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Alzheimer's disease (AD) is a widespread multifactorial aging-related pathology, which includes cholinergic deficit among its main causes. Following a multi-target design strategy, the structure of the approved drug donepezil was taken as the starting point for generating some new potential multi-functional compounds. Therefore, a series of twenty molecular hybrids were synthesized and assayed against three different enzymes, namely the well-established targets acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and the innovative one fatty acid amide hydrolase (FAAH). In silico studies confirmed the interaction of benzylpiperidine and the benzylpiperazine isostere with the catalytic anionic site (CAS) of AChE, while the aryloxycarbonyl portion appeared to be important for the interaction with the peripheral site (PAS). A QSAR study was carried out on AChE inhibition data, which revealed that the inhibition potency seems to depend upon the length of the spacer and the number of polar atoms. The docking poses of selected compounds within BChE and FAAH were also calculated. Furthermore, pharmacokinetics and drug-likeness properties were assessed by chemoinformatic tools. Several piperidine derivatives (in particular compound 10) showed interesting profiles as multi-target directed agents, while the lead piperazine derivative 12 (SON38) was found to be a more potent and selective AChE inhibitor (IC

The present study investigated the dietary and urinary OTA occurrence among 44 Lebanese children. Relying on HPLC-FLD analysis, OTA was found in all the urine samples and in 46.5% and 25% of the 24 h duplicate diet and dinner samples, respectively. The means of OTA levels in positive samples were 0.32 ± 0.1 ng/g in 24 h diet, 0.32 ± 0.18 ng/g in dinner and 0.022 ± 0.012 ng/mL in urines. These values corresponded to margin of exposure (MOE) means of 7907 ± 5922 (neoplastic) and 2579 ± 1932 (non-neoplastic) calculated from positive 24 h diet, while 961 ± 599 (neoplastic) and 313 ± 195 (non-neoplastic) calculated from the urine. Since the MOE levels for the neoplastic effect were below the limit (10,000), a major health threat was detected and must be addressed as a health institutions’ priority. Besides, the wide difference between PDIs and MOEs calculated from food and urine suggests conducting further OTA’s toxicokinetics studies before using urine to measure OTA exposure.

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 mycotoxin limits are in force for herbs. A total of 132 samples of spices (94) and herbs (38) purchased from Beirut in Lebanon 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) ranged from 0.1 μg/kg (ZEA) to 20.5 μg/kg (DON) and limits of quantification (LOQ) ranged from 0.3 μg/kg (ZEA) to 68.2 μg/kg (DON). 80% of analysed samples were contaminated by 1–11 mycotoxins. Total aflatoxins and ochratoxin A were detected in 19 and 30% of spices, 8 and 11% of herbs, respectively. Mean levels of total aflatoxins and ochratoxin A were 168.1 and 7.1 μg/kg in positive spices, 36.1 and 7.0 μg/kg in positive herbs, respectively. 78 and 10% of positive spice samples contained aflatoxin and ochratoxin A at levels higher than the limits, 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). Within the non-regulated mycotoxins FB1 was the most occurring (60% in spices, 55% in herbs) followed by FB2 (35% in spices, 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 AFG2 were never detected. Mean levels of FB1, FB2, ZEA and DON in positive samples of spices were 6432.3, 203.2, 30.6, 1751.4 μg/kg, respectively; in positive samples of herbs they were 2826.3, 214.9, 2.8, 589.7 μg/kg, respectively. The whole results demonstrate the higher susceptibility of spices to mycotoxin contamination with respect to herbs. Comparison of results obtained for samples produced with (81) and without (51) HACCP and GMP showed that the implementation of HACCP and GMP practices seems to be effective in reducing the occurrence of regulated mycotoxins but was ineffective for the non-regulated ones. The samples analysed in this study originated from at least 15 Countries and the results obtained gives indications about 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 study highlight the problem of mycotoxin contamination in spices and herbs consumed in Lebanon. The occurrence of high levels of aflatoxins and OTA in some non-regulated spices and herbs suggests the addition of these matrices in the list of regulated ones. The high number of positive samples and the high levels of fumonisins observed in this study suggest the inclusions of these mycotoxins in the list of regulated mycotoxins for these matrices.

Relazione tecnica delle attività espletate durante il primo anno del progetto con primi risultati ottenuti nei diversi workpackage e task del progetto.

Spices are susceptible to mycotoxin contamination which can cause gastrointestinal and adverse central nervous symptoms in humans, which highlights the importance of assessing the risk of their consumption on a daily basis. The aim of this study was to assess the risk of mycotoxin intake from spices in routinely prepared Lebanese dishes. 150 households were interviewed about their usage of 27 type of spices and 6 routinely prepared Lebanese dishes. Results showed a high variability in consumption levels. Among the investigated dishes, the minimum number of spices that were consumed in a dish was 13 while the maximum was 18. The mean intake of one spice ranged from 0.26 g/portion observed for cloves to 5.37 g/portion for cinnamon, with its intake per portion more than 1 g in 2/3 of dishes. 20% of portion sizes of coriander, cinnamon and fennel, had an intake exceeding 5 g/portion. Ochratoxin A (OTA) Probable Daily Intake (PDI) had a mean of 0.11 ng/kg-bw/day. Mean PDI of fumonisin B1 (FB1) was 79.3 ng/kg-bw/day. Aflatoxin B1 (AFB1) PDI had a mean of 1.55 ng/kg-bw/day. The Margin of Exposure (MOE) of AFB1 ranged from 108.10 to 4444.44. The present study showed that the risk of AFB1 from spices is a matter of concern while the risk of OTA and FB1 is limited with the exception of FB1 from garlic and onion.

Spices are susceptible to mycotoxin contamination which can cause gastrointestinal and adverse central nervous symptoms in humans, which highlights the importance of assessing the risk of their consumption on a daily basis. The aim of this study was to assess the risk of mycotoxin intake from spices in routinely prepared Lebanese dishes. 150 households were interviewed about their usage of 27 type of spices and 6 routinely prepared Lebanese dishes. Results showed a high variability in consumption levels. Among the investigated dishes, the minimum number of spices that were consumed in a dish was 13 while the maximum was 18. The mean intake of one spice ranged from 0.26 g/portion observed for cloves to 5.37 g/portion for cinnamon, with its intake per portion more than 1 g in 2/3 of dishes. 20% of portion sizes of coriander, cinnamon and fennel, had an intake exceeding 5 g/portion. Ochratoxin A (OTA) Probable Daily Intake (PDI) had a mean of 0.11 ng/kg-bw/day. Mean PDI of fumonisin B

Aflatoxins are fungal metabolites extensively produced by many different fungal species that may contaminate a wide range of agricultural food products. They have been studied extensively because of being associated with various chronic and acute diseases, especially immunosuppression and cancer, and their presence in food is strictly monitored and regulated worldwide. Aflatoxin detection and measurement relies mainly on chemical methods usually based on chromatography approaches, and recently developed immunochemical based assays that have advantages but also limitations, since these are expensive and destructive techniques. Nondestructive, optical approaches are recently being developed to assess presence of contamination in a cost and time effective way, maintaining acceptable accuracy and reproducibility. In this paper are presented the results obtained with a simple portable device for nondestructive detection of aflatoxins in almonds. The presented approach is based on the analysis of fluorescence spectra of slurried almonds under 375 nm wavelength excitation. Experiments were conducted with almonds contaminated in the range of 2.7–320.2 ng/g total aflatoxins B (AFB1 + AFB2) as determined by High Performance Liquid Chromatography with Fluorescence Detection (HPLC/FLD). After applying pre-processing steps, spectral analysis was carried out using a binary classification model based on Support Vector Machine (SVM) algorithm. A majority vote procedure was then performed on the classification results. In this way we could achieve, as best result, a classification accuracy of 94% (and false negative rate 5%) with a threshold set at 6.4 ng/g. These results illustrate the feasibility of such approach in the great challenge of aflatoxin detection for food and feed safety.

Ochratoxin A (OTA) occurrence in grapes is caused by black Aspergilli (Aspergillus carbonarius followed by A. niger) vineyards contamination. It depends on climatic conditions, geographical regions, damage by insects, and grape varieties. Good agricultural practices, pesticides, and fungicides seem adequate to manage the problem during low OTA risk vintages, but the development of new strategies is always encouraged, especially when an extremely favourable condition occurs in the vineyard. Electrolysed oxidising water (EOW) has become an interesting alternative to chemicals in agriculture, mainly during the post-harvest phase. This study tested the fungicidal efficacy of EOW generated by potassium chloride, in vitro, on black Aspergilli conidia, and detached grape berries infected by A. carbonarius. Then, during field trials on Primitivo cv vineyard treated with EOW, A. carbonarius contamination, and OTA levels were compared with Switch® fungicide treatment (0.8 g/l). Black Aspergilli conidia were killed on plate assay after 2 min of treatment by EOW containing >0.4 g/l of active chlorine. EOW (0.6 g/l active chlorine) treatment reduced the rate of A. carbonarius infections in vitro of about 87–92% on detached berries and, more than half in the field trials, although Switch® showed better performance. A significant reduction in the OTA concentration was observed for the EOW and Switch® treatments in vitro (92% and 96%, respectively), while in the field trials, although the average decrease in OTA was recorded in the treated grapes, it was not statistically significant. These results highlighted that EOW could be considered effective, as a substitute for fungicides, to reduce the contamination of A. carbonarius and OTA on grapes.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) were screened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were used for the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1 and 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). Frequency of 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 of mycotoxin 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 mycotoxins in each sample ranged from 2 to 16 mycotoxins (mean 7). No statistical correlation was found between moulds and their mycotoxins occurrence. Within the four groups of peppers collected herein (fresh, dried, grounded and fried) higher percentages of contamination and mycotoxin levels were measured in grounded peppers, whereas much lower values were observed for fried peppers. The high percentages of positive samples and the high levels of some mycotoxins observed in this study confirm the susceptibility of peppers to mycotoxin contamination and claims for an improvement of the conditions used during production and drying process.

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 24 h (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., Highlights • A single dose of mycotoxins mixture (DON, AFB1, FB1, ZEN, and OTA ) could produce serious impairments of gut microbiota of piglets. • The explorative metabolomic analysis on urine piglets, fed with boluses contaminated with mycotoxins mixtures, was performed. • NMR-based MVA resulted in a higher urinary concentrations of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentrations of betaine and TMAO after treatment with respect to own controls.

The emerging Alternaria mycotoxins tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tentoxin (TTX) are not yet regulated worldwide awaiting for more data on their toxicity and natural occurrence. A total of 131 samples of spices (94) and herbs (37) were collected in Beirut (Lebanon) markets and analysed for TeA, AOH, AME, ALT and TTX by using a UPLC-MS/MS method based on ‘extract, dilute and shoot’ approach. High percentage (89%) of samples were contaminated by 1–5 mycotoxins whereas 14% of samples were contaminated with a mixture of TeA, AOH, AME and TTX. TeA was the predominant mycotoxin with the highest percentages of positive samples (76%), followed by AME (46%), TTX (37%), AOH (34%) and ALT (5%) with overall mean/max levels of 2453.2/106792.8, 17.1/306, 14.8/179.4, 35.0/636.4 and 2.8/22.1 μg/kg, respectively (middle bound approach). Higher percentage of positive samples and mean mycotoxin levels were observed in spices as compared to herbs. The spices with higher levels of mycotoxin contamination were, in descending order, red chili, paprika, caraway, coriander seeds, onion powder, white pepper and garlic powder whereas within the herbs they were corn silk, violets and marjoram. Based on the mean levels of TeA measured in different spices and herbs, the daily consumption of >3.3 g red chili, >3.7 g paprika, >16.7 g caraway or >36 g of corn silk will exceed the threshold of toxicological concern (TTC) of TeA (1,500 ng/kg bw per day). For AOH or AME the TTC (2.5 ng/kg bw/day) will be exceeded by a daily consumption >0.5 g of white pepper, >0.9 g of onion powder, > 1.4 g of garlic powder, >1.7 g of black pepper, >1.9 g of sage, >3.3 g of violets or > 4.4 g of thyme leaves. This study provide large information on the natural occurrence of Alternaria mycotoxins in spices and herbs useful for the human exposure assessment and future regulation.

Aflatoxin detection currently relies mainly on chemical methods usually based on chromatography approaches, and recently developed immunochemical based assays that are fairly accurate, however, they are time-consuming, expensive and destructive. Non-destructive, optical approaches are recently being developed in order to assess the presence of contamination in a cost and time-effective way, maintaining high levels of accuracy and reproducibility, but are usually based on the benchtop and expensive instruments. Here we will present the evolution of results of the analysis of fluorescence spectra of contaminated almond samples during the development of an optical multi-sensor device in the framework of PhasmaFOOD project. The aim of the project was to develop a low cost and portable instrument comprising multispectral and imaging capabilities, conjugated with a cloud reference database and analysis toolbox for food features analysis. One of the use cases of the project is the fast, reliable and non-destructive detection of mycotoxin (in particular aflatoxin) contamination in food products. For this use case, we used in particular fluorescence spectroscopy and different approaches to data analysis. After the first feasibility tests in the range of mg/g contamination range with a simple and effective analysis that led to highly reliable results, the work was focused on the detection limits with samples (almond) in the range of 0-291 ng/g acquired with a simple portable device and excitation light at 365 nm wavelength. An ad hoc processing strategy based on a feature selection steps coupled with a nonlinear classifier has been developed and test with two different datasets collected one month from the other another. The system performances have been evaluated training the classification model with one dataset and testing its with the other. The results have shown an accuracy higher than 80% with a threshold lower than 10ppb as contamination level.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal , shows a high similarity to a halogenase that is located in the OTA cluster of A. niger . The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps , indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius . IMPORTANCE Ochratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius . The encoding gene, AcOTAhal , is contiguous to biosynthetic genes that we have already described ( nrps and pks ), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius , which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.

Pancreatic lipase, a key enzyme for lipid absorption, is one of the most important targets for the treatment of obesity, while natural compounds have recently attracted much interest as potential inhibitors of this enzyme. Here, in an attempt to find new effective agents, the methanolic extract from Moricandia arvensis (L.) DC. and its sub-extracts were investigated for their potential inhibitory activity. The ability to inhibit pancreatic lipase was verified through the in vitro evaluation of the prevention of p-nitrophenyl caprylate hydrolysis. The antioxidant activity was also verified by means of DPPH and &beta, carotene bleaching tests. Compositional profiling revealed that flavonoid glycosides were the main specialized metabolites present in the methanolic extract from the aerial parts of the plant with kaempferol and quercetin representing the two O-glycosylated aglycones. Kaempferol-3-O-&beta, (2&Prime, O-glucosyl)-rutinoside and kaempferol-3-O-a-arabinosyl-7-O-rhamnoside were the most abundant flavonols. The crude methanolic extract and the dichloromethane and ethyl acetate sub-extracts showed a strong lipase inhibitory activity, with IC50 values of 2.06 &plusmn, 0.02, 1.52 &plusmn, 0.02 and 1.31 &plusmn, 0.02 mg/mL, respectively. The best capacity to scavenge DPPH radical was detected for the ethyl acetate sub-extract (IC50 = 171.9 &plusmn, 1.0 &micro, g/mL), which was also effective in protecting linoleic acid from peroxidation (IC50 = 35.69 &plusmn, 2.30 &micro, g/mL). Obtained results support the hypothesis that M. arvensis can be a source of bioactive phytochemicals for the pharmacological inhibition of dietary lipids absorption.

Twelve different approaches commonly used for the 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 cereals and feed materials. They comprised different extraction solvents, types of cleanup [solid-phase extraction, QuEChERS, and immunoaffinity (IMA)], and calibration approaches (external or matrix-matched). The percentage of mycotoxins with acceptable recovery, according to Regulation (EC) No. 401/2006, ranged from 9 to 100%. The approach giving the highest percentage of acceptable results was selected and further tested for corn, rice, and feed spiked at three different mycotoxin levels (low, medium, and high). The method is based on extraction with MeOH–water (70 + 30, v/v) and cleanup with two multiantibody IMA columns. For corn and rice spiked at low mycotoxin levels, a significant matrix effect was observed and was compensated by using 13C calibration. At higher mycotoxin levels (medium and high), matrix effects were negligible as no significant differences were observed for the majority of recovery results calculated by 13C calibration and external calibration. Although the proposed method still needs improvement in terms of accuracy and, to a lesser extent, precision, it was successfully tested with four proficiency tests in buckwheat, corn, rice, and feed, giving acceptable z-scores for 97% (34 out of 35) of results.

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

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.

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 B1 (FB1), fumonisin B2 (FB2), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), ?-zearalenol (?-ZOL), ?-zearalenol (?-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.

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 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. References 1. 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.

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

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.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal 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 with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean ± standard deviation 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 indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with b-glucuronidase and 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 in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

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 the 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, β-zearalenol and zearalenone, 87% for α-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.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 μg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 μg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 μg/kg in good maize, respectively, and of 14 and 5.8 μg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 μg/kg in good maize, respectively, and of 111 and 135 μg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

International audience; 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 mu g of fumonisin B, (FB1), 275 mu g of deoxynivalenol (DON), 29 mu g of zearalenone (ZEN), 6.5 mu g of aflatoxin B-1 (AFB1()) and 6.6 mu g of ochratoxin A (OTA) corresponding to 2.2, 0.8, 0.08, 0.02, and 0.02 mu 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 AFB(1) (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.

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. 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) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB1 + AFB2, whereas AFG1 and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a 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 in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB1 and from 0.06 to 1.79 μg/kg for total aflatoxins.

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M(1) (AFM(1)), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B(1) (FB(1)) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL(-1), using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.

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

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.

A liquid chromatographic method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn-based foods for infants and young children was subjected to an interlaboratory validation study involving 11 laboratories. Five blind duplicate sample pairs of each matrix were analyzed to establish the accuracy, repeatability, and reproducibility of the method. Mass fractions in the baby food samples ranged from 89.1 to 384.4 microg/kg FB1 and from 22.5 to 73.6 microg/kg FB2. The method involved a warm extraction with citrate phosphate buffer-methanol-acetonitrile (50 + 25 + 25, v/v/v), a cleanup through an immunoaffinity column, and an end-determination of fumonisins by LC after automated precolumn derivatization with o-phthaldialdehyde reagent. RSDs for within-laboratory repeatability (RSDr) ranged from 6.8 to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDs for between-laboratory reproducibility (RSDR) ranged from 15.4 to 26.2% for FB1 and 21.6 to 36.3% for FB2. Mean FB1 recoveries from baby foods spiked at 100.0 and 250.0 microg/kg were 89 and 96%, respectively; for FB2 spiked foods at 25.0 and 62.5 microg/kg recoveries were 90 and 85%, respectively. HorRat values ranged from 0.8 to 1.2 for FB1, whereas for FB2 they ranged from 0.9 to 1.4 when calculated according to Horwitz, and from 1.0 to 1.7 when calculated according to Thompson, indicating an acceptable among-laboratory precision for all matrixes (HorRat values2).

A liquid chromatographic method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn-based foods for infants and young children was subjected to an interlaboratory validation study involving 11 laboratories. Five blind duplicate sample pairs of each matrix were analyzed to establish the accuracy, repeatability, and reproducibility of the method. Mass fractions in the baby food samples ranged from 89.1 to 384.4 μg/kg FB1 and from 22.5 to 73.6 μg/kg FB2. The method involved a warm extraction with citrate phosphate buffer–methanol–acetonitrile (50 + 25 + 25, v/v/v), a cleanup through an immunoaffinity column, and an end-determination of fumonisins by LC after automated precolumn derivatization with-o-phthaldialdehyde reagent. RSDs for within- laboratory repeatability (RSDr) ranged from 6.8 to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDs for between-laboratory reproducibility (RSDR) ranged from 15.4 to 26.2% for FB1 and 21.6 to 36.3% for FB2. Mean FB1 recoveries from baby foods spiked at 100.0 and 250.0 μg/kg were 89 and 96%, respectively; for FB2 spiked foods at 25.0 and 62.5 μg/kg recoveries were 90 and 85%, respectively. HorRat values ranged from 0.8 to 1.2 for FB1, whereas for FB2 they ranged from 0.9 to 1.4 when calculated according to Horwitz, and from 1.0 to 1.7 when calculated according to Thompson, indicating an acceptable among- laboratory precision for all matrixes (HorRat values

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 µg/kg FB1 and 20-200 µg/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 µg/kg for FB1 and 2.2 µg/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 µg/kg.