Ochratoxin A and Sterigmatocystin in Long-Ripened Grana Cheese: Occurrence, Wheel Rind Contamination and Effectiveness of Cleaning Techniques on Grated Products.

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

Alternaria toxins in tomato products in Northern Italy in the period 2017-2019.

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.

Modelling Fungal Growth, Mycotoxin Production and Release in Grana Cheese.

No information is available in the literature about the influence of temperature (T) on Penicillium and Aspergillus spp. growth and mycotoxin production on cheese rinds. The aim of this work was to: (i) study fungal ecology on cheese in terms of T requirements, focusing on the partitioning of mycotoxins between the rind and mycelium; and (ii) validate predictive models previously developed by in vitro trials. Grana cheese rind blocks were inoculated with A. versicolor, P. crustosum, P. nordicum, P. roqueforti, and P. verrucosum, incubated at different T regimes (10-30 °C, step 5 °C) and after 14 days the production of mycotoxins (ochratoxin A (OTA); sterigmatocystin (STC); roquefortine C (ROQ-C), mycophenolic acid (MPA), Pr toxin (PR-Tox), citrinin (CIT), cyclopiazonic acid (CPA)) was quantified. All the fungi grew optimally around 15-25 °C and produced the expected mycotoxins (except MPA, Pr-Tox, and CIT). The majority of the mycotoxins produced remained in the mycelium (~90%) in three out of five fungal species (P. crustosum, P. nordicum, and P. roqueforti); the opposite occurred for A. versicolor and P. verrucosum with 71% and 58% of STC and OTA detected in cheese rind, respectively. Available predictive models fitted fungal growth on the cheese rind well, but validation was not possible for mycotoxins because they were produced in a very narrow T range.

Aspergillus flavus and Fusarium verticillioides Interaction: Modeling the Impact on Mycotoxin Production.

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 order to improve effective prediction of growth and mycotoxin production by such fungi.

LC-MS/MS and LC-UV Determination of Moniliformin by Adding Lanthanide Ions to the Mobile Phase.

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 ß-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.

Correction: Mauro, A., et al. Biological Control Products for Aflatoxin Prevention in Italy: Commercial Field Evaluation of Atoxigenic Aspergillus flavus Active Ingredients. Toxins 2018, 10, 30.

The authors wish to make the following correction to their paper [...].

Mitigation measures for acrylamide reduction in dough-based potato snacks during their expansion by frying.

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.

Fate of mycotoxins and related fungi in the anaerobic digestion process.

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.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

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.

Biological Control Products for Aflatoxin Prevention in Italy: Commercial Field Evaluation of Atoxigenic Aspergillus flavus Active Ingredients.

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 Aspergillusflavus 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-X1™.

Survey on acrylamide in roasted coffee and barley and in potato crisps sold in Italy by a LC-MS/MS method.

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.

Sterigmatocystin Occurrence in Paddy and Processed Rice Produced in Italy in the Years 2014-2015 and Distribution in Milled Rice Fractions.

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 µg·kg-1. 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 µg·kg-1; for parboiled rice, the incidence was 90.9% and the maximum level was 1.09 µg·kg-1. 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.

Modeling Growth and Toxin Production of Toxigenic Fungi Signaled in Cheese under Different Temperature and Water Activity Regimes.

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, awopt = 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.

Co-occurrence of aflatoxins, ochratoxin A and citrinin in "egusi" melon (Colocynthis citrullus L.) seeds consumed in Ireland and the United Kingdom.

The natural co-occurrence of aflatoxins (AFs), ochratoxin A (OTA) and citrinin (CIT) in melon seed samples obtained from retailers and households in Ireland and the United Kingdom (UK) was evaluated. AFs and OTA were determined by HPLC with fluorescence detection while CIT was analysed by HPLC-MS/MS. AFB1 was detected in all (100%) samples (mean = 9.7 µg kg(-1); range = 0.2-66.5 µg kg(-1)). Mean total AFs was 12.0 µg kg(-1) (range = 0.3-82 µg kg(-1)). Commercially retailed samples showed a significantly higher AFB1 contamination (p < 0.05) than the household samples. OTA occurred in 3 (13.6%) samples, while 4 (18.2%) were contaminated with CIT at very low levels. In this study, 68% of the melon seed samples were contaminated above the 2 µg kg(-1) EU limit for AFB1 in oilseeds. These results highlight the need for the development of strategies to reduce AF contamination in "egusi" for human consumption.

Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese.

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.

Analysis of sterigmatocystin in cereals, animal feed, seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification.

A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.

Co-occurrence of type A and B trichothecenes and zearalenone in wheat grown in northern Italy over the years 2009-2011.

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.

Nutrition and Ageing.

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.

Structure of an Aspergillus flavus population from maize kernels in northern Italy.

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.

Plasma ochratoxin A levels, food consumption, and risk biomarkers of a representative sample of men and women from the Molise region in Italy.

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: OTA was detected in 99.1% of plasma samples (LOD 25 ng/L); the mean ± SD value was 0.229 ± 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 ± 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 (ß = 0.20 ± 0.08; P = 0.015 and ß = 0.25 ± 0.08; P = 0.001, respectively); however, the association was present in men but not in women. CONCLUSIONS: 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.