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An unprecedented, environmentally friendly, and faster method for the determination of Ochratoxin A (OTA) (a mycotoxin produced by several species of Aspergillus and Penicillium and largely widespread in nature, in wheat and derived products) has, for the first time, been set up and validated 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 extraction solvents. This also reduces worker exposure to toxic chemicals. Results are comparable to those obtained using conventional, hazardous and volatile organic solvents (VOCs) typical of the standard and official methods. OTA recovery from spiked durum wheat samples, in particular, was to up to 89% 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 column was excellent as it was able to retain up to 96% of the OTA. Recovery and repeatability for durum wheat, bread crumbs, and biscuits proved to be within the specifications required by the current European Commission (EC) regulation. Good results in terms of accuracy and precision were achieved with mean recoveries between 70% (durum wheat) and 88% (bread crumbs) and an RSDr between 2% (biscuits) and 7% (bread).

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.

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.

This review summarises developments in the determination of mycotoxins over a period between mid-2014 and mid-2015. In tradition with previous articles of this series, 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 new methods used to analyse botanicals and spices and newly developed LC-MS based multi-mycotoxin methods. The critical review aims to briefly discuss the most important developments and trends in mycotoxin determination as well as to address shortcomings of current methodologies.

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.

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.

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.

Introduction The aim of socio-economic research in the MoniQA Network of Excellence is to develop a toolbox to support impact assessment of proposed food safety regulatory changes. Objectives The scope of this contribution is to present an initial version of such a toolbox with its application to a case study, which concerns a proposal on setting maximum levels of T-2 and HT-2 toxins in cereals and cereal products. A regulatory proposal of ‘strict’ maximum limits is compared with two alternative options: the ‘do nothing’ option and the ‘soft’ regulatory option. Methods The proposed toolbox involves a preliminary qualitative assessment of the likely impacts of each of the policy options considered, with a coding/ scoring procedure, in order to identify the greatest impacts. A feasibility filter subsequently considers the availability of data necessary for impact quantification. The subsequent quantitative assessment is performed with different methodologies for the most important impacts. Finally, a multi-criteria analysis approach – which allows for a combination of qualitative and quantitative measurements – is used to arrive at a ranking of policy options. Results The outcome of this assessment exercise is that the ‘do nothing’ option is clearly the most preferable option, and, between the two regulatory options (options 2 and 3), the setting out of ‘strict’ maximum limits is preferable to ‘soft’ maximum limits. Conclusion This case study shows the potentialities of the toolbox as support to policy makers, which will be improved and tested with additional case studies in the remaining years of the MoniQA Network of Excellence.

Introduction The occurrence of major Fusarium toxins in maize samples collected in the Black Sea region of Turkey and fate of these toxins during traditional Turkish maize bread production was investigated. Materials and methods Twenty maize samples were analysed for deoxynivalenol, zearalenone, fumonisins, T-2 and HT-2 toxins. Contamination profiles of maize samples were determined in order to choose the toxins to be monitored during bread production. Breads were produced from low or uncontaminated maize samples and from the mixture of highly contaminated ones. After baking, crust and crumb of the breads were separated and analysed by high-performance liquid chromatography standard methods with ultraviolet or fluorescence detection. Results and conclusions In total, toxin levels in 30% of the maize samples were found to be higher than the limits established by the 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 relevant maize bread only 2.1%, 0.1% and 3.1% of the total initial amounts of deoxynivalenol, zearalenone and fumonisins, respectively, were lost. Breads were not analysed for T-2 and HT-2 as their levels in maize were quite low (o50mgkg 1 ).

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2008 and mid-2009. It covers the major mycotoxins: aflatoxins, alternaria toxins, cyclopiazonic acid, fumonisins, ochratoxin, patulin, trichothecenes and zearalenone. Developments in mycotoxin analysis continue, with emphasis on novel immunological methods and further description of LC-MS and LC-MS/MS, particularly as multimycotoxin applications for different ranges of mycotoxins. Although falling outside the main emphasis of the review, some aspects of natural occurrence have been mentioned, especially if linked to novel method developments.

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 microg/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.

Food chemical contaminants are chemicals that are not normally found in a particular food, their ingredients or the original raw material. Their presence is only acceptable when the levels that they occur at do not compromise public safety and are unavoidable after exercise of best practice. Mycotoxins, the toxic metabolites of certain filamentous fungi, are one group of contaminants and specific legislation, regulating permitted levels of contamination exists for a number of mycotoxin/food combinations. In many cases there is now considerable knowledge not only of the conditions conducive to their formation but also of the mechanisms that need to be in place to prevent or limit their occurrence. In a number of cases this knowledge has been converted into guidelines or other strategies, which both farmers and the food industry have adopted. Assessing levels of compliance and efficacy of these measures is achieved through appropriate audit/inspection and chemical-analytical regimes.

The genus Alternaria includes both plant-pathogenic and saprophytic species, which may affect crops in the field or cause harvest and postharvest decay of plant products. The taxonomy of the genus Alternaria is not well-defined yet. A polyphasic approach based on morphological features, phylogeny and toxin profiles could be the key to a correct identification at species level and the evaluation of mycotoxin risks associated with fungal contamination. Species of Alternaria are known to produce many metabolites, mostly phytotoxins, which play an important role in the pathogenesis of plants. However, certain species, in particular the most common one A. alternata, are capable of producing several mycotoxins in infected plants and/or in agricultural commodities. The major Alternaria mycotoxins belong to three structural classes: the tetramic acid derivative, tenuazonic acid; the dibenzopyrone derivatives, alternariol, alternariol monomethyl ether and altenuene; and the perylene derivatives, the altertoxins. The toxic effects of the Alternaria toxins have not yet received the same attention as the biological activities of other mycotoxins. However, the Alternaria mycotoxins should not be underestimated since they are produced by several Alternaria species frequently associated with a wide range of diseases in many plants of a high agrifood value. The major problems associated with Alternaria mycotoxin contamination of agricultural products are illustrated by focusing on various crops and their relevant diseases, e.g. black rot of tomato, olive, and carrots; black and grey rot of citrus fruits; black point of small-grain cereals; and Alternaria diseases of apples.

A key requirement of any food safety management system is the process of demonstrating its continuous effectiveness (verification). Increasingly, rapid test methods are being used by food businesses as a tool to assist them in this process. This includes management of hazards presented by mycotoxins. These compounds provide a significant challenge to both food processors and regulatory agencies, since they are usually present in the raw material on receipt and subsequent process steps have only limited efficiency in effecting their removal. Within the commercial environment it is critical for those purchasing raw materials to satisfy themselves that any delivery meets regulatory and specification requirements. Decisions to accept or reject deliveries usually need to be taken within short time periods (minutes rather than hours). This requires the use of rapid and - at the same time - reliable analytical methodology; furthermore, the methods need to be of minimal complexity. Within the European Union (EU), analytical methods to determine mycotoxin levels for regulatory purposes must fulfil to certain performance criteria. This principle also holds true for rapid test kits. Current activities within the EU-funded project MoniQA have identified a number of areas in relation to the use of rapid test kits which need to be addressed. These include the criteria which determine robustness of such kits, operator competence and the issues relating to the fate of the rejected lots. Given the diverse stakeholder (e.g. regulator, food business and consumer) interest in the data generated from rapid test kits, a harmonised rationale for their use and also the actions taken upon the results obtained, is desirable.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2007 and mid-2008. It covers the major mycotoxins: aflatoxins, Alternaria toxins, cyclopiazonic acid, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. Some aspects of natural occurrence, particularly if linked to novel aspects of analytical methods, are also included. The review demonstrates the rise of LC-MS methods, the continuing interest in developing alternative and rapid methods and the modification of well-established mycotoxin analytical methods by individual laboratories to meet their own requirements.

The main source of ochratoxin A (OTA) in the wine food chain is the infection of grapes by “black aspergilli” in the field. OTA-producing black aspergilli include principally Aspergillus carbonarius, followed by A. niger and possibly A. tubingensis. They are opportunistic fungi that develop particularly on damaged berries at ripening, although they may occur and form OTA on grapes from veraison to harvest. Climatic conditions (high humidity and temperature) and geographical location are important factors favouring OTA accumulation in grape berries. The severity of aspergillus rot is influenced by excessive irrigation and rainfall prior to harvest, which causes berry splitting. In addition, berry wounds caused by insect attack provide preferential entries for black aspergilli. High OTA levels occur in grapes severely damaged by the grape moth, Lobesia botrana, particularly in Mediterranean areas. Some grape varieties display greater susceptibility to aspergillus rot due to intrinsic genetic characteristics and bunch conformation (i.e. compact > sparse). Control measures for toxigenic mycoflora in the vineyards must consider these critical control points. Proper fungicidal and insecticidal treatments can reduce OTA contamination. Nevertheless, knowledge about the fate of OTA and its distribution in wine and winery by-products is important to manage OTA risk in contaminated stock. In our wine-making experiments, only 4% of the OTA present in grapes remained in the wine-the majority is retained in pressed grape pomaces. OTA concentration remained unchanged in wine after a 1-year aging as well as in all liquid fractions collected during vinification (i.e. must, free run wine, and wine after first and second decantation). Activated carbon can reduce OTA levels in wine but negatively affects wine quality

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.

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 environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental 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 on almonds. The objective of this study was to determine the influence of different combinations of temperature (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 two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28 °C and 0.96 aw; no fungal growth and AFB1 production were observed at 20 °C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37 °C AFB1 production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28 °C) and minimum (20 °C and 37 °C) AFB1 production. Conversely the two structural genes (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 is strictly 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 most likely 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 processing chain to suppress aflatoxin production in this important product.

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

Fumonisins are mycotoxins found primarily in corn and corn products that are produced by Fusarium verticillioides, F. proliferatum, and several other Fusarium species. The toxicity of fumonisin B₁ (FB) from culture material with and without activated carbon was evaluated using weanling piglets. Fifty-six weanling pigs were assigned to one of four treatments diets based on BW. The treatment diets were 1) control = corn-soybean basal diet with

In the EU, all major draft laws must be subject to a regulatory impact assessment. The aim of socio-economic research in the MoniQA Network of Excellence is to develop a toolbox for impact assessment of food safety regulatory proposals. The aim of this contribution is to present a draft of such toolbox with its application to a case study, regarding a proposal on setting maximum levels of T-2 and HT-2 toxins in cereals and cereal products. Such toolbox is based on multi-criteria analysis rather than cost-benefit analysis, which cannot be complete and reliable in most situations due to major difficulties: poor data availability; difficulty in isolating confounding factors like weather and market forces; probabilistic outcome of some actions; uncertainty in compliance levels; different timing in the occurrence and discounting of costs and benefits. We argue that a multi-criteria analysis method dealing with both fuzzy linguistic and stochastic quantitative variables - which take into account uncertainty - is the best way forward to an ex ante comparison of alternative policy options, especially in the food safety area, where the effect on public health - not precisely quantifiable - is the most important impact as giving the rationale for a regulatory proposal. The proposed toolbox involves a preliminary qualitative assessment of the likely impacts of each of the policy options considered, with a coding/scoring procedure, in order to identify the greatest impacts. Then, a feasibility filter considers availability of data necessary for impact quantification, in terms of monetary and time constraints for data collection. The subsequent quantitative assessment is performed with different methodologies for the most important impacts. Finally, a fuzzy multi-criteria analysis approach – which allows for a combination of qualitative and quantitative measurements - is used to arrive to a ranking of the policy options. The alternative policy options considered for the case study are: 1) maintaining the status quo; 2) setting ‘soft’ maximum levels; 3) setting ‘strict’ maximum levels. Cereals susceptible to T2/HT2 contamination are wheat (durum and soft), oats, barley, maize, and rye, but the wheat and oats are the most susceptible. This type of regulation would prominently imply impacts on public health, conduct of businesses, public authorities, and SMEs. The application of multi-criteria analysis shows a dominance of the ‘status quo’ option. All data used for this example are based on expert consultation and statistical databases, but, due to time constraints and the main aim of the research (testing the draft evaluation toolbox), the outcome of the analysis should not be taken as relevant for a real policy decision process. This case study shows the potential of the toolbox, which will be improved and tested with additional case studies in the remaining years of the MoniQA project.

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.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2012 and mid-2013. It covers the major mycotoxins: aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone. A wide range of analytical methods for mycotoxin determination in food and feed were developed last year, in particular immunochemical methods and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-based methods. After a section on sampling and sample preparation, due to the rapid spread and developments in the field of LC-MS/MS multimycotoxin methods, a separate section has been devoted to this area of research. It is followed by a section on mycotoxins in botanicals and spices, before continuing with the format of previous reviews in this series with dedicated sections on method developments for the individual mycotoxins.

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.

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.

Sphinganine/sphingosine (SA/SO) ratio, a biomarker of fumonisin exposure, has been measured in urine, kidney and liver of male Wistar rats exposed to fumonisin-contaminated diet with and without the addition of activated carbon (AC). The latter was previously shown to adsorb fumonisin B-1 in vitro. Rats were fed either control diet or fumonisin-contaminated diet (4 mug FB1+FB2/g) or fumonisin-contaminated diet mixed with 20 mg AC/g diet for 1 week. In rats fed fumonisin-contaminated diet, the SA concentration and SA/SO ratio increased significantly and reversibly in kidney, while urine and liver did not show a significant increase of SA/SO ratio. The addition of AC to the fumonisin-contaminated diet did not alter the change of SA/SO biomarker for fumonisin exposure. This provides indications that the use of AC to reduce the toxicity of fumonisins is unlikely to be effective in vivo. (C) 2001 Elsevier Science Ltd. All rights reserved.

In order to optimize the analytical method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in different maize products, five materials (maize flour, corn flakes, extruded maize, muffins and infant formula) were investigated under a variety of experimental conditions organized in a ruggedness test according to a factorial design. The influence of five factors (extraction solvent, extraction mode, volume of extraction solvent, test sample size and clean-up) on method performances was tested by four laboratories using spiked materials (0.5 microgram/g and 1.5 micrograms/g FB1 + FB2) and naturally contaminated materials (ca 1.5 micrograms/g with FB1 + FB2). The end determination step was performed by high-performance liquid chromatography analysis of the o-phthaldialdehyde derivatized extracts. The ruggedness test permitted identification of two critical factors in the analysis of fumonisins in the above products, namely 'extraction solvent' and 'clean-up procedure'. In particular, the use of acetonitrile (ACN)-water (1 + 1, v + v) as extraction solvent and immunoaffinity column for clean-up provided better recovery of fumonisins and chromatographic resolution as compared with methanol (MeOH)-water (3 + 1, v + v) and strong anion exchange (SAX), respectively. However, phase separation occurring after extraction with ACN-water may have given incorrect results. Based on the information obtained with the present study it was possible to develop a new method horizontally applicable to all the above mentioned maize-based food matrices.

Several adsorbent materials were tested at I mg/ml for their in vitro capacity to adsorb fumonisin B1(FB1) from aqueous solutions. Cholestyramine showed the best adsorption capacity (85% from a solution containing 200 microg/ml FB1) followed by activated carbon (62% FB1). Bentonite adsorbed only 12% of the toxin from a solution containing 13 microg/ml FB1, while celite was not effective even at the lowest tested FB1 concentration (3.2 microg/ml). Cholestyramine was tested in vivo to evaluate its capacity to reduce the bioavailability of fumonisins (FBs) in rats fed diet contaminated with toxigenic Fusarium verticillioides culture material. Rats were exposed for one week to FBs-free diet, FBs-contaminated diet containing 6 or 20 microg/g FB1 + FB2 and the same FBs-contaminated diet added of 20 mg/g cholestyramine. The increase of sphinganine/sphingosine (SA/SO) ratio in urine and kidney of treated rats was used as specific and sensitive biomarker of fumonisin exposure. The addition of cholestyramine to the FBs-contaminated diets consistently reduced the effect of FBs by reducing significantly (P < 0.05) both urinary and renal SA/SO ratios.

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.

The isolation, chemical characterization and biological activity of two phytotoxic metabolites of Phomopsis helianthi Munt-Cvet et al. is reported. These compounds were identified by spectroscopic methods (UV, IR, H-1 and C-13 NMR, and MS) as trans-4,6-dihydroxymellein (trans-3-methyl-4,6,8-trihydroxy-3,4-dihyroisocoumarin) and cis-4,6-dihydroxymellein (cis-3-methyl-4,6,8-trihydroxy-3,4-dihydroisocoumarin). This is the first report of the isolation of trans-4,6-dihydroxymellein from fungal cultures and of the production of cis- and trans-4,6-dihydroxymelleins by P. helianthi. Rice was found to be a good substrate for the production of the dihydroxymelleins. Culture extracts of some Italian and French strains of P, helianthi showed different degrees of phytotoxicity towards sunflower leaves and seedlings. The minimum effective doses of trans- and cis-4,6-dihydroxymelleins with different bioassays were 76 and 135 mu g per spot (leaf puncture bioassay), 3 and 5 mu mol g(-1) fresh tissue (absorption by leaf cutting) and 5 and 2 mu mol g(-1) fresh tissue (absorption by cut seedlings), respectively. These compounds may contribute to the severity of the sunflower disease caused by P. helianthi. Copyright (C) 1999 John Wiley & Sons, Ltd.

Fusariumsambucinum(F. trichothecioides),F. avenaceum andF. crookwellense, were the species most frequently isolated from lots of potato tubers imported to Southern Italy in 1985. Isolates of these species were tested for the production of zearalenone, zearalenols, trichothecenes and moniliformin, on three solid media (autoclaved kernels of maize or rice, and fresh potato tubers), incubated at different conditions. The toxigenic potential of the isolates was expressed only on rice and maize, while they were non capable to produce toxins on fresh potato under any conditions. In particular,F. crookwellense produced zearalenone and/or zearalenols (diastereomeric mixture) on maize, andF. avenaceum produced moniliformin both on maize and on rice; while no toxins were produced byF. sambucinum.

Fusarium tricinctum strain (KF 260) isolated from wheat in Poland, produced on maize cultures a compound (C11H14O5, MW226) toxic toArtemia salina. The compound, identified by various spectroscopical tecniques as 5-hydroxy-4-methoxy-6,8a-dimethyl-6, 7-dihydro-2H, 8aH-pyrano/2, 3-b/pyran-2-one, was identical to chlamydosporol, a mycotoxin recentlydiscovered in cultures ofF._ chlamydosporum isolated from rice. The compound showed inhibitory effect on human lymphocyte proliferation at concentration of 25 μg/mL. LD50 onA. salina was 56 μg/mL.

Liquid chromatography coupled with single or tandem mass spectrometry (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 the European Committee for Standardization and the Association of Analytical Communities. A proficiency test (PT) was conducted for the simultaneous determination of up to 11 mycotoxins (aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 ), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B 1 (FB 1 ) and fumonisin B 2 (FB 2 )) 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. Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB 1 , AFB 2 , AFG 1 and AFG 2 , OTA, DON, T-2, HT-2, ZEA, FB 1 and FB 2 ); and 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. 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%, FB 1 50% and 30%, FB 2 52% and 38%, ZEA 68% and 64%, T-2+HT-2 toxins 82% and 85%, OTA 58% and 60%, AFB 1 56% and 62%, AFG 1 73% and 84%, AFB 2 40% and 78%, AFG 2 64% and 78%, respectively. The poorest performance (|z| >3) was obtained for FB 1 (31%), FB 2 (32%), AFB 1 (32%) and AFB 2 (32%) in contaminated maize and for DON (35%), FB 1 (63%) and FB 2 (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 FB 1 and 163% for FB 2 ). 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 inter-laboratory validation. The results of this PT and the relevant method’s details can be useful to identify methodology strengths and weaknesses.

Fumonisins are mycotoxins mainly produced by Fusarium moniliforme, one of the most prevalent seed-borne fungi of maize. Strains of Fusarium moniliforme isolated from cereals in Europe produced in cultures high levels (up to 4 mg/g) of fumonisins B1 and B2 (FB1 and FB2). FB1 and FB2 have been found in maize and maize-based foods and feeds in most European countries. In Italy these mycotoxins have been directly involved in a fatal outbreak of equine leukoencephalomalacia, and have been found at different levels in most maize-based food products, including polenta--a staple food in a region with high incidence of esophageal cancer. The European Commission, Community Bureau of Reference (BCR) and Measurements and Testing Programme (M&T), has undertaken two consecutive intercomparison studies, involving over 20 laboratories from 12 European countries, to improve the quality of fumonisins analysis at European level. The first study consisted in the evaluation of the determination of fumonisins in an unknown solution, while the second one involved the analytical methodology for fumonisins in contaminated and blank maize samples. A definite improvement in the performance of the different laboratories has been observed in the second study. In view of the production of reference materials certified for their fumonisins contents, new analytical procedures need to be developed.

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 α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, 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 α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

Acuminatopyrone, a recently described metabolite of Fusarium acuminatum was shown to be produced by several strains of Fusarium chlamydosporum and F. tricinctus isolated from various substrates in different countries. The structure of acuminatopyrone has been revised on the basis of NOEDIF and HETCOR nmr experiments, and shown to be 4-methoxy-7,8-dimethyl-2H-pyrano[4,3-b]pyridin-2-one. This structure is biosynthetically consistent with that of chlamydosporol and related compounds that are produced by the same fungal species

A novel compound, trivially named visoltricin, has been isolated from cultures of Fusarium tricinctum on corn. Its structure has been established by chemical reactions and spectroscopic methods (UV, IR, MS, 1 H NMR, and 13 C NMR) as 3- [1-methyl-4-(3-methyl-2-butenyl)imidazol-5-yl]-2-propenoic acid methyl ester (molecular formula C 13 H 18 N 2 O 2 ; molecular weight 234.297). It is the first imidazole derivative produced by a Fusarium species. About 30% of the tested isolates (33) of F. tricinctum from various substrates and geographic areas produced visoltricin when grown on corn. None of the 14 isolates tested from the United States produced the compound

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).

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2009 and mid-2010. It covers the major mycotoxins aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. New and improved methods for mycotoxins continue to be published. Immunological-based method developments continue to be of wide interest in a broad range of formats. Multimycotoxin determination by LC-MS/MS is now being targeted at the specific ranges of mycotoxins and matrices of interest or concern to the individual laboratory. Although falling outside the main emphasis of the review, some aspects of natural occurrence have been mentioned, especially if linked to novel method developments.

This deliverable describes the results obtained from the expression analysis of some genes involved in the biosynthesis of aflatoxin B1 (AFB1) in Aspergillus flavus grown on almond medium under physical stress (aw and T°). In particular, the aim was to study the effect of varying (various?) water activity (aw) conditions on the activation of the two regulatory genes, aflR and aflS, located in the biosynthesis cluster, during the growth of the fungus, in relation to aflatoxin production. In addition, the relation to the expression of some structural genes (aflD, aflM, aflO, aflP, and aflQ) of the biosynthesis pathway during the time course experiment was assessed. The analyses were performed on fungal mycelium harvested in early days of incubation (seven time points, every 24 hours, for all the conditions tested) and then after 10 days and 15 days of growth. Fungal growth, AFB1 production and relative quantification of the expression for each gene during the growth for each aw value were evaluated. Additionally, at days 5 and 7 among the different aw, relative expression of each gene and the expression levels of all the genes considered were compared. Mycelial growth and AFB1 production resulted delayed with the lowering of aw values. During the early stages of incubation, they were especially reduced at 0.90 aw. At this condition, all the tested biosynthesis genes exhibited a reduced level of transcription, with aflS showing a lower expression level than aflR. At 0.99 and 0.96 aw, aflR and aflS were transcribed from the beginning, whereas at 0.93 aw their expression started later, and reached the maximum just in the proximity of the peak of AFB1 accumulation, at 15 days of growth. Finally, the comparison of the relative expression of all the genes at different aw showed the highest transcription levels at 5 days for aw 0.99, and at 7 days for 0.96 aw.

The molecular regulation of aflatoxin biosynthesis is complex and influenced by several environmental conditions. A lot of studies have been conducted on the variation of the capability of aflatoxin production in A. flavus, when an abiotic stress is applied. In the current study, for the first time, the influence of two important factors, such as water activity and temperature, was evaluated during the infection of almond by A. flavus. To this aim an agarized almond medium was used and the expression of some of the aflatoxin biosynthetic genes was analysed during the fungal growth. The best growth rate, in terms of quantity of fungal biomass, and the higher production of AFB1 were observed when A. flavus grew at 28°C and at aw value of medium set at 0.96, even though the accumulation process was slower and the maximum was reached later in comparison with 0.99 aw. Both an increase and a reduction of temperature cause a lowering of AFB1 production when the medium presents a aw standard condition (0.99), with the higher temperature (37°C) affecting more than the low temperature (20°C). Likewise, at standard conditions of temperature (28°C) the decrease of aw at the lowest values results in a remarkable loss of production capability. In Fig. 6 the concurrent effects of the two factors considered in this work is represented. When the two parameters are contemporaneously changed, their influence is greater, so that at 0.96 aw and at 20°C mycotoxin production is reduced compared to 0.99 aw at the same temperature. When the aw is set at the lowest values (0.93 and 0.90 aw), the growth is completely inhibited at 20°C, while at 37°C the production is repressed at 0.90 aw, and at 0.93 aw is slightly higher than at 0.96 aw. It can be observed how, with a prolonged time of incubation, the risk of OTA contamination can increase with lowering of aw values. Regarding the expression of biosynthetic genes, the evidence is that at optimal conditions of production (0.96 and 0.99 aw, and 28°C) the two regulatory genes (aflR and aflS) are activated at their highest levels in the early days, much more and sooner than the structural ones, which show a maximum level of transcription, even greater than aflR and aflS, just before the peak of AFB1 accumulation. Generally, when conditions become unfavourable for aflatoxin production, the expression levels decrease, above all those of structural genes which never exceed the levels of aflR and aflS. The regulatory genes are always expressed, also when no AFB1 accumulation is detected, and with the lowering of aw, in the very early days of fungal growth, even in a greater manner than at more favourable conditions, Moreover, at 28°C with the decrease of aw, the ratio of expression aflR:aflS is higher than at optimal conditions at the beginning of incubation. In addition, when the conditions become less permissive, this ratio is subject to notable fluctuations with the passage of time, while when temperature and aw values are most favourable for AFB1 production, the ratio tends to change in a lesser extent. This aspect is worth of further investigation to better understand the mechanism of regulation based on the two genes of aflatoxin cluster.

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.

Aspergillus carbonarius is considered the main fungus responsible for ochratoxin A (OTA) contamination in grapes. OTA is a potent nephrotoxin and a possible human carcinogen with a polyketide derived structure. Fungal polyketide synthases (PKSs) have recently been demonstrated to be involved in OTA biosynthesis in both Penicillium and Aspergillus species. We report here on the identification and characterisation of part of a novel polyketide synthase gene, ACpks from A. carbonarius. The sequence appears to encode conserved ketosynthase and acyl transferase domains, which are characteristic of previously characterised PKS enzymes. Expression of the ACpks gene is differentially regulated, with transcription being observed when the fungus was grown on synthetic grape medium and on OTA permissive medium (MM) whereas no transcription was detected when the fungus was grown on OTA restrictive medium (YES). ACpks expression was also observed when A. carbonarius was grown at low pH, with concomitant increases in OTA production. This correlation between ACpks gene expression and OTA production suggests the likely involvement for the product of this gene in ochratoxin A biosynthesis in the fungus. From a preliminary screening of Aspergillus isolates with ACpks specific primers, ACpks gene homologues appear to be present in A. sclerotioniger and A. ibericus, two species of section Nigri which are closely related to A. carbonarius.

MoniQA (Monitoring and Quality Assurance in the total food supply chain) is an EU‐funded Network of Excellence that involves experts from around the globe collaborating to harmonize worldwide food quality and safety monitoring and control strategies. The initial network of over 155 scientists from 20 countries has grown to over 400 experts from over 35 countries from five continents in the first 18 months. MoniQA focuses on validation of and performance criteria/requirements for methods used to analyse foods and food products for safety and quality, with the main focus being on rapid methods and their applicability and reliability in routine testing. The work involves validation guidelines, validation studies, design and development of reference materials/testing materials and validation guidelines. MoniQA will play an important role in integrating European and worldwide food quality and safety research by creating a virtual laboratory for joint research, training, dissemination and mobility programmes. It will allow and actively promote sharing of data and knowledge, as well as of equipment, materials and personnel through creation of a global platform for food Q&S researchers. MoniQA will enable shared access to the world's best research facilities, technological platforms, databases, analytical tools and knowledge. MoniQA's harmonized food quality and safety control strategies will add value in the food chain and will improve consumer confidence worldwide. MoniQA's socio‐economic impact assessment will enhance the effectiveness and efficiency of new food quality and safety regulations within the EU and worldwide.

Mycotoxins (the poisonous metabolites of certain filamentous fungi) are potential contaminants of staple food commodities and, if uncontrolled, may present a significant public health hazard. In many jurisdictions, questions relating to mycotoxin contamination are addressed at both generic and specific levels by food-safety legislation. Key to the successful management of the mycotoxin question, both in terms of verifying food-safety measures by the agri-food businesses and ensuring compliance with statutory limits by enforcement agencies, is the use of reliable sampling and analytical methodology. Evidence from European Union Rapid Alert System for Food and Feed data suggest that harmonization of methodologies used to determine the mycotoxin content of foods would contribute to improved compliance at both regulatory and commercial levels.

The effectiveness of seven phenolic compounds (esculetin, ferulic acid, quercetin, resveratrol, scopoletin, scoparone and umbelliferone) in controlling Penicillium expansum growth and patulin accumulation was evaluated by both in vitro and in vivo studies. The in vitro screening showed that quercetin and umbelliferone were the most effective compounds in controlling Penicillium growth and patulin accumulation, respectively. Quercetin and umbelliferone, resulted to be effective also in the in vivo screening, were further tested (alone and in combination) on Granny Smith and Golden Delicious apples. The efficacy in controlling Penicillium growth was better expressed on Golden Delicious than on Granny Smith apples, with quercetin providing a better control of both incidence of decay and disease severity as compared to umbelliferone. Both compounds exerted a considerable control of patulin accumulation on the two apple cultivars. Quercetin and umbelliferone can be considered as natural compounds to be used as alternative strategy to chemical fungicides in postharvest control of P. expansum infections on apples.

The selective enzymatic deacetylation of T-2 toxin to give HT-2 toxin has been investigated in aqueous crude extracts of different cereals and exploited to develop an analytical method for the determination of the sum of T-2 and HT-2 toxins. The method has been validated for the analysis of total T-2 and HT-2 toxins in maize, wheat, and oats, showing recoveries from 72 to 97% for maize, from 67 to 84% for wheat, and from 61% to 87% for oats, at spiking levels of 20-400 microg/kg, with relative standard deviation lower than 10%. Liquid chromatography-tandem mass spectrometry was used for quantitative toxin determination. The potential biological role of this enzymatic conversion and its perspectives for application in the development of antibody-based analytical techniques are discussed.

A liquid chromatographic method for the determination of ochratoxin A in grapes, dried vine fruits, and winery byproducts was developed. A mixture of either acetonitrile/water or acetonitrile/water/methanol was used as an extraction solvent mixture. After immunoaffinity column cleanup, the final extract was analyzed by high-performance liquid chromatography (HPLC) with a fluorometric detector (FLD). Mean recoveries from grapes, grape pomace, and lees samples spiked in the range of 1-200 microg/kg were 78, 86, and 88%, respectively, with a detection limit of 0.1 microg/kg and within-laboratory repeatability ranging from 6 to 15%. Tested on naturally contaminated samples of grapes, grape pomace, and sultanas, the method showed better performances as compared to two other methods also based on immunoaffinity cleanup and HPLC/FLD determination. Ochratoxin A was detected in samples of grape pomace (levels ranging from 34.2 to 456.8 microg/kg) and lees (levels ranging from 48.3 to 602.5 microg/kg) derived from the wine making of red grapes of 2004 and 2005 vintages in southern Italy. After distillation of contaminated grape pomace in a pilot-scale equipment to produce grappa, the toxin remained unchanged in the exhausted pomace and was not detected in any of the distilled fractions (detection limit of 0.02 microg/L).