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.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Recently, the biosynthetic mechanism of this mycotoxin has been mainly elucidated by experiments of knocking out of the key biosynthetic genes. The mutant strains of A. carbonarius, in which the AcOTAnrps gene had been disrupted, was unable to produce OTA but retained its ability to degrade OTA into OT? when it was grown in presence of exogenous OTA. Microbial degradation of OTA is due to the enzymatic cleavage of the amide bond between L-?-phenylalanine and OT? by proteolytic proteins. Then, an in silico screening has been made on the available genome sequence of A. carbonarius ITEM 5010 to identify genes encoding proteases and to investigate their involvement in the OTA degrading activity of A. carbonarius. Preliminary transcriptomic analysis allowed selecting eight protease encoding genes that were expressed at increased level during OTA production. From the analysis of functional domains of the deduced protein sequences, four identified genes encode for aspartic proteases, three of them encode for serine proteases and one for a metalloprotease. Wild type and three mutant strains of A. carbonarius ITEM 5010 (?AcOTAnrps, ?AcOTApks, ?AcOTAhal) previously obtained and resulted to be unable to produce OTA, have been incubated in presence of OTA under different conditions and time of growth. Expression levels during growth and activation rate of the selected protease genes are under investigation in order to establish their involvement in the degradation activity of A. carbonarius strains.

Grape pomace (pulp and skins) was investigated as a new biosorbent for removing mycotoxins from liquid media. In vitro adsorption experiments showed that the pomace obtained from Primitivo grapes is able to sequester rapidly and simultaneously different mycotoxins. Aflatoxin B-1 (AFB(1)) was the most adsorbed mycotoxin followed by zearalenone (ZEA), ochratoxin A (OTA), and fumonisin B-1 (FB1), whereas the adsorption of deoxynivalenol (DON) was negligible. AFB(1) and ZEA adsorptions were not affected by changing pH values in the pH 3-8 range, whereas OTA and FB1 adsorptions were significantly affected by pH. Equilibrium adsorption isotherms obtained at different temperatures (5-70 degrees C) and pH values (3 and 7) were modeled and evaluated using the Freundlich, Langmuir, Sips, and Hill models. The goodness of the fits and the parameters involved in the adsorption mechanism were calculated by the nonlinear regression analysis method. The best-fitting models to describe AFB(1), ZEA, and OTA adsorption by grape pomace were the Sips, Langmuir, and Freundlich models, respectively. The Langmuir and Sips models were the best models for FB1 adsorption at pH 7 and 3, respectively. The theoretical maximum adsorption capacities (mmol/kg dried pomace) calculated at pH 7 and 3 decreased in the following order: AFB(1) (15.0 and 15.1) > ZEA (8.6 and 8.3) > OTA (6.3-6.9) > FB1 (2.2 and 0.4). Single- and multi-mycotoxin adsorption isotherms showed that toxin adsorption is not affected by the simultaneous presence of different mycotoxins in the liquid medium. The profiles of adsorption isotherms obtained at different temperatures and pH and the thermodynamic parameters (Delta G degrees, Delta H degrees, Delta S degrees) suggest that mycotoxin adsorption is an exothermic and spontaneous process, which involves physisorption weak associations. Hydrophobic interactions may be associated with AFB(1) and ZEA adsorption, whereas polar noncovalent interactions may be associated with OTA and FB1 adsorption. In conclusion, this study suggests that biosorption of mycotoxins onto grape pomace may be a reasonably low-cost decontamination method.

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.

Zearalenone (ZEN), a mycotoxin with high estrogenic activity in vitro and in vivo, is a widespread food contaminant that is commonly detected in maize, wheat, barley, sorghum, rye and other grains. Human exposure estimates based on analytical data on ZEN occurrence in various food categories and food consumption data suggest that human exposure to ZEN and modified forms of ZEN may be close to or even exceed the tolerable daily intake (TDI) derived by the European Food Safety Authority (EFSA) for some consumer groups. Considering the inherent uncertainties in estimating dietary intake of ZEN that may lead to an under- or overestimation of ZEN exposure and consequently human risk and current lack of data on vulnerable consumer groups, there is a clear need for more comprehensive and reliable exposure data to refine ZEN risk assessment. Human biomonitoring (HBM) is increasingly being recognized as an efficient and cost-effective way of assessing human exposure to food contaminants, including mycotoxins. Based on animal and (limited) human data on the toxicokinetics of ZEN, it appears that excretion of ZEN and its major metabolites may present suitable biomarkers of ZEN exposure. In view of the limitations of available dietary exposure data on ZEN and its modified forms, the purpose of this review is to provide an overview of recent studies utilizing HBM to monitor and assess human exposure to ZEN. Considerations are given to animal and human toxicokinetic data relevant to HBM, analytical methods, and available HBM data on urinary biomarkers of ZEN exposure in different cohorts.

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.

Blue mold is one of the most important postharvest diseases of pome fruit in all producing countries. Its causal agent, Penicillium expansum, is also known to produce the mycotoxin patulin, with mutagenic, immunotoxic, and neurotoxic properties. Aims of the present study were to identify Penicillium isolates associated with blue mould decay of pome fruits in Apulia region (South Italy), verify if their genetic potential to produce patulin corresponded to actual toxin contamination, and compare their in vitro and in vivo toxigenicity. Twenty-nine isolates of Penicillium spp. were recovered from apples and pears with blue mold symptoms. Fruits were analyzed for patulin content and results were compared with in vitro toxin production. In general, patulin production was more conspicuous in vivo (particularly on Golden Delicious apples) than in vitro, although the stronger in vivo producer did not correspond to the stronger in vitro producer. Isolate identification was based on both morphological characters and DNA analysis by PCR amplification with P. expansum species-specific primers and sequencing of beta-tubulin gene. Furthermore, fungal isolates were tested for the occurrence of gene (patN) coding the enzyme isoepoxydon dehydrogenase (IDH), involved in the patulin metabolic pathway and considered an useful indicator of critical control points for patulin contamination. All 25 isolates identified as P. expansum were patN and patulin production positive. Moreover, 4 pear isolates belonging to other Penicillium spp. were found, whose identification is being confirmed. They were positive for the patN gene, but only two actually produced patulin. It can be concluded that blue mold of pome fruits in Apulia is mainly associated with toxigenic P. expansum isolates, thus a rapid detection is important to avoid patulin contamination beyond the regulatory limits. Nevertheless, it seems that patN gene alone cannot be considered a predictive assay for production of patulin. An evaluation of its expression level should be carried out.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meat derived products, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species belonging to the genus Aspergillus and Penicillium. OTA is the primary mycotoxin risk in wine and dried vine fruits, its maximum level is regulated by law. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. In particular, Aspergillus carbonarius has been identified as the major cause of contamination in grape berries. This contamination is strongly related to climatic conditions, geographical regions, grape varieties, damage by insects, growing season; in particular great variations may occur from one year to another. So, climate represents an important key-factor in the agro-ecosystem, influencing fungal colonization and ochratoxin A production in grapes. Climate change is expected to have a profound effect on our landscape worldwide, and also to have an important impact on sustainable food production system. Recent studies have reported how the climate change may affect mycotoxins production in the fields and the relevant risk on economically important crops. In this regard, the interacting effect of water stress (aw 0.99-0.93) and different day/night climate conditions simulating nowadays (18-31 and 15-28°C) and climate change scenarios (18-34 and 20-37 °C) in high OTA risk area of Southern Italy during the ripening season, were studied. Mycelial growth rate, OTA production and molecular expression of key genes (PKS, NRPS, Hal, p450, bZIP) of OTA biosynthetic cluster by A. carbonarius ITEM 5010 were measured. Our results showed that, in water stress conditions (0.93 aw), no OTA production was observed and, except at 20-37°C, the growth rate was slower compared to 0.99 aw. A significantly higher amount of OTA was observed at 0.99 aw and 18-34°C climate change scenario. Gene expression, monitored by quantitative real time RT-PCR, gave evidence of the high expression levels of OTA biosynthetic genes in this condition, in particular NRPS and Hal genes were strongly expressed. These preliminary and new results on A. carbonarius in a climate change scenario suggest that a possible slight increase of temperature may lead to higher OTA contamination and to a possible expansion of the risk area in the Mediterranean basin.

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.

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

Deep Eutectic Solvents (DESs) represent an emerging class of biorenewable solvents usuallyprepared by mixing two or three safe and inexpensive components able to form an eutecticmixture with a melting point further below that of the individual components. Due to theirlow toxicity and biodegradability they are progressively replacing conventional volatileorganic compounds (VOCs) in several fields of science. An increasing number of studies, inparticular, have reported the feasibility of extracting bioactive compounds from naturalsources (Ruesgas-Ramón et al., JAFC, 2017, 65, 3591). Our efforts of late have focused onthe use of these media in the extraction of contaminants, such as mycotoxins, from foodmatrices.MethodsAn analytical method for the determination of the mycotoxin Ochratoxin A (OTA) in wheatand some processing products has been developed and validated using choline chloride(ChCl)-based solvent as privileged and biodegradable extraction media (Piemontese et al.Molecules, 2017, 22, 121). We have then implemented the method to include a largerscreening on many solid food matrices using several DESs with different physical-chemicalcharacteristics.ResultsUsing the ChCl/urea (1:2)/water (+ 40% w/w) mixture, the analytical performances, in termof recovery and repeatability for durum wheat, bread crumbs, and biscuits, proved to becomparable to those obtained with conventional and hazardous VOCs, which are typicallyemployed according to the standard and official methods. The tunability of DESs ispromising to extend the use of the validated method to other food commodities. Preliminaryresults obtained with the ChCl/lactic acid (1:2)/water (+25% w/w) mixture have revealedgood extraction performances on a wide number of solid matrices.

An unprecedented, environmentally friendly, and faster method for the determination ofOchratoxin A (OTA) (a mycotoxin produced by several species of Aspergillus and Penicillium andlargely widespread in nature, in wheat and derived products) has, for the first time, been set up andvalidated using choline chloride (ChCl)-based deep eutectic solvents (DESs) (e.g., ChCl/glycerol(1:2) and ChCl/ urea (1:2) up to 40% (w/w) water) as privileged, green, and biodegradable extractionsolvents. This also reduces worker exposure to toxic chemicals. Results are comparable to thoseobtained using conventional, hazardous and volatile organic solvents (VOCs) typical of the standardand official methods. OTA recovery from spiked durum wheat samples, in particular, was to up to89% versus 93% using the traditional acetonitrile-water mixture with a repeatability of the results(RSDr) of 7%. Compatibility of the DES mixture with the antibodies of the immunoaffinity columnwas excellent as it was able to retain up to 96% of the OTA. Recovery and repeatability for durumwheat, bread crumbs, and biscuits proved to be within the specifications required by the currentEuropean Commission (EC) regulation. Good results in terms of accuracy and precision wereachieved with mean recoveries between 70% (durum wheat) and 88% (bread crumbs) and an RSDrbetween 2% (biscuits) and 7% (bread).

Aflatoxins can be found as contaminants in a wide range of foods, such as nuts, cereals, dried fruits and milk. Due to their hepatotoxic and carcinogenic effects, the maximum allowed concentration ofaflatoxins is nowadays regulated in many countries, with levels up to 50 ?g/kg. In routine analysis, the main methods used to determine aflatoxins are based on high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) [1]. Despite the very high sensitivity of these methods, they are destructive, expensive, time consuming and not appropriate for real time control, e.g., online. Consequently, the development of fast, non destructive and economic methods for aflatoxins detection and monitoring in food industry is becoming more and more important.Our studies showed that manual sorting of dark or spotted apricot kernels removed 97.3-99.5% of total aflatoxins [2]. However, discolored seeds could be visually identified only after removing theskins from each seed by means of a time-consuming operation. For these reasons, in this work we investigated the possibility to use NIR-HSI for the fast and nondestructive automated identification of aflatoxin contaminated unpeeled apricot kernels.On the whole 9 hyperspectral images, each one containing 48 kernels, were acquired in the 900-1700 nm range. After image acquisition, the kernels were peeled to identify the dark or spotted kernels and subjected to HPLC analysis for AFB1 quantification. Classification models were then calculated on a training set of NIR spectra extracted from a representative number of non-contaminated and dark seeds, selected on 5 images on the basis of HPLC analysis results as well as of the visual evaluation of the peeled kernels. The remaining 4 images were instead used as independent test set for model validation. Since dark seeds were found to have a higher concentration of AFB1 than spotted seeds, the latter ones were not included in the training set.Different iPLS-DA classification models, built using different signal preprocessing methods and different interval size values, were then evaluated in terms of classification efficiency in cross validation of the training set pixels, in order to select the optimal conditions. The results were reported under the form of predicted probability maps, and for each single kernel the contamination was estimated as the percentage of pixels assigned to the "contaminated" class by the iPLS-DA model.

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.

Fungi cause hazards such as mildew, spoilage, and mycotoxins, so the Chinese government has formulated maximum level limits (MLs) for fungi in spices. However, the current standard culture-based detection method is time-consuming (5-7 days) and unsuitable for the rapid on-site screening of fungal contamination to facilitate immediate control measures. Therefore, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid (<4 h) screening of fungi at levels greater than or equal to those of Chinese MLs (104 cfu/g) in spices, without requiring any expensive instruments. Contaminated samples could be identified based on colour change that was visible to the naked eye. The specificity based on spiked sample experiments and comparisons with the traditional culture-based method demonstrated that this LAMP method could be applicable to the on-site testing of pepper and paprika powder samples, and it could also be used by laymen. Furthermore, this method could be applied to the rapid screening of fungal contamination in other spices, foods and feeds. In addition,Tween-20 was included in the assay to enhance the collection of fungus from sample suspensions.

This review summarises developments in the determination of mycotoxins over a period between mid-2016 and mid-2017. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone are covered in individual sections. Advances in proper sampling strategies are discussed in a dedicated section, as are methods used to analyse botanicals and spices and newly developed LC-MS based multi-mycotoxin methods. This critical review aims to briefly discuss the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

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.

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

Il deossinivalenolo (DON) è una micotossina importante che si ritrova spesso nelle granaglie ed è frequentemente associato alla sua forma modificata DON-3-glucoside (DON-3-Glc). L'ozono (O3) è un potente disinfettante e ossidante che degrada il DON in soluzione acquosa e potrebbe essere efficace se usato direttamente sulle cariossidi. Questo studio riporta l'efficacia dei trattamenti con ozono gassoso nel ridurre DON, DON-3-Glc, batteri, funghi e lieviti in grano duro naturalmente contaminato. Un prototipo è stato utilizzato per erogare l'ozono in modo continuo e omogeneo a diverse concentrazioni e tempi di esposizione. Sono state identificate le condizioni ottimali che non influiscono sui parametri chimici e reologici del grano duro, della semola e della pasta (55 g di O3 h-1 per 6 ore). L'ozonizzazione ha prodotto una riduzione significativa (p<0,05) del conteggio totale (CFU/g) di batteri, funghi e lieviti, di DON (29%) e DON-3-Glc (44%).

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.

Blue mould, caused by Penicillium expansum, is one of the most economically damaging postharvest diseasesof pome fruits, although it may affect a wider host range, including sweet cherries and table grapes. Severalreports on the role of mycotoxins in plant pathogenesis have been published, but few focussed on theinfluence of mycotoxins on the variation in host preference amongst producing fungi. In the present studythe influence of the host on P. expansum pathogenicity/virulence was investigated, focussing mainly on therelationship with patulin production. Three P. expansum strain groups, originating from apples, sweet cherries,and table grapes (7 strains per host) were grown on their hosts of isolation and on artificial media derivedfrom them. Strains within each P. expansum group proved to be more aggressive and produced more patulinthan the other two groups under evaluation when grown on the host from which they originated. Table grapestrains were the most aggressive (81% disease incidence) and strongest patulin producers (up to 554 ?g/g).The difference in aggressivenessamongst strainswas appreciable only in the presence of a living host, suggestingthat the complex pathogen-host interaction significantly influenced the ability of P. expansum to cause thedisease. Incidence/severity of the disease and patulin production proved to be positively correlated, supportingthe role of patulin as virulence/pathogenicity factor. The existence of genetic variation amongst isolates wasconfirmed by the High Resolution Melting method that was set up herein, which permitted discrimination ofP. expansum from other species (P. chrysogenum and P. crustosum) and, within the same species, amongst thehost of origin. Host effect on toxin production appeared to be exerted at a transcriptional level.

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.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the beta-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production. (C) 2014 Elsevier B.V. All rights reserved.

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.

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

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.

Maximum permitted levels for aflatoxins in dried fruits such as almonds and apricot kernels are higherin kernels intended for further processing as compared to ready-to-eat derived products. The effect ofelectronic sorting, peeling and manual colour sorting on apricot kernels was tested to check levels anddistribution of aflatoxins. The fate of aflatoxins during transformation processes of almonds into nougat,pastries and almond syrup (blanching, roasting, water infusion and cooking) was also evaluated. Themass balance approach was used to determine levels and distribution of aflatoxins in each fractioncollected during processing steps. Experiments were conducted on naturally contaminated apricotkernels and almonds inoculated with a toxigenic strain of Aspergillus flavus. An improved HPLC-FLDmethod was used for aflatoxin determination in all the matrices considered in this study. Highly variableresults were obtained with electronic sorting experiments because rejected fractions contained 13-59%of total aflatoxins. The manual colour sorting of peeled apricot kernels gave excellent results because theremoval of discoloured kernels removed 97.3-99.5% of total aflatoxins. Blanching processes (bysteaming or boiling in water) did not reduce aflatoxin levels in blanched almonds and apricot kernelswhereas the removal of skins removed only 7-10% of total aflatoxins. Negligible amounts of aflatoxins(<1%) were found in boiling water analysed after blanching contaminated almonds or apricot kernels.Almond pastries were prepared by mixing almond paste, eggs and sugar that were cooked at 140°C for30 min, 160°C for 20 min or 180°C for 15 min. Aflatoxins were substantially stable during preparationand cooking of pastries and a slight reduction of aflatoxins (10%) was observed only at 180°C. For thepreparation of nougat the peeled almonds were roasted at 150°C for 30 min then mixed with caramel orcaramel+honey at 105°C until cooked, then shaped into small heaps. Roasting produced about 50%reduction of aflatoxins. Higher aflatoxin reduction (82%) was observed by roasting at 150°C for 120min, but almonds lost their organoleptic characteristics. The preparation and cooking of nougat produceda further consistent reduction of 54-70% of aflatoxins. Almond syrup was prepared from peeled almondpaste that was infused in water for 5 hours. After infusion the exhausted almonds were discarded and theinfuse was sugared and boiled until reaching the consistency of syrup. The whole process of almondsyrup preparation produced a marked increase of total aflatoxins probably due to the involvement ofenzymes during the infusion step that released free aflatoxins from masked aflatoxins. About 15-22% oftotal aflatoxins passed in the final syrup during the whole process of almond syrup preparation. Thesenew information could be useful for food producers to keep aflatoxin contamination under control andimprove the safety of almond

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.

A few symptomatic drugs are currently available for Alzheimer's Disease (AD) therapy, but these molecules are only able to temporary improve the cognitive capacity of the patients if administered in the first stages of the pathology. Recently, important advances have been achieved about the knowledge of this complex condition, which is now considered a multi-factorial disease. Researchers are, thus, more oriented toward the preparation of molecules being able to contemporaneously act on different pathological features. To date, the inhibition of acetylcholinesterase (AChE) and of ?-amyloid (A?) aggregation as well as the antioxidant activity and the removal and/or redistribution of metal ions at the level of the nervous system are the most common investigated targets for the treatment of AD. Since many natural compounds show multiple biological properties, a series of secondary metabolites of plants or fungi with suitable structural characteristics have been selected and assayed in order to evaluate their potential role in the preparation of multi-target agents. Out of six compounds evaluated, 1 showed the best activity as an antioxidant (EC50 = 2.6 ± 0.2 ?mol/µmol of DPPH) while compound 2 proved to be effective in the inhibition of AChE (IC50 = 6.86 ± 0.67 ?M) and A?1-40 aggregation (IC50 = 74 ± 1 ?M). Furthermore, compound 6 inhibited BChE (IC50 = 1.75 ± 0.59 ?M) with a good selectivity toward AChE (IC50 = 86.0 ± 15.0 ?M). Moreover, preliminary tests on metal chelation suggested a possible interaction between compounds 1, 3 and 4 and copper (II). Molecules with the best multi-target profiles will be used as starting hit compounds to appropriately address future studies of Structure-Activity Relationships (SARs).

According to the theory of the multifactorial origin of Alzheimer's Disease (AD), in recent years many scientists focused their studies on multi-target agents, acting on features such as the inhibition of acetylcholinesterase (AChE), monoamine oxidases (MAOs) or ?-amyloid (A?) aggregation, the N-methyl-D-aspartate (NMDA) receptor antagonism, and the antioxidant activity, which have been recognized as important at the onset of the pathology [1, 2]. However, recent evidence has shown that the removal and/or redistribution of metal ions at the level of the nervous system can significantly reduce the formation of A? and thus of reactive oxygen species, which are typical of AD and other neurodegenerative diseases. Therefore, the chelation of copper and/or zinc cations is a new fascinating, attractive target for innovative therapies [3].Five secondary metabolites of plants or fungi (1 tenuazonic acid, 2 mycophenolic acid, 3 2-epiradicinol, 4 radicinin, 5 6-methoxymellein) with suitable structural characteristics have been selected and assayed in order to preliminary evaluate their metal chelating properties (Figure 1). These molecules have been also tested on other targets, considering that many natural compounds are able to inhibit the A? aggregation as well as possess an anti-oxidant and anticholinesterase activity [4]. In this Communication we will present the promising results obtained with some selected hit compounds which will address our future studies of Structure-Activity Relationships.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin ?, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin ?, the dechloro analog of ochratoxin ?, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin ? is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

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.

Blue mould is one of the most important postharvest diseases of pome fruit in all producing countries. It is mainly associated to Penicillium expansum that produces the mycotoxin patulin, although other species might be involved. The aim of the present study was to characterise Penicillium isolates associated with blue mould decay of pome fruit marketed in Apulia region (southern Italy), and verify their ability to produce patulin in vitro. Twenty-nine isolates of Penicillium spp. were recovered from pome fruit showing visible blue mould symptoms, and analysed for patulin production. After fungal isolation, the fruits were singularly analysed for patulin content. In general, the isolates proved to produce patulin and most of the pome fruit contained significant amounts of patulin, but there was no quantitative correspondence between in vitro and in vivo toxin accumulation. Isolate identification at species level was based on DNA analysis by P. expansum species-specific primers and sequencing of ?-tubulin gene. Furthermore, fungal isolates were tested for the occurrence of the patN gene coding the enzyme isoepoxydon dehydrogenase (IDH), involved in patulin metabolic pathway and considered a useful indicator of critical control points for patulin contamination. All 26 isolates identified as P. expansum were positive for patN and produced patulin. Moreover, three pear isolates belonging to other Penicillium species were found. They were positive for patN, but only two actually produced patulin. It can be concluded that toxigenic P. expansum isolates are associated with blue mould of pome fruit marketed in Apulia, thus a rapid detection is important to avoid patulin contamination beyond regulatory limits. Nevertheless, the presence of patN gene alone cannot be considered a predictive assay for patulin production. An evaluation of its expression level should be carried out.

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.

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

A proficiency test (PT) was organized for LC-MS/(MS) multi-mycotoxin methods for the determination of eleven mycotoxins (aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins, zearalenone, and fumonisins B1 and B2) in maize. This PT was earlier described by M. Solfrizzo+ and A. De Girolamo?.Although the different performance criteria for mycotoxins stated in the Commission Regulation (EC) no 519/2014 are based more or less on single-mycotoxin methods, the performance of the multi-mycotoxin methods was assessed by DUCARES against the 'single-mycotoxin methods' requirements of the European Commission.The data assessment, with the requirements of the European Commission as the target standard deviation (?p) values, was made on a naturally contaminated maize sample and a spiked maize sample. The z-score performance per mycotoxin was divided in three categories, namely z <=2 satisfactory, 2< z <=3 questionable, z >3 unsatisfactory. For the naturally contaminated maize sample the percentage of satisfactory z-score values (z <=2) was for ten mycotoxins between 68% and 89%. For one mycotoxin (HT-2 toxin) a satisfactory performance of 97% was obtained. For the spiked maize sample the percentage of satisfactory z-score values (z <=2) was between 43% and 94% for eight mycotoxins. For three mycotoxins a satisfactory performance was obtained of 100% (T-2 toxin), 97% (HT-2 toxin) and 97% (aflatoxin G2).The LC-MS/(MS) multi-mycotoxin methods do not meet the performance criteria for most of the mycotoxins in both maize samples in view of the Commission Regulation (EC) no 519/2014.+Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS), M. Solfrizzo et al, Quality Assurance and Safety of crops & foods, March 2013; 5 (1): 15-48?Critical evaluation of LC-MS-based methods for simultaneous determination of deoxynivalenol, ochratoxin A, zearalenone, aflatoxins, fumonisins and T-2/HT-2 toxins in maize, A. De Girolamo et al, World Mycotoxin Journal, August 2013; 6 (3): 317-334

Some of the secondary metabolites produced by Alternariaspecies have been recognized potentially harmful for humanhealth due to their toxicity and occurrence in foodcommodities. Currently, there are no regulations worldwide forthese mycotoxins with the exception of Bavarian health andfood safety authority who decided to limit at 500 mg/kg thetenuazonic acid content in sorghum/millet-based infant food.The European Food Safety Authority (EFSA) performed a riskassessment for four of the known Alternaria mycotoxins(alternariol, alternariol monomethyl ether, tenuazonic acid andtentoxin) and established the threshold for toxicologicalconcern (TTC) for these mycotoxins.In this review recent dataon the biological activity, toxicokinetics, human exposure,modified forms and reduction strategies of the prevalentAlternaria mycotoxins are reported.

Ochratoxin A (OTA) undergoes to enzymatic biodegradation by proteolytic enzymes able to hydrolyze its amide bond with consequent formation of ochratoxin ? (OT?) and L-?-phenylalanine. This mechanism can be regarded as a detoxification method since OT? and L-?-phenylalanine are considered as less and non-toxic, respectively. Different microorganisms belonging to bacterial, yeast and fungal species have been reported to degrade OTA. Several enzymes may be involved in microbiological degradation of OTA, such as carboxypeptidase A, lipase, and acid proteases. Also Aspergillus carbonarius, one of the most important fungal producer of OTA and the major responsible of OTA contamination of grapes, wine and by-products, turned out to be able to degrade OTA. In the attempt to identify the enzyme able to degrade OTA in this microorganism, a protease encoding gene, located in the genomic region recognized as OTA cluster, has driven our attention. In particular, this gene, namely Acap1 of A. carbonarius strain ITEM 5010, encodes for an aspartic protease and is located downstream of the core genes involved in OTA biosynthesis. Acap1gene was isolated and cloned for its characterization. The gene is 1367 bp long and the in silico analyses of the deduced protein sequence of 421 aa revealed that the AcAP1 protein shows the functional typical structure of aspartic protease enzymes. Aspartyl proteases are a highly specific family of proteases that tend to cleave dipeptide bond and they are optimally active at acidic pH. Heterologous recombinant production of the AcAP1 protein has been carried out in order to verify the involvement of AcAP1 in the ability of A. carbonarius in OTA degradation and to analyze its structural and functional properties for a potential biotechnological use of the enzyme. Acap1 gene was cloned in two expression vectors (p426 and pYES), carrying a constitutive and an inducible promoter, respectively, in fusion with a sequence encoding for a His-tag at the 3'-terminus. Three different strains of Saccharomyces cerevisiae, carrying diverse genotypes, have been transformed. Data concerning the protein expression by yeast, evaluation of the protease activity, and purification of the recombinant protein will be produced.

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.

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.

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

Introduction The occurrence of major Fusarium toxins in maize samples collectedin the Black Sea region of Turkey and fate of these toxins during traditional Turkishmaize bread production was investigated. Materials and methods Twenty maizesamples were analysed for deoxynivalenol, zearalenone, fumonisins, T-2 and HT-2toxins. Contamination profiles of maize samples were determined in order tochoose the toxins to be monitored during bread production. Breads were producedfrom low or uncontaminated maize samples and from the mixture of highlycontaminated ones. After baking, crust and crumb of the breads were separatedand analysed by high-performance liquid chromatography standard methods withultraviolet or fluorescence detection. Results and conclusions In total, toxin levelsin 30% of the maize samples were found to be higher than the limits established bythe European Union. After bread processing no significant reduction of deoxynivalenol,zearalenone and fumonisins was measured in the bread crust and crumb.According to the mass balance of mycotoxins measured in maize flour and relevantmaize bread only 2.1%, 0.1% and 3.1% of the total initial amounts of deoxynivalenol,zearalenone and fumonisins, respectively, were lost. Breads were notanalysed for T-2 and HT-2 as their levels in maize were quite low (o50 mg kg1).

Nine recently described Aspergillus species and four known species in section Versicolores were tested for their ability to produce sterigmatocystin on two liquid media, Czapek w/20% Sucrose Broth and Yeast Extract Broth grown in the dark for 1 week at 25 °C. Detection and quantification of ST were performed by reversed-phase liquid chromatography coupled with electrospray ionization ion trap mass spectrometry. Limit of detection was 3 ng/mL and limit of quantification was 10 ng/mL. Nine newly described Aspergillus species from various substrates, A. amoenus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus in section Versicolores were found to produce sterigmatocystin. Production was confirmed in recently collected isolates of A. protuberus and A. versicolor. A. austroafricanus and A. tabacinus did not produce sterigmatocystin.

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).

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.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meats, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species of genus Aspergillus and Penicillium. OTA is the primarily mycotoxin risk in wine and dried vine fruits. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. Nine different black Aspergillus species have been identified on grapes with different secondary metabolites profiles. These species are often difficult to be identified by means of classical methods. The polyphasic approach used in our studies led to characterization of three new non toxigenic species occurring on grapes: A. brasiliensis, A. ibericus and A. uvarum. However, the main source of OTA contamination in grapes is A. carbonarius, followed by A. niger and A. welwitschiae. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, growing season (high susceptibility from early veraison to harvest, with a peak at ripening), and great variations may occur from one year to another. Differently from other mycotoxins, the genes and the enzymatic stages involved in OTA biosynthesis pathway have remained unknown for long. In last years, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key steps of OTA biosynthesis. Genome sequencing of A. carbonarius led us to predict OTA cluster and to elucidate the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) and the order of the enzymatic steps of the biosynthesis pathway. Other predicted genes in the cluster have been identified and analysed, such as a p450 monooxygenase and a transcription factor gene, likely involved in the structural and regulatory mechanisms of OTA production. Furthermore, transcriptomic analyses are in progress to study and clarify the complex genetic picture of the fungus during OTA biosynthesis at a deeper level. Interestingly, recent studies on climate change effects evidenced the influence of raising temperature and CO2 levels on OTA production increase. Managing OTA contamination to reduce risks in grapes implies several strategies, such as implementations of good agricultural practices and risk maps, in association with the use of insecticides and fungicides when favourable climatic conditions occur. In addition, corrective actions can be adopted in wineries.

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.