piper spp., Myristica fragrans,Zingiber officinale, Curcuma longa) and mixtures of spices containing one or more of these spices. No mycotoxinlimits are in force for herbs. A total of 132 samples of spices (94) and herbs (38) purchased from Beirut inLebanon were analysed for 12 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, FB1,FB2, HT-2, T-2, ZEA, DON, NIV)by using a UPLC-MS/MS method based on 'dilute and shoot' approach. The limits of detection (LOD) rangedfrom 0.1 ?g/kg (ZEA) to 20.5 ?g/kg (DON) and limits of quantification (LOQ) ranged from 0.3 ?g/kg (ZEA) to68.2 ?g/kg (DON). 80% of analysed samples were contaminated by 1-11 mycotoxins. Total aflatoxins andochratoxin A were detected in 19 and 30% of spices, 8 and 11% of herbs, respectively. Mean levels of totalaflatoxins and ochratoxin A were 168.1 and 7.1 ?g/kg in positive spices, 36.1 and 7.0 ?g/kg in positive herbs,respectively. 78 and 10% of positive spice samples contained aflatoxin and ochratoxin A at levels higher than thelimits, respectively. Total aflatoxin levels higher than the European limits were also measured in some non-regulated spices (allspice, cloves, coriander, fenugreek) and some herbs (rosemary, sage and oregano). Withinthe non-regulated mycotoxins FB1was the most occurring (60% in spices, 55% in herbs) followed by FB2(35% inspices, 18% in herbs), ZEA (30% in spices, 3% in herbs), DON (12% in spices, 3% in herbs), T-2 and HT-2 toxins(3-5%), whereas NIV and AFG2were never detected. Mean levels of FB1,FB2, ZEA and DON in positive samplesof spices were 6432.3, 203.2, 30.6, 1751.4 ?g/kg, respectively; in positive samples of herbs they were 2826.3,214.9, 2.8, 589.7 ?g/kg, respectively. The whole results demonstrate the higher susceptibility of spices to my-cotoxin contamination with respect to herbs. Comparison of results obtained for samples produced with (81) andwithout (51) HACCP and GMP showed that the implementation of HACCP and GMP practices seems to beeffective in reducing the occurrence of regulated mycotoxins but was ineffective for the non-regulated ones. Thesamples analysed in this study originated from at least 15 Countries and the results obtained gives indicationsabout the occurrence of mycotoxins in relation to the Country of origin of the samples.The high percentages of positive samples and the high levels of some mycotoxins observed in this studyhighlight the problem of mycotoxin contamination in spices and herbs consumed in Lebanon. The occurrence ofhigh levels of aflatoxins and OTA in some non-regulated spices and herbs suggests the addition of these matricesin the list of regulated ones. The high number of positive samples and the high levels of fumonisins observed inthis study suggest the inclusions of these mycotoxins in the list of regulated mycotoxins for these matrices.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxinLC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancerregion, Transkei, South Africa. Fifty-three female participants donated part of their maize-based eveningmeal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker)and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery withLOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean ± standarddeviation 0.342 ± 0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4 ± 49.4 ng/mg creatinine)after hydrolysis with b-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicateddeoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with b-glucuronidaseand immunoaffinity clean-up determined zearalenone (100%; 0.529 ± 1.60 ng/mg creatinine), FB1(96%; 1.52 ± 2.17 ng/mg creatinine), a-zearalenol (92%; 0.614 ± 1.91 ng/mg creatinine), deoxynivalenol(87%; 11.3 ± 27.1 ng/mg creatinine), b-zearalenol (75%; 0.702 ± 2.95 ng/mg creatinine) and ochratoxin A(98%; 0.041 ± 0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods inmeasuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinarydeoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

Red grape pomaces are rich in natural polyphenols that comprise numerous pigments. Thanks to their antioxidant properties, they maintain and promote health and reduce the risk of cardiovascular diseases. Grape pomaces are increasingly being used as starting material to produce plant food supplements (PFS), food coloring agents and tartrates. But these byproducts could be naturally contaminated by ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic effects. In this study, we analyzed 24 commercial PFS, 13 food coloring samples, and 4 leavening agents derived from Vitis vinifera using an improved HPLC-FLD method for OTA determination.1Materials and methodsCommercially products derived from Vitis vinifera were purchased from retail stores in South Italy or from e-commerce for a total of 41 samples. Each sample was extracted with organic solvents, purified with immunoaffinity column and analysed by HPLC/FLD.1 The identity and concentration of OTA in the purified extracts of two food supplements containing low levels of OTA were confirmed by LC-MS/MS. The method was robust, precise, accurate and applicable to all tested samples. Results of recovery and repeatability experiments were in the range of 87-102% and 2-4%, respectively; the values of limit of detection (LOD) and limit of quantitation (LOQ), calculated as signal-to-noise ratios of 3:1 and 6:1, were 0.50 ?g/kg and 1.16 ?g/kg, respectively.1ResultsOTA was found in 75% of PFS samples at levels of <1.16-20.23 ?g/kg and in 69% of food coloring samples at levels of <1.16-32.00 ?g/kg. The four commercial leavening agents containing tartrates were negative for OTA.1ConclusionsIn Europe the maximum levels of OTA in several food commodities are in force2 but not for PFS and food coloring agents derived from Vitis vinifera. The high incidence of OTA contamination in these products suggests an amendment of the regulation to include them. We previously reported that OTA can occur at high levels (up to 849.10 µg/kg) in grape pomaces.3 These findings makes imperative to control for OTA contamination all grape pomaces destined to the preparation of edible products.References1Solfrizzo et al. J. Agric. Food Chem.2015, DOI: 10.1021/acs.jafc.5b00326. 2EC No.1881/2006 of 19 December 2006 and subsequent amendments. 3Solfrizzo et al. J. Agric. Food Chem. 2008, 56, 11081-11086.

In the nineties fungal secondary metabolites (SMs), such as antibiotics and mycotoxins, started to be genetically characterized. Then, the clustered arrangement of genes involved in the biosynthesis of a single SM was studied. In the pre-genomic era, gene cluster discovery in fungi was complex and time-consuming, involving cumbersome traditional molecular methods. Genomics has revolutionized the research on SM biosynthesis pathways, allowing the bypass of such approaches. The breakthrough of next-generation sequencing (NGS) technologies and the advent of Bioinformatics have opened a new era in the study of biological systems. NGS technologies contributed significantly to the increasing availability of fungal genomes and bioinformatic analysis lead to the identification of SM clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown microbial metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis. Here, we present the example of how the genomic approach has led to the identification of biosynthetic genes and their role in ochratoxin A (OTA) production by Aspergillus carbonarius. From the genome sequencing and the subsequent prediction of OTA cluster, we demonstrated by gene knock-out approach the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) in the OTA biosynthesis. Single gene knock-out mutant allowed us to elucidate the order of the enzymatic steps in the biosynthesis pathway. Other predicted genes in the cluster, such as a p450 monooxygenase and a transcription factor gene, need to be investigated for the full knowledge of the structural and regulatory mechanisms of toxin production. Furthermore, transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.

Deoxynivalenol (DON) occurs frequently in wheat grains and it is often associated with significant levels of its modified form DON-3-glucoside (DON-3G). Several approaches were tested in the past to prevent the formation of DON in the field or reduce its level in the grains. Ozone (O3) is a powerful disinfectant and oxidant that reacts quite easily with specific compounds including mycotoxins and pesticides. It was demonstrated to degrade these contaminants in aqueous solution and it has the potential to be also effective for decontamination of grains. Ozone in gaseous form as a fumigant gave also promising results against insects and a wide range of microorganisms including bacteria, fungi, viruses, protozoa and fungi spores that may grow and survive on wheat during storage. Moreover, it is classified as GRAS (Generally Recognised As Safe) which makes very attractive its use in food industry. However, ozone is unstable and decays naturally into inactive diatomic oxygen (O2) and it must be continuously added in the mass of the grain to maintain the active concentrations. We have tested the efficacy of gaseous ozone treatments for reduction of mycotoxins, pesticides, heavy metals, bacteria, fungi and yeasts in durum wheat grain naturally contaminated with low levels of these contaminants. A prototype was tested to continuously and homogeneously dispense ozone in 2 kg aliquots of durum wheat. Levels and distribution of contaminants were measured before and after ozone treatments (different concentrations and time of exposure), as well as in all fractions obtained during wheat processing. The optimal conditions of ozone treatments were identified in order to maximize the reduction of contaminants and maintain unaffected chemical and rheological parameters of durum wheat. Therefore, optimal ozonation conditions were applied to 20 kg of durum wheat that were successively processed into semolina and pasta. A significant reduction of the levels of some contaminants was obtained, with minor influence on qualitative parameters of durum wheat. The scale up at industrial level for ozone treatment of wheat grains will be discussed.

Ochratoxin A (OTA) is an extremely harmful mycotoxin, having nephrotoxic, immunosuppressive, teratogenic and carcinogenic properties. Aspergillus carbonarius was identified as the main species responsible for OTA accumulation in grape and derived products. During winemaking, 95% of OTA originally present in grapes remains adherent onto pomaces, the main by-product. Grape pomaces are increasingly being used as starting material in the industrial production of plant food supplements (PFS), food colouring and tartrates. The occurrence of OTA in commercially available PFS (24 samples), food colouring (13 samples) and leavening agents containing tartrates (4 samples), all derived from Vitis vinifera was investigated in this study by using an improved HPLC-FLD method. The occurrence of OTA in 32 samples of grape pomaces collected from vineries of Apulia and Basilicata during 2013 and 2014 was also investigated. OTA was found in 75% of commercial PFS samples and 69% of food colouring samples at levels of 0.50-20.23 ?g/Kg and 0.50-32.00 µg/kg, respectively. The four commercial leavening agents containing tartrates were negative for OTA. Ninety-six percent of grape pomaces samples collected in 2013 contained OTA at levels of 3.60-140.90 µg/kg. All samples collected in 2014 contained OTA at level of 2.80-47.30 µg/kg. Higher levels of OTA (up to 849.10 µg/kg) were measured in samples of grape pomaces collected in Apulia in the past. The high incidence of positive samples as well as the high variability of OTA levels in grape pomaces makes it imperative to check the OTA level in this starting material if used for production of PFS and food colouring agents. Maximum permitted level(s) of OTA should be established in commercial PFS and food colouring agents derived from V. vinifera due to the high incidence of OTA contamination in these products.

Objectives. Almonds and apricot kernels have several health benefits. They are a good source of proteins, fiber, unsaturated fats, vitamins and minerals. They are consumed as raw nuts or as ingredients of derived products. However they can be contaminated by aflatoxins (AFs), well known carcinogenic mycotoxins. We evaluated the evolution of AFs during transformation processes of almonds into traditional Italian products, such as pastries, nougat and syrup which involves blanching, peeling, cooking, roasting and water infusion. The efficacy of sorting to segregate AFs contaminated apricot kernels was also assessed. Materials and MethodsExperiments were conducted on naturally contaminated apricot kernels and almonds inoculated with a toxigenic strain of Aspergillus flavus. A robust and horizontal HPLC-FLD method was optimized and used for AFs determination in all the matrices considered in this study1.ResultsBlanching processes (by steaming or boiling in water) did not reduce AFs levels in peeled almonds and apricot kernels. Standard roasting conditions produced up to 50% reduction of AFs. During nougat preparation it was observed a 54-70% AFs reduction due to the caramelisation of sugar on almond surface. AFs were instead stable during cooking of pastries. During almond syrup preparation 18-25% of AFs passed in the final syrup and the whole process produced a marked increase of total AFs. This increase was probably due to the water infusion of ground peeled almonds that activates endogenous almond enzymes that release free AFs from masked AFs1. To reduce AFs contamination in apricot kernels we applied the manual colour sorting to peeled kernels and obtained excellent results: 97-99% AFs reduction by removing only discoloured kernels.ConclusionsRoasting, caramelisation and manual sorting of peeled nuts were identified as effective processes to reduce AFs in nuts. We observed, for the first time, a marked increase of AFs during almond syrup preparation which suggests the existence of masked AFs never reported before. The separation of discolored apricot kernels is a feasible strategy to remove almost all AFs in contaminated peeled kernels. These information are extremely useful and can be exploited by food producers to improve the safety of nuts and derived products.1 Zivoli et al. 2014. Effect of Almond Processing on Levels and Distribution of Aflatoxins in Finished Products and By-products. J. Agric. Food Chem. 62: 5707-5715.

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels wasassessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched,peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) fromhealthy ones. The samples obtained from the two sorting approaches were ground, homogenized, andanalysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure thedistribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitatedin all collected fractions at levels ranging from 1.7 to 22,451.5 g/kg of AFB1 + AFB2, whereas AFG1and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernelssince the removal of discolored kernels (2.6%-19.9% of total peeled kernels) removed 97.3%-99.5% oftotal aflatoxins. The combination of peeling and visual/manual separation of discolored kernels isa feasible strategy to remove 97%-99% of aflatoxins accumulated in naturally-contaminated samples.Electronic optical sorter gave highly variable results since the amount of AFB1 + AFB2 measured inrejected fractions (15%-18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improvedimmunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins(0.01-0.05 g/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercialproducts containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levelswere found in 38% of the tested samples and ranged from 0.06 to 1.50 g/kg for AFB1 and from 0.06to 1.79 g/kg for total aflatoxins.

L'ocratossina A (OTA) è la micotossina più diffusa nei vari prodotti agroalimentari la cui formazione è dovuta a diverse specie fungine del genere Aspergillus e Penicillium, in particolare A. carbonarius. Quest'ultima è stata identificata come la specie fungina maggiormente responsabile della contaminazione da OTA delle uve, vini, succhi d'uva e uva passa. Di particolare rilevanza è la contaminazione delle uve rosse coltivate in alcune aree geografiche ad elevato rischio di infezione da A. carbonarius che colonizza le uve durante le fasi di maturazione. La presenza e la diffusione di questo fungo e il conseguente accumulo di OTA nei vigneti è fortemente influenzata dall'area geografica, da fattori climatici e ambientali, dalla suscettibilità varietale e da eventuali lesioni degli acini che favoriscono l'ingresso e la diffusione del fungo nelle uve [1]. Con il Regolamento CE n.123/2005, il limite massimo consentito è stato fissato a 2 ?g/L per vino, mosto e succo d'uva [2], e pertanto sono state studiate diverse strategie per il controllo e la riduzione della contaminazione di OTA, sia in campo (con l'uso di fungicidi e agenti di biocontrollo) che in cantina (con l'uso di carbone attivo enologico e tecniche di ripasso). Il ripasso breve di mosti/vini contaminati su vinacce fresche o stabilizzate è un approccio naturale che rimuove l'OTA conservando o migliorando le caratteristiche organolettiche di mosti/vini trattati [3]. Recentemente sono stati sviluppati un prototipo ed un processo di stabilizzazione delle vinacce, validati anche in cantina, che permettono di eseguire in automatico il processo di ripasso in sole 5 ore [4]. In questo studio è stata analizzata la variazione, rispetto al controllo (A), dei profili metabolomici (ottenuti mediante analisi 1H NMR) di vini Primitivo trattati con metodo del ripasso su vinacce sempre di cultivar Primitivo (fresche, B, stabilizzate, C) e su vinacce fresche di Aglianico (D) o trattati con carbone attivo enologico (E).

The increasing availability of fungal genomes and bioinformatic tools have led to the identification of clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis (1). The genome sequencing of Aspergillus carbonarius has advanced the knowledge of the molecular mechanism of biosynthesis of ochratoxin A (OTA), one of the most important mycotoxin contaminating several commodities. Differently from other mycotoxins, the elucidation of the genetic background of OTA biosynthesis has remained uncompleted for a long time. Aspergillus carbonarius is the major responsible of OTA contamination of wine and other grape products in the Mediterranean area, constituting a great health risk and cause of important economic losses (2). The analysis of A. carbonarius genome has revealed the presence of a great number of PKSs and NRPSs, enzymes having an essential role in the synthesis of fungal secondary metabolites. Subsequently, the identification of the PKS putatively involved in the biosynthesis of OTA has led to an extensive study of the adjacent genomic region, in the attempt to identify other genes involved and to define the OTA biosynthesis cluster. The roles of three key genes -AcOTApks, AcOTAnrps and AcOTAhal - have been demonstrated by gene knock-out approach and the order of the fundamental enzymatic steps in the biosynthesis pathway of OTA has been clarified. These studies demonstrated that the enzymatic step involving the addition of phenilalanine to the polyketide ring takes place before the chlorination step. Moreover, it was demonstrated that OT? is not a precursor of OTA but rather a product of OTA hydrolysis (3, 4). Other predicted genes in the cluster need to be further investigated to fully clarify the structural and regulatory mechanisms of toxin production, among which the genes coding a p450 monooxygenase, a transcription factor, a transporter protein and an aspartyl protease. Transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.References1. Brakhage A.A., 2013. Nature Reviews Microbiology 11.1: 21-32.2. Perrone G. et al., 2008. Aspergillus in the genomic era, Academic Publishers, Wageningen, 2008, 179-212.3. Gallo A. et al., 2012. Appl. Environ. Microbiol., 78 (23), 8208-8218.4. Ferrara M. et al., 2016. Appl. Environ. Microbiol., 82 (18), 5631-5641.

Deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) are toxicologically relevant mycotoxins frequently occurring in cereals and cereal-based feeds and recently quantified in vivo in piglets urines in dose dependent manner [1]. The possible metabolic effects due to mycotoxins exposure were evaluated by 1H-NMR spectroscopy and MVA (Multivariate Analysis) of exposed piglets urine. Four piglet groups (four animals per group) received feed boluses containing DON, AFB1, FB1, ZEA, and OTA at four increasing doses (one bolus per piglet). The 24 h post dose urines were collected, analysed and results were compared with the controls (urines from the same piglets sampled the day before administration). Interestingly, the urine from piglets fed with high contaminated boluses showed higher content of p-cresol glucoronide, phenyl acetyl glycine and creatinine and lower level of TMAO with respect to the control.[1]. Gambacorta L, Solfrizzo M, Visconti A, Powers S, Cossalter AM, Pinton P, Oswald IP, Validation study on urinary biomarkers of exposure for aflatoxin B1, ochratoxin A, fumonisin B1, deoxynivalenol and zearalenone in piglets. World Mycotoxin Journal. 2013 6(3): 299 - 308.

The multi-biomarker approach was used to validate urinary biomarkers in piglets administered with boluses contaminated with different doses of a mixture of deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA). Boluses contaminated with target levels of mycotoxins were prepared by slurrying with water and freeze-drying feed material fortified with extracts of culture materials of selected toxigenic fungi. Piglets were individually placed in metabolic cages to collect urine before gavage and in 24 h urine post dose. Urine samples were hydrolysed with ?-glucuronidase enzyme and analysed by a multi-biomarker LC-MS/MS method developed and validated to identify and measure biomarkers of FB1, OTA, DON, ZEA and AFB1. Urinary levels of FB1, OTA, DON + de-epoxy-deoxynivalenol (DOM-1), ZEA + alpha-zearalenol (?-ZOL) and aflatoxin M1 (AFM1), were selected as biomarkers of FB1, OTA, DON, ZEA and AFB1, respectively. Mean percentages of dietary mycotoxins excreted as biomarkers in the 24 h post-dose urines were 36.8% for ZEA, 28.5% for DON, 2.6% FB1, 2.6% for OTA and 2.5% for AFB1. A good correlation was observed between the amount of mycotoxins ingested and the amount of the relevant biomarkers excreted in 24 h post-dose urines. Linear dose-response correlation coefficients ranged between 0.68 to 0.78 for the tested couples mycotoxin/biomarker. The good sensitivity of the LC-MS/MS method and the good dose-response correlations observed in this study permitted to validate the selected mycotoxin biomarkers in piglet at dietary levels close to the maximum permitted levels reported in the directive 2003/100/EC for AFB1 and the guidance values reported in the recommendation 2006/576/EC for DON, ZEA, OTA and FB1.