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A survey on the occurrence of ochratoxin A (OTA) and sterigmatocystin (STC) in grated cheese products obtained from hard grana-type cheeses was carried out, where 107 grated products were collected in retail outlets and analysed. OTA and STC were found in 48.6% and 94.4% of the samples, in a range from

The occurrence of tenuazonic acid (TeA), alternariol, alternariol monomethyl and tentoxin in tomato-based products was surveyed over the years 2017–2019. A total of 120 samples were collected from retail outlets and tomato-based food producers located in Northern Italy. After extraction and purification through prepacked columns, the mycotoxins were analysed using LC-MS/MS. A widespread contamination of TeA was found in tomato-based products, particularly in concentrated tomato paste. Other Alternaria toxins were not detected. The incidence of TeA was 78.5%, 47.4%, 55.5%, and 76.9% in concentrated tomato paste (maximum value 5955 µg kg−1), tomato sauce, tomato pulp, and ketchup, respectively. The mean level was 243 ± 725 µg kg−1 in concentrated tomato paste and below 30 µg kg−1 in the other tomato products. The contamination varied from year to year. Alternaria spp strains isolated from fresh tomatoes produced mainly TeA. This study provides further Alternaria toxins occurrence data, useful for future risk assessments.

In this study, the possibility to manage maize contaminated with aflatoxins and fumonisins for the production of biogas was considered. This is a priority in the climate change scenario that is expected to increase the occurrence of aflatoxins in maize. The results clearly underline how the anaerobic digestion process used in biogas plants is able to reduce aflatoxin contamination, mainly when highly contaminated maize is used for feeding the biodigestors without affecting the efficiency of methane production. In particular, the higher aflatoxin contamination is, the higher is mycotoxin reduction during biodigestion, with reductions up to 95% in digestate. The co-occurring mycotoxins, fumonisins, were also reduced by around 15%. The vitality of mycotoxin producing fungi was also significantly reduced. Biogas production is therefore suggested as a good alternative use for uncompliant maize.

A survey on the occurrence of acrylamide (AA) in roasted coffee, barley, and potato crisps was carried out using an intra-lab validated liquid chromatography (LC)–MS (mass spectrometry)/MS method. Over the years 2015–2016, 66 samples of coffee, 22 of roasted barley, and 22 of potato crisps were collected from retail outlets in Italy. AA was detected in almost all samples. In roasted coffee, the level exceeded 450 µg kg−1, the limit recommended by the European Commission (EC), in 36.4% of the samples. In roasted barley, mean contamination was slightly lower than in coffee and no sample exceeded the EC limit of 2000 µg kg−1. The AA contamination in potato crisps was remarkable. A percentage of 36.4 (n = 8) showed a value higher than the EC limit of 1000 µg kg−1. Considering the average consumption of coffee and potato crisps by Italian people, AA exposure is significant and should be decreased.

The aim of this study was to investigate in vitro and model the effect of temperature (T) and water activity (aw) conditions on growth and toxin production by some toxigenic fungi signaled in cheese. Aspergillus versicolor, Penicillium camemberti, P. citrinum, P. crustosum, P. nalgiovense, P. nordicum, P. roqueforti, P. verrucosum were considered they were grown under different T (0–40 °C) and aw (0.78–0.99) regimes. The highest relative growth occurred around 25 °C; all the fungi were very susceptible to aw and 0.99 was optimal for almost all species (except for A. versicolor, aw opt = 0.96). The highest toxin production occurred between 15 and 25 °C and 0.96–0.99 aw. Therefore, during grana cheese ripening, managed between 15 and 22 °C, ochratoxin A (OTA), penitrem A (PA), roquefortine-C (ROQ-C) and mycophenolic acid (MPA) are apparently at the highest production risk. Bete and logistic function described fungal growth under different T and aw regimes well, respectively. Bete function described also STC, PA, ROQ-C and OTA production as well as function of T. These models would be very useful as starting point to develop a mechanistic model to predict fungal growth and toxin production during cheese ripening and to help advising the most proper setting of environmental factors to minimize the contamination risk.

An innovative chromatographic analysis was developed for the determination of moniliformin (MON). Because of its ionic nature, MON is weakly retained in reversed-phase chromatography and the separation may be tricky. Nevertheless, this technique is normally used either with the formation of ion pairs or employing specific RP columns for polar compounds, or combining anion exchange and hydrophobic interactions. Hydrophilic interaction chromatography (HILIC) was also used, but a non-negligible peak tailing was observed. Besides its ionic nature, MON is a di-ketone and di-ketones, mainly -di-ketones, can easily form complexes with lanthanide ions. Then, in this work the addition of lanthanide ions to the mobile phase was investigated, aiming at improving peak shape and MON separation. La3+, Tb3+ or Eu3+ aqueous solutions were used as mobile phase and MON was chromatographed using a LC-NH2 column. The probable formation of coordination complexes lanthanide-MON in the HPLC mobile phase allowed to obtain a symmetrical peak shape and a satisfactory chromatographic separation by both mass spectrometry (MS/MS) and UV detection. Finally, a suitable extraction and purification method for MON determination in cereal samples was developed.

The influence of climate change on agricultural systems has been generally accepted as having a considerable impact on food security and safety. It is believed that the occurrence of mycotoxins will be greatly affected by future climate scenarios and this has been confirmed by recent data. Temperature (T) and CO2 increases, variation in rain intensity and distribution, as well as extreme weather events, affect the dominant fungal species in different ways, depending on their ecological needs. Therefore, the aim of this work was to study Aspergillus flavus (Af) and Fusarium verticillioides (Fv) co-occurrence in vitro in order to collect quantitative data on the effect of fungal interaction on growth and mycotoxin production and develop functions for their description. Experimental trials were organized with the cited fungi grown alone or together. They were incubated at different T regimes (10–40°C, step 5°C) for 21 days. Fungal growth was measured weekly, while AFs and FBs were quantified at the end of the incubation period. Temperature and incubation time significantly affected fungal growth both for Af and Fv (p ≤ 0.01), and a significant interaction between T and the presence of one versus both fungi influenced the amount of AFs and FBs produced. Each fungus was affected by the presence of the other fungus; in particular, Af and Fv showed a decrease in colony diameter of 10 and 44%, respectively, when they were grown together, compared to alone. The same influence was not found for mycotoxin production. In fact, the dynamics of toxin production in different temperature regimes followed a comparable trend with fungi grown alone or together, but a significant impact of inoculum × temperature interaction was highlighted. Fungal growth and toxin production in different T regimes were well described, both for AFs and FBs, by a Bete function. These results are the first attempt to model mycotoxigenic fungal co-occurrence under several T regimes; this is essential in or

Based on the EFSA proposal “Survey on sterigmatocystin in food” (GP/EFSA/CONTAM/2013/02), this study provides a survey on the occurrence of this mycotoxin. A total of 1259 samples of cereal grains (429), cereal products (713), beer (53) and nuts (64) were analysed for the presence of sterigmatocystin (STC). Samples were mainly collected at processing plants, storage facilities, wholesale and retail between August 2013 and November 2014, in nine European countries, mostly Greece, Italy, the Netherlands and the United Kingdom. The products originated from 27 European countries and 18 other countries. All samples were analysed by methods based on liquid chromatography coupled with tandem mass spectrometry. The limit of quantification (LOQ) was 0.5 µg/kg and the limit of detection (LOD) was in the range 0.05-0.15 µg/kg (0.005-0.01 µg/L for beer). Overall, STC was identified in 10% of the samples; it was not detected in either beer or nut samples. More than 50% of the contaminated samples contained levels between LOD and LOQ; in the other cases, levels were between 0.5-6 µg/kg with one exception (33 µg/kg in oats). In cereal grains, rice and oats seemed the cereals most prone to STC contamination (100% unprocessed rice, 22% oats grains); however the number of rice samples was limited (n=28) and the samples were collected almost exclusively in Italy and Greece. In cereal products, levels were lower than in cereal grains. The highest incidence was in processed rice (21%) and breakfast cereals (19%), while for the other cereal products this was between 5-7%. In the contaminated cereal products, rice and oats were often present as ingredients.

The occurrence of sterigmatocystin (STC) in paddy and processed rice samples produced in Italy was surveyed. After extraction and purification, STC was analysed using HPLC-MS/MS. STC was detected in all paddy rice samples (n = 49), in the range 0.29–15.85 gkg1. As regards processed rice, a widespread contamination was found in brown and parboiled rice. All the brown rice samples were contaminated between 0.12 and 1.32 gkg1; for parboiled rice, the incidence was 90.9% and the maximum level was 1.09 gkg1. The contamination in white rice was significantly lower (p < 0.01). The STC distribution in different rice fractions, obtained by the de-hulling and polishing processes, was evaluated. After de-hulling, the STC percentage remaining in brown rice was in the range 21.2%–30.8%. The polishing process, from brown to white rice, caused another remarkable decrease of contamination; the STC remaining in white rice was 2.2%–8.3% of the amount found in paddy rice.

The extraction of aflatoxin M1 (AFM1) from cheese is generally carried out using chlorinated organic solvents. In this study, two innovative methods were developed, based on an enzyme-assisted (EA) extraction using proteolytic enzymes (pepsin or pepsin–pancreatin). After purification through an immunoaffinity column, AFM1 is determined by HPLC–FLD. A comparison between the new EA methods and an established chloroform (CH) method was carried out on 24 cheese samples. The results showed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheeses. The simpler (pepsin) of the two methods has been adopted by our laboratory for routine analysis of AFM1 in cheese. In comparison with the CH method, the pepsin-HCl (P-HCl) method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results.

Fumonisins B (FBs) can occur in maize as free forms and as hidden FBs (interaction between FBs and matrix macro-constituents). The analytical methods to detect free FBs in maize and derived products usually involve extraction with aqueous methanol and/or acetonitrile; for hidden FBs, alkaline hydrolysis (2MKOH) and following CH3CN extraction is used. Recently, we proposed a simple phosphate buffer (PB) extraction to detect free FBs in masa and maize samples. This study aimed at evaluating if this method is suitable to detect also hidden FBs; then, a comparison between the most common solvents, PB, and alkaline hydrolysis was carried out. For maize samples, the results showed that 0.4 M PB can extract both free and most hidden FBs; however, this method was not suitable for some categories of maize-based processed food products, in particular cornflakes, chips, and crispbreads. To solve this drawback, a modified PB extraction, which entails the addition of Carrez solutions in order to precipitate proteins, was tested and satisfactory results were obtained. Finally, the alkaline hydrolysis of maize-based food extracts showed that bound FBs can be co-extracted together with free forms by 0.4 M PB and quantified as hydrolyzed FBs.

The fate of AFM1 during production of a long maturing cheese (Parmesan cheese) was assessed. Different levels of AFM1 contamination and of the fat/casein (F/C) ratio of milk were considered, in order to evaluate if these factors can influence the enrichment factor (EF) of AFM1 in cheese. For this purpose, 24 cheese-makings were carried out using naturally contaminated milk at 3 different AFM1 levels and at 2 F/ C ratios. AFM1 analysis was performed by HPLC in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing. The mass balances of the cheese-making processes were close to 100%; in whey, AFM1 concentration was about 40% less than the concentration in cauldron milk. The EF in curd was between 4.0 and 5.2, with an average value of 4.7 ± 0.4; this factor was not significantly affected by either AFM1 contamination level or F/C ratio. During maturation, AFM1 concentration and consequently EF increased from curd to 16 ageing months; successively, AFM1 slightly decreased at 24 months and consequently the EF. At 3, 9, 16 months of maturation, the EF was significantly higher for cheeses prepared using milk with low F/C than those with high F/C milk; on the contrary, EF was not significantly influenced by the AFM1 contamination level. In cheeses, EF values were between 4.7 and 6.3; from these results, the maximum admissible level for AFM1 in Parmesan cheese should be about 0.275 mg kg1.

MycoKey, an EU-funded Horizon 2020 project, includes a series of “Roundtable Discussions” to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

Acrylamide (AA) can occur in fried and baked food products which contain reducing sugars and free asparagine. Recently, the European Commission established mitigation measures and benchmark levels for the reduction of AA in food. The content of reducing sugars in raw materials and the temperature and time of the expansion process by frying were considered in this study of the preparation of dough-based potato snacks, mainly destined for children. Final moisture and bulk density were also evaluated. An increase from 0.15 to 1.0% in reducing sugar content, due to the addition of micro-ingredients in the dough, caused a remarkable AA increase of five- to six-fold. During frying at temperatures between 175 and 195°C, AA was produced after only a few seconds; the AA content was affected more by process time than by temperature. The best temperature/time conditions for expansion by frying were 185°C for 8 s.

Since 2003, non-compliant aflatoxin concentrations have been detected in maize produced in Italy. The most successful worldwide experiments in aflatoxin prevention resulted from distribution of atoxigenic strains of Aspergillus flavus to displace aflatoxin-producers during crop development. The displacement results in lower aflatoxin concentrations in harvested grain. The current study evaluated in field performances of two atoxigenic strains of A. flavus endemic to Italy in artificially inoculated maize ears and in naturally contaminated maize. Co-inoculation of atoxigenic strains with aflatoxin producers resulted in highly significant reductions in aflatoxin concentrations (>90%) in both years only with atoxigenic strain A2085. The average percent reduction in aflatoxin B1 concentration in naturally contaminated maize fields was 92.3%, without significant differences in fumonisins between treated and control maize. The vegetative compatibility group of A2085 was the most frequently recovered A. flavus in both treated and control plots (average 61.9% and 53.5% of the A. flavus, respectively). A2085 was therefore selected as an active ingredient for biocontrol products and deposited under provisions of the Budapest Treaty in the Belgian Co-Ordinated Collections of Micro-Organisms (BCCM/MUCL) collection (accession MUCL54911). Further work on development of A2085 as a tool for preventing aflatoxin contamination in maize produced in Italy is ongoing with the commercial product named AF-X1TM.

Occurrence of aflatoxins, ochratoxin A, sterigmatocystin, cyclopiazonic acid, citrinin, roquefortine C and mycophenolic acid in fresh chestnuts and dried chestnut products was surveyed. A total of eighty-two samples were collected from retail outlets located in northern Italy. After specific extraction and purification through immunoaffinity or prepacked columns, mycotoxins were analysed using HPLC-FLD or HPLC-MS/MS. In fresh chestnuts, mycotoxins were rarely detected, while a widespread contamination was found in dried products, particularly in chestnut flour. The incidence of aflatoxin B1 was 92.0 and 40.0% in chestnut flour (maximum value 58.6 µg kg-1) and dried chestnuts, respectively; in chestnut flour, the percentage of samples exceeding the value of 2.0 µg kg-1 for aflatoxin B1 (maximum limit fixed by EC Regulation 165/2010 in dried fruits) was 24.0%. Chestnut flour was also often contaminated with ochratoxin A, citrinin, roquefortine C and mycophenolic acid, showing sometimes high values (particularly for mycophenolic acid); in 80% of samples, more than four mycotoxins were detected. To the best of our knowledge, no data are available from previous studies about the occurrence of citrinin, roquefortine C and mycophenolic acid in chestnuts and derived products. These results showed that probably the contamination occurred in the post-harvest, during exsiccation, storage and sorting; the high incidence and concentration found in chestnut flour are probably related to the practice of producing it by grinding of the product coming from sieving of dried chestnuts.

Mycotoxins represent the main health risk from food, in terms of exposure and severity of chronic disease, especially cancer. Maize is a focus crop in this context, being susceptible to several fungi, co-existing on the ears in several environments and meteorological conditions. The main concern in northern Italy regards Fusarium verticillioides and Aspergillus flavus, fumonisin and aflatoxin producers, respectively. A multifaceted approach was followed from 2000 to prevent grain contamination in field, including breeding for resistance with focus on F. verticillioides, and biocontrol with atoxigenic strains of A. flavus for aflatoxin reduction. A wide range of maize lines was screened by artificial inoculation, allowing to select those more promising for possible commercial development. Several genomic approaches (microarrays, RNASeq, GBS) were used in order to understand the molecular basis of the maize resistance and to identify QTLs for resistance. A consolidated collaboration with IITA-Nigeria is in force. This country faces the same mycotoxin problems, but with more severe contamination and related risks for the population. Few promising lines, potentially suitable for growing in Africa, were shared with IITA. Final data are not yet available but this is an important contribute in knowledge sharing to jointly reduce the human and animal exposure to mycotoxins.

Chemical hazards may occur in any phases of the different livestock production systems. Aim of this review is to address an update about the key issues related to the risk of contamination in foods of animal origin by environmental contaminants linked to industrialisation or urbanisation (e.g., heavy metals and persistent organic pollutants), and natural contaminants (e.g., mycotoxins). This review deals with current issues and future perspectives on the complex issue of the safety of feeds and foods of animal origin, by taking into account the estimation of the occurrence of chemical residues in food, the hazard identification and characterisation of mycotoxins in animal feeds, and the analysis of feedstuffs as a tool to control and evaluate food safety.

The occurrence of the most widespread type A and B trichothecenes and of zearalenone was surveyed in soft and durum wheat produced in northern Italy. A total of 293 wheat fields, grown in the years 2009–2011, were surveyed; for each field, weather and cropping system data were collected. The results indicated a high deoxynivalenol incidence, with durum always more contaminated than soft wheat; in 2010, the percentage of durum wheat samples exceeding the European Commission legal limit was 39.6%. As regards type A trichothecenes, widespread contamination was observed in 2010. In soft wheat, an incidence of 70% and 85% was found for T-2 and HT-2 toxins, respectively; all the durum wheat samples were contaminated. The trichothecene contamination was affected by weather conditions; copious rainfall and high relative humidity (RH) during flowering occurred in 2010, when the highest contamination of both type A and B trichothecenes was found.

The world elderly population is rapidly increasing. This demographic change represents a new challenge for the society and demands for a multisectorial intervention to promote a long, healthy, and active life span. Between the factors that contribute in fostering a long healthy life, the nutritional regime plays a central role and is recognized as a major factor in the onset of chronic diseases. A better understanding of the interaction between nutrition and ageing is essential to unravel the mechanisms responsible for these positive/negative effects and to identify diet components promoting the quality of life in the old age and to contribute to the prevention of late-life disabilities. At Università Cattolica del Sacro Cuore, the research activity in food science is focusing on four main objectives: food quality, food safety, functional foods and diet balancing. These objectives are the target of multidisciplinary ongoing and future research activities for a better understanding of the link between diet and ageing. Briefly, the different activities are addressed to the study of the following subjects: the most relevant factors affecting food choices and habits of old aged persons; the effects of long term low dose supplementation of conjugated linoleic acid in mouse; the use of low glycemic index and high resistant starch foods to prevent diabetes and obesity; the adjuvant effect of food bacteria for vaccination; the role of food ingredients in disease; the immunosuppression effect of mycotoxins, and its relevance in ageing people; the production of sustainable and natural antioxidant ingredients to encourage a healthy diet. Our research projects emphasize an holistic and integrated approach that, by bringing together complementary research groups, can combine the collective expertise and thus provide a comprehensive assessment of the role of nutrition in healthy ageing people.

Introduzione. Legislazione. Campionamento. Metodi analitici.

The aim of this study was to evaluate the contribution of direct and indirect contamination on ochratoxin A (OTA) levels in different ripened pork products. A total of 24 Large White pigs were fed naturally OTA contaminated diets (4 different contamination levels, from 0.40 to 171 mg kg 1) for 2 weeks. Typical Italian pork products (dry sausage, dry-cured pork neck, dry-cured streaky bacon and dry-cured ham)were prepared using the contaminated tissues and ripened in 3 dry-cured ham manufacturing plants. Plasma, organs, tissues and ripened pork products were analysed for OTA content. As regards the animals fed at a level slightly less than 50 mg kg -1, the guidance value recommended by the Commission of European Communities, the OTA levels in muscle and in the ripened products were close to 1 mg kg -1 (range of the mean values 0.65e1.62 mg kg -1), the guideline value in meat products recommended by the Italian Ministry of Health. Dry sausage and dry-cured pork neck showed significantly higher OTA concentrations than dry-cured ham and dry-cured streaky bacon. OTA was partially degraded during the long ripening time of dry-cured ham. In dry sausage and dry-cured pork neck direct contamination was low and it was not detected in dry-cured streaky bacon; on the contrary, very high OTA levels were detected in several outer dry-cured ham samples (maximum OTA value: 314 mg kg-1). These results confirmed that direct contamination should be mainly monitored in dry-cured ham, whereas indirect contamination may eventually be relevant in other ripened pork products.

In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy between 2003 and 2010, were unable to produce detectable levels of aflatoxins. The genetic diversity of the population was assessed by analysis of vegetative compatibility groups (VCGs) and presence or absence of several aflatoxin biosynthesis genes. Forty-eight VCGs were identified through complementation between nitrate non-utilizing mutants. Twenty-five VCGs contained only atoxigenic isolates, and the remaining 23 only aflatoxin producers. Members of the largest atoxigenic VCG (IT6) were found in 4 of the 5 districts sampled. Six deletion patterns of genes in the aflatoxin biosynthesis gene cluster were detected. No deletions in the cluster were detected for twelve atoxigenic isolates and 10 had the entire cluster deleted. One isolate had a deletion pattern only seen once before in Nigeria. The basis for initial selection of endemic atoxigenic strains of A. flavus for biological control of aflatoxin contamination of maize in Italy is provided.

The occurrence of aflatoxins and ochratoxin A in chestnut products made in Italy was surveyed. Thirty-seven samples of chestnut flour and fourteen of dried chestnuts were collected from retail outlets located in northern Italy. The mycotoxin contamination found in this survey was widespread and remarkable. As regards aflatoxins, the incidence of aflatoxin B1 was 62.2 and 21.4 % in chestnut flour and dried chestnuts, respectively; in the same products, the percentage of samples exceeding the value of 2.0 µg kg-1 for aflatoxin B1 (maximum limit fixed by EC Regulation 165/2010 in dried fruits) was 24.3 % and 7.1 %. A widespread and high incidence of AFG1 was also found (40.5 %). The maximum values for aflatoxin B1 and total aflatoxins were 67.88 and 188.78 µg kg-1, respectively (chestnut flour sample). Ochratoxin A occurred in all samples, showing very high values (mean 12.38 and 13.63 µg kg-1 for chestnut flour and dried chestnuts, respectively); the percentages of samples exceeding the limit of 10 µg kg-1 (EU limits for dried vine fruit) were 64.9 and 42.8 % for chestnut flour and dried chestnuts, respectively. The maximum value was 65.84 µg kg-1 (dried chestnut sample).

Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood, due to its long half-life. Plasma OTA detection represents a good parameter for evaluating the exposure at the population level. The relation between plasma OTA levels, dietary habits, and specific disease risk biomarkers (body mass index (BMI), C-reactive protein (CRP), and cardiovascular risk score) was investigated. The study involved 327 subjects (150 men and 177 women) aged between 38 and 48 years. Food consumption was evaluated by means of the EPIC questionnaire; plasma OTA was measured by HPLC; CRP was determined in fresh serum samples by a latex particle-enhanced immunoturbidimetric assay. OTA was detected in 99.1% of plasma samples (LOD 25 ng/L); the mean +/- A SD value was 0.229 +/- A 0.238 ng/mL. However, only 5.2% of samples exceeded 500 ng/L, considered the threshold for a possible pathogenic activity. The estimated mean daily dietary intake of OTA resulted 0.452 +/- A 0.468 ng/kg body weight (bw)/day, markedly lower than the tolerable daily intake set by EFSA (17.1 ng/kg bw/day). Processed and mutton/lamb meat were found to contribute most to plasma OTA variance. Nevertheless, cereals, wine, beer, and jam/honey consumption correlated positively with OTA levels. Plasma OTA showed a significant positive association with CRP and cardiovascular risk score (beta = 0.20 +/- A 0.08; P = 0.015 and beta = 0.25 +/- A 0.08; P = 0.001, respectively); however, the association was present in men but not in women. Even if the hypothesis of a possible hepatic toxicity of OTA in humans is yet to be verified, the positive association between plasma OTA and CRP may indicate a possible role of OTA in inflammation status and consequently in the genesis of cardiovascular diseases and cancer.

The occurrence of principal mycotoxins in maize produced in northern Italy in 2008 was surveyed. Some easily available information related to each sample, such as hybrid maturity class, maize growth location and mean daily meteorological data were considered in order to explain the observed wide variability in mycotoxin contamination. In addition, a previously developed simple predictive model for aflatoxin B-1 contamination in maize was tested using the 2008 data. 197 samples were collected at harvest and meteorological data (mean daily temperature, daily rainfall) were collected from 40 weather stations located near the sampling regions during the period 1 June to 30 September 2008. The results indicated that aflatoxin contamination is of concern in a restricted geographic region (south-east Po Valley). In this region, about 75.0% of the maize samples showed aflatoxin levels higher than 0.5 mu g/kg. After a slight modification, the predictive model could indicate a possible high contamination risk. Moreover, the study confirmed that maize harvested in northern Italy was contaminated with fumonisins; throughout the northern Italian plain contamination occurred at significant levels. On the other hand, contamination with trichothecenes and zearalenone was generally low. It has been observed that FAO class 300-400 maize hybrids can be more susceptible to aflatoxin contamination and less to Fusarium mycotoxins than later maturing hybrids.

The temporal efficacy of three different chemical fungicides (Folicur®, Proline®, Sportak 45EW®) and a biocontrol bacterium (Serenade, B. subtilis) in reducing growth and toxin production by isolates of F. verticillioides and A. flavus was studied in vitro under different water activity regimes (0.99, 0.98 and 0.95). All the fungicides significantly inhibited mycelial growth compared with the control; the most effective treatment, both against F. verticillioides and A. flavus, was Sportak 45EW® (approx. 99%). The inhibitory effect of all fungicides generally improved with increasing concentration. Serenade always decreased fungal growth, with optimal results at concentrations of 104 and 106 (70‒75% reduction). All the fungicide treatments resulted in a significant reduction in both FB1+FB2 and AFB1 production when compared to the control, at the end of the incubation period and with the 2 concentrations used (approx. 99%). A threshold concentration inoculum of at least 104 CFUs of B. subtilis per g was required to achieve a significant control of mycotoxin production. Sportak 45EW® and Serenade gave the best control of mycotoxin production with a reduction of 95% compared to the controls. Use of Serenade in the field should include due consideration to its sensitivity to low water activities, when compared to the target pathogens.

The aim of this work was to investigate the dynamics of fungi and related mycotoxins during cereal storage in silo bags. A 2-year trial was organised (2009-2011); in each year, two silo bags, filled with maize and durum wheat respectively, were prepared . During storage, meteorological data were collected from a close station and temperature, CO2 and O2 were measured inside the silos. Grain was sampled from silo filling (September) every 40 days until June (7/8 samples per silo per year). Water activity of grain, colony forming units (CFU) and mycotoxin content (fumonisins, aflatoxins and ochratoxin in maize, deoxynivalenol and ochratoxin in wheat) were determined. Temperature inside the silo bags followed the trend of external temperature, with a more limited variation. A decrease of O2 was observed from 16.4% to 2.0% in maize and from 15.7% to 15.1% in wheat; the decrease was balanced by CO2 increase. Minor variations were registered in grain water activity and CFU and mycotoxin content did not change significant throughout the storage period; the results were consistent in the two years. On the basis of these results, silo bags prove to be suitable for safe cereal storage.

AFs were determined by HPLC with fluorescence detection and FBs by HPLC coupled with mass spectrometry. Aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) were found in 57% and 21% of flours from white yam with concentrations ranging from <0.02 (limit of detection, LOD) to 3.2 g kg-1 (mean = 0.4 g kg-1) and from <0.05 to 3.5 g kg-1, respectively. AFB1 was the only aflatoxin detected in samples from water yam, contaminating 32% of the samples with values ranging from

Aflatoxin (AF) contamination in maize is of worldwide importance. Aspergillus flavus and A. parasiticus are the principal fungi responsible for AF production. Based on the current literature, AFs are not considered a problem in wheat and rice at harvest and no data were found on aspergilliwheat/ rice interactions in the field. Data on the effects influencing the development of A. flavus and A. parasiticus on maize and maize kernel at harvest were collected; however data on A. parasiticus and AFB2-G1-G2 were not sufficient for further use in predictive modelling. Thus, a model was developed to predict the risk of AFB1 contamination, due to A. flavus, in maize at harvest and further adapted to wheat and rice as host crops. The Joint Research Centre of the EC provided a database with mean daily temperatures during emergence, flowering and harvesting of maize, wheat and rice. Meteorological data (temperature, relative humidity and rain) obtained from the LARS weather generator, were used as input for the modelling of crop phenology and A. flavus behaviour. The output was designed at a 50 x 50 km scale over the European territory and generated over 100 years, in three different climate scenarios (present and A2 and B2 storylines, or +2°C and +5°C scenarios, proposed by the Intergovernmental Panel on Climate Change). Predictions showed a reduction in season length and an advance in flowering and harvest dates leading to an enlargement of the crop growing areas towards north EU, mainly for maize and rice, because earlier ripening could occur in these areas. The risk of A. flavus contamination was expected to increase in maize, both in the +2°C and +5°C scenarios, to be very low in wheat and to be absent in rice. Results were discussed and recommendations were made on data collection and prevention measures on AF risks.

The occurrence of ochratoxin A, trichothecenes, fumonisins and aflatoxins in a sample of 106 beers produced in several European countries, was investigated. Aflatoxins were not detected in any samples, whereas ochratoxin A, trichothecenes and fumonisins were found in a relatively high number of samples. Their presence was at low levels in all samples; however, some differences were observed between the European countries. As regards ochratoxin A, beer samples from southern Europe showed levels always lower than 0.040 µg l-1, while the samples from other European countries showed significantly higher values, up to 0.189 µg l-1 (P<0.001). For fumonisins, the levels of Italian beers were significantly higher compared to the samples from other countries (P=0.006).

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12 µgkg-1, respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p<0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.

MYCORED is a large collaborative project focused on developing strategic solutions to reduce contamination by mycotoxins of major concern in economically important food and feed chains, through mycotoxin research joint actions. The work package 3 of the project is focused on the development and validation of predictive models for mycotoxin producing fungi. Deoxynivalenol (DON) and zearalenone (ZEA) in cereals are considered as an health problem worldwide. They are secondary metabolites associated to Fusarium head blight (FHB), a ear disease caused by a complex of Fusaria, with F. graminearum considered as the main fungus involved. Predictive models and decision support systems (DSS) are useful tools to rationalise both the cropping system and the survey of contamination in order a) to minimise consumers exposure; b) to describe the risk of contamination at harvest and rationalise the harvest/post-harvest logistic; c) to draw different scenarios based on real and simulated meteorological data. The aim of this work is to validate a predictive model developed by Rossi et al (2003) and the related DSS (Rossi et al., 2007) to forecast risk level associated with FHB in wheat, in different geographic areas. Four countries were involved (Italy, Russia, Egypt, and Mexico), where 133 wheat samples with related cropping system and meteorological data were collected in 2009 according to defined protocols. DON and ZEA contamination at harvest was determined in all samples. Meteorological data were used as input in the model and the predicted risk of DON and ZEA in wheat at harvest was obtained as output. Predicted and observed data were compared to validate the model. The model validation gave good results in almost all the geographic areas monitored; correct predictions varied between 75 and 100% in almost all data sets, except for those collected in central-southern Italy. As a global result, correct predictions were 69%; the absence of underestimates, which could represent a

Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood and milk. The link between the nutritional habits of pregnant women both of Italian and foreign nationality resident in Italy and the presence of ochratoxin A in cord blood and in maternal milk was investigated. The study involved 130 pregnant women. Food consumption during pregnancy was evaluated by means of the EPIC questionnaire; OTA content was determined in cord serum and maternal milk by HPLC. The mean daily dietary intake of OTA was 1.02 +/- A 1.20 and 0.87 +/- A 0.78 ng/kg of bodyweight for Italian and non-Italian women, respectively, but this difference was not statistically significant. The incidence of positive milk samples was 73.0 and 85.0% among the Italian and non-Italian mothers, respectively. Pork meat, soft drinks, sweets and red wine showed a significant relationship with OTA level in serum. As far as milk is concerned, a positive relationship resulted for pork meat, sweets, soft drinks and seed oils. A positive relationship between serum OTA level and the ratio serum/milk OTA was found. The intake of OTA had no effect on the cord blood creatinine level. This study confirms that OTA is widely present in human milk and therefore could pose a risk for the newborn.

The objective of this study was to compare the effect of Bt corn and isogenic corn on the growth of weaned piglets. One hundred and twenty-eight weaned piglets weighing 8.8 ±1.27 kg live weight were randomly assigned to 4 groups of 32 animals each (16 castrated males and 16 females). Bt corn (line MON810) and isogenic corn were produced at two farms located in the provinces of Lodi and Venice (northern Italy). The Bt corn had the same chemical composition as the isogenic corn but a lower content of fumonisin B1 (FB1). The experimental period (35 days) was in two phases: 0-14 d and 15-35 d. There was no significant difference in average daily gain (ADG) among groups during the first feeding phase. Compared to animals fed isogenic corn, the piglets fed Bt maize gained more weight during the second feeding phase (Bt: 464.1 g/d, isogenic: 429.1 g/d; P < 0.05). Also, the ADG over the entire trial was higher in piglets fed Bt corn versus piglets fed isogenic corn (Bt: 396.4 g/d, isogenic: 374.1 g/d; P < 0.05). The ADG of the whole period decreased linearly (P <0.05) with respect to the FB1 content of the diet. Final weight was higher in piglets fed the diet containing Bt corn (Bt: 22.68 kg, isogenic: 21.83 kg; P < 0.05). No differences in feed intake and in the feed:gain ratio were observed, although a linear response between FB1 and feed:gain ratio in first 14 days of the experiment was detected.

The aim of this study was to define quantitative relationships between temperature and water activity (aw), fungal growth and aflatoxin B1 (AFB1) production. A strain of A. flavus isolated from maize in North Italy, and previously tested and found positive for AFB1 production, was used for these experiments. The optimum temperature for AFB1 production was at 25°C, slightly lower with respect to results obtained in other countries. 0.83 aw was the limit for growth of this strain of A. flavus after 60 days incubation at the optimum temperature. The solutes used to modify aw, glycerol and NaCl, influenced both growth and secondary metabolite production. Media modified with glycerol resulted in more AFB1 production when compared to the non-ionic solute NaCl added media. Maize based media, prepared with flour obtained from kernels at different ripening stages, only slightly influenced growth rates of A. flavus. The quantitative relationship obtained between fungal growth and AFB1 production in diverse temperature and aw levels were used to develop a valid predictive model for A. flavus presence and AFB1 production in the field.

The extraction procedure for aflatoxin determination in maize is based on a methanol-water (8+2 v/v) or an acetone-water (85+15 v/v) mixture. Initially, the extraction efficiency of two solvents was evaluated for each aflatoxin. Significantly higher levels of aflatoxin B1 were obtained by acetone-water, on the contrary higher levels of aflatoxin G2 were achieved by methanol-water. Then, acetone-water mixtures in different proportions (7+3, 6+4 and 5+5 v/v) were tested to improve the extraction of aflatoxin G2. Applying these extraction mixtures, the values both of aflatoxin B1 and of other aflatoxins were generally higher compared to those obtained by acetone-water 85+15; moreover, acetone-water (6+4) and (7+3) showed the best extraction efficiency for all aflatoxins.

BACKGROUND: A six year study was conducted to evaluate the possible support of pesticides in mitigating mycotoxin contamination in maize grown in northern Italy. Different pesticides to control Ostrinia nubilalis (ECB), Fusarium verticillioides and Aspergillus flavus were considered to check their efficacy and the best schedule to reduce fumonisin and aflatoxin contamination. RESULTS: Fumonisin B1 (FB1) contamination at harvest has been reduced by ECB control while the addition of ingredients targeted to F. verticillioides gave a limited advantage, mainly with low FB1 contamination; nevertheless, fusaria control is relevant in high risk conditions, when FB1 is likely to exceed the legal limit. The combination of fungal and pest control in a single spray at silk browning stage (BBCH 67) is the best way of application. These results are robust because obtained in a 6 year trial, with relevant differences in meteorological conditions, ECB and fungal presence and mycotoxins contamination. CONCLUSION: Insecticides have been shown to give advantages in their application, while the low FB1 contamination in the final years of the study did not lead to conclusive data on the advantage of adding a fungicide. Chemicals applied never modified A. flavus incidence nor aflatoxin contamination (only detected in trace amounts).

The heterogeneous three-dimensional spatial distribution of mycotoxins has proven to be one of the main limitations for the design of effective sampling protocols. Current sample collection protocols for mycotoxins have been designed to estimate the mean concentration and fail to characterise the spatial distribution of the mycotoxin concentration due to the aggregation of the incremental samples. Geostatistical techniques have been successfully applied to overcome similar problems in many research areas. However, little work has been developed on the use of geostatistics for the design of sampling protocols for mycotoxins. This paper focuses on the analysis of the two and three-dimensional spatial structure of fumonisins B1 (FB1) and B2 (FB2) in maize in a bulk store using a geostatistical approach and on how results help determine the number and location of incremental samples to be collected. The spatial correlation between FB1 and FB2, as well as between the number of kernels infected and the level of contamination was investigated. For this purpose, a bed of maize was sampled at different depths to generate a unique three-dimensional data set of FB1 and FB2. The analysis found no clear evidence of spatial structure in either the two-dimensional or three-dimensional analyses. The number of Fusarium infected kernels was not a good indicator for the prediction of fumonisin concentration and there was no spatial correlation between the concentrations of the two fumonisins.

The extraction efficiency of aflatoxin B1 (AFB1) in cattle feed containing nine adsorbents (ADSs) was investigated using two organic/aqueous solvents composed of methanol/water (80/20 v/v; MeOH) and acetone/water (85/15 v/v; AC). Samples were obtained including a highly AFB1-contaminated (HC) and a low-level AFB1-contaminated (LC) feedstuff (15.33 and 7.57 mu g kg-1, respectively), nine ADSs (four clay minerals; one yeast cell wall-based product; one activated carbon and three commercial ADS products) at two different levels of inclusion (10 and 20 g kg-1). After solvent extraction and immunoaffinity column clean-up, all samples were analysed for AFB1 by high-performance liquid chromatography (HPLC) with fluorescence detection. For each contamination level (HC and LC), the data obtained were analysed using a factorial arrangement in a completely randomized design. Means were compared with the correspondent controls using the Dunnett's test. No statistical difference was found in AFB1 levels of feedstuffs not containing ADSs when extracted with AC or MeOH, even if numerically higher values were obtained with AC. A dose-dependent effect (p 0.01) of ADSs inclusion was observed on AFB1 recoveries that were lower when the higher ADS level (20 g kg-1) was included in the HC and LC feedstuffs. Higher AFB1 recoveries were obtained using AC compared with MeOH, both in HC (75.0% versus 12.0%, respectively) and in LC (84.0% versus 22.8%, respectively) ADSs containing feedstuffs. However, when the activated carbon and the sodium bentonite were included in feeds, lower AFB1 concentrations with respect to control values (p 0.001 and 0.05, respectively) were obtained also using AC.

Undesirable substances such as mycotoxins and natural toxicants can be present in plants and derived products thereof. Depending on the nature and the concentration levels of the compound, these might be of concern for human and/or animal health. Risk assessments on the natural plant toxicant morphine in poppy seeds and the selected mycotoxins: alternaria toxins, moniliformin, diacetoxyscirpenol, sterigmatocystin and phomopsins in food and feed, ergot alkaloids in food and nivalenol in feed have not been carried out at the European level. Therefore it is expected that the European Commission may ask EFSA to assess the risks to human and animal health related to these substances in the near future. To carry out these risk assessments to the highest standards and in an efficient way scientific background information e.g. chemistry, occurrence, toxicokinetics and toxicity on these selected mycotoxins and plant toxicant are needed. The project CFP/EFSA/CONTAM/2008/01 “Scientific information on mycotoxins and natural plant toxicants” reports this information in the present report. The report presents information regarding a plant toxicant, morphine, in poppy seeds, and some mycotoxins, in particular Alternaria toxins in food and feed, Ergot alkaloids in food, moniliformin in food and feed, nivalenol in feed, diacetoxyscirpenol in food and feed, sterigmatocystin in food and feed and phomopsins in food and feed. After a short introduction, fungi involved in the production of each specific mycotoxin are described. Physico-chemical characteristics, biosynthesis and chemical analisis are then discussed for all toxins. Occurrence data are presented, taking into account all products in which the mycotoxins have been reported. Mitigation, intended as all actions that can be taken in the pre- and post-harvest periods to reduce the contamination of the final products are discussed. Pharmacokinetic aspects (absorption, distribution and excretion, metabolism and carry-over), as well

Ochratoxin A (OTA) is a mycotoxin suspected to exert toxic effects on liver cell: in our research, we investigated the role of OTA on the genesis of liver disease and HCC in a case-control study. 43 subjects with chronic liver disease (case group) were matched for age and sex with 62 volunteers without liver disease (control group). In order to avoid any confounding effect of alcohol intake, all subjects consumed less than 20 g/day of alcohol for men and 10 g/day for woman. A blood sample was taken from each subject and analyzed for OTA, liver transaminases, ALP. Bilirubin, CRP and creatinine. In our study, OTA intake was low (0.039 ng/kg b.w./day - 0.065 ng/kg b.w./day) and 49% (51/104) of subjects had plasma OTA levels lower than LOD (25 ng/l). Only 10 (%) subjects exceeded the value of 200 ng/l; no differences were found between serum OTA concentrations of control and case groups. In the case group, the CRP levels were linearly related with bilirubin levels (r = 0.298; P < 0.05) and in subjects with liver disease and positive serum OTA concentration, this relationship improved (r = 0.638; P < 0.01). Although OTA did not affect the prevalence of liver disease, among the 29 cirrhotic patients, the prevalence of HCC was higher in OTA positive subjects than in OTA negative ones (P < 0.05). Our results do not clearly support the role of OTA as a risk factor for HCC or cirrhosis, however the observed prevalence of HCC in OTA positive subjects requires further investigation.