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A total of 64 samples of sorghum (37 Tunisian sorghum samples and 27 Egyptian sorghum samples) were collected during 2011-2012 from markets in Tunisia. Samples were analysed for contamination with aflatoxin B1, ochratoxin A and zearalenone by High-Performance Liquid Chromatography Coupled with Fluorescence Detection (HPLC-FLD). Aflatoxin B1 was found in 38 samples in the range 0.03-31.7 µg kg -1 . Ochratoxin A was detected in 24 samples with concentrations ranging from 1.04 to 27.8 µg kg -1 . Zearalenone was detected in 21 samples and the concentration varied between 3.7 and 64.5 µg kg -1 . ANOVA analysis of the influence of the country of origin on the incidence and concentration of mycotoxins in the samples studied showed no significant difference (P > 0.05) between the two batches of samples for each of the three mycotoxins studied. The studied mycotoxins contaminate sorghum and may also co-exist because of the diversity of the mycobiota in this cereal.

Ochatoxin A (OTA) is one of the most important mycotoxins based on its toxicity. The oral route is the main gateway of entry of OTA into the human body, and specialized epithelial cells constitute the first barrier. The present study investigated the in vitro cytotoxic effect of OTA (5, 15 and 45 μM) and production of OTA metabolities in Caco-2 and HepG2 cells using a co-culture Transwell System to mimic the passage through the intestinal epithelium and hepatic metabolism. The results derived from MTS cell viability assays and transepithelial electrical resistance measurements showed that OTA was slightly cytotoxic at the lowest concentration at 3 h, but significant toxicity was observed at all concentrations at 24 h. OTA metabolites generated in this co-culture were ochratoxin B (OTB), OTA methyl ester, OTA ethyl ester and the OTA glutathione conjugate (OTA-GSH). OTA methyl ester was the major metabolite found in both Caco-2 and HepG2 cells after all treatments. Our results showed that OTA can cause cell damage through several mechanisms and that the OTA exposure time is more important that the dosage in in vitro studies. OTA methyl ester is proposed as an OTA exposure biomarker, although future studies should be conducted., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The major objective of this study was to describe the effect of water activity and temperature on radial growth and production of ochratoxin A (OTA) and zearalenone (ZEA) on sorghum grains of three Aspergillus tubingensis and three Fusarium incarnatum isolates. The water activity range was 0.91-0.99 a w for F. incarnatum isolates and 0.88-0.99 a w for A. tubingensis isolates. Temperatures of incubation were 15, 25 and 37°C for both species. Mycotoxin production was determined after 7, 14, 21 and 28days depending on the growth rate of the six isolates. Maximum growth rates (mm/day) were observed at 37°C and 0.99 a w for A. tubingensis isolates and at 0.99 a w and 25°C for F. incarnatum isolates. A. tubingensis was able to grow at 15°C only at the highest a w levels (0.97 and 0.99 a w ). However, at this temperature F. incarnatum grew at 0.94 a w . Optimum ochratoxin A production was observed at 0.97 a w ×37°C whereas optimal conditions for ZEA production varied from one isolate to another. Moreover, isolates of F. incarnatum from Tunisia do not require high a w and temperature levels to yield maximum levels of ZEA. In general, our results showed that there is no correlation between the growth and production of ZEA in the case of F. incarnatum. This is the first study on the water activity and temperature effect on growth rate and ZEA production of F. incarnatum. Our results show that sorghum grains not only support growth but also OTA and ZEA production by A. tubingensis and F. incarnatum, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The stability of deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-glucoside), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), de-epoxy-deoxynivalenol (DOM-1) and ochratoxin A (OTA) during thermal processing has been studied. Baking temperature, time and initial mycotoxin concentration in the raw materials were assayed as factors. An improved UPLC-MS/MS method to detect DON, DON-3-glucoside, 3-ADON, 15-ADON and DOM-1 in wheat baked products was developed in the present assay. The results highlighted the importance of temperature and time in mycotoxin stability in heat treatments. OTA is more stable than DON in a baking treatment. Interestingly, the DON-3-glucoside concentrations increased (>300%) under mild baking conditions. On the other hand, it was rapidly reduced under harsh conditions. The 3-ADON decreased during the heat treatment; while DOM-1 increased after the heating process. Finally, the data followed first order kinetics for analysed mycotoxins and thermal constant rates (k) were calculated. This parameter can be a useful tool for prediction of mycotoxin levels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The contamination of food commodities by fungal toxins has attracted great interest because many of these mycotoxins are responsible for different diseases, including cancer and other chronic illnesses. Ochratoxin A (OTA) is a mycotoxin naturally present in food, and long-term exposure to food contaminated with low levels of OTA has been associated with renal cancer. In the present study, the cytotoxicity, cytostaticity, and genotoxicity of OTA (0.075-15 µM) in human lymphocytes were evaluated. A comet assay, a modified comet assay (DNA repair assay), which uses N-hydroxyurea (NHU) to detect non-repaired lesions produced by OTA, and a cytokinesis-blocked micronucleus assay were used. Treatments with OTA were not cytotoxic, but OTA caused a cytostatic effect in human lymphocytes at a concentration of 15 µM. OTA (0.075-5 µM) produced a slight increase in the percentage of DNA in the comets and a delay in the DNA repair capacity of the lymphocytes. Micronucleus (MN) induction was observed at OTA concentrations of 1.5 and 5 µM. Our results indicate that OTA induces DNA stable damage at low doses that are neither cytotoxic nor cytostatic, and OTA delays the DNA repair kinetics. These findings indicate that OTA affects two pivotal events in the carcinogenesis pathway.

The aflatoxins (AFs), deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based raw materials. Usually, the higher mycotoxins concentrations in cereals are found in the external layers of the grain (bran). Nowadays bran is increasingly consumed for its high fibre concentration. The objectives of this study were determining the concentration of these mycotoxins in bran samples intended for direct human consumption and to study the influence of some characteristics of the samples that may affect the mycotoxins content, there are not studies about fibre for direct human consumption. 67 bran samples from shops and supermarkets from two different Spanish cities were analyzed, being 37 samples of wheat bran and the remaining of oat bran. The results showed a major presence of DON in the analyzed samples, with levels above the EU legislation in some samples. Presence of DON was more frequent in wheat samples, compared to oats ones (p<0.05). Extruded or toasted samples, subjected to a heat treatment during processing, presented a significantly lower concentration of OTA, and differences between the organically and conventionally produced samples were also detected in OTA, which showed higher levels in the organic samples. Co-occurrence was frequently found between the Fusarium mycotoxins (ZEA and DON). Due to the high levels of DON in the analyzed samples, a calculation of DON intake has been made and it has been demonstrated that bran can account for an important percentage of DON exposure in the total diet., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess the impact of suboptimal environmental conditions on the intraspecific variability of A. carbonarius growth and OTA production using thirty isolates of A. carbonarius. Three a(w)/temperature conditions were tested, one optimal (0.98a(w)/25°C) and two suboptimal: 0.90a(w)/25°C and 0.98a(w)/37°C as suboptimal water activity and temperature, respectively, which might take place through over ripening and dehydration of grapes. For each condition, 12 Petri dishes were inoculated, and colony growth and OTA production were measured over time. ANOVA revealed significant differences among μ and λ within the 30 assayed isolates. Coefficients of variation (CV%) revealed a wider dispersion of growth rates at 0.90a(w)/25°C compared to 0.98a(w)/25°C, and a more than 4-fold higher CV at 0.98a(w)/37°C compared to 0.98a(w)/25°C. However, dispersion of lag phases was similar at 0.98a(w)/25°C and 0.90a(w)/25°C and wider at 0.98a(w)/37°C. There were significant differences (p<0.05) among OTA levels (ng/mm(2)) for the different conditions, values being lower under marginal conditions, and particularly at 0.98a(w)/37°C. Coefficients of variation (CV%) revealed a wider dispersion of OTA production at 0.90a(w)/25°C compared to 0.98a(w)/25°C, while CV at 0.98a(w)/37°C was similar to that at 0.98a(w)/25°C. In order to address the strain variability in growth initiation and prove the well-established notion of reducing OTA in foods by preventing fungal growth, a greater number of strains should be included when developing models for conditions that are suboptimal both for a(w) for OTA production and temperature levels for growth., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The effect of three alternating temperatures cycles (20/30, 20/37 and 25/42 degrees C) and photoperiod on growth and Ochratoxin A (OTA) production of six isolates of Aspergillus carbonarius on synthetic nutrient medium were investigated. The different temperature regimes affected significantly both the mycelial growth and the OTA production. The best growth and OTA production were recorded at 20/30 degrees C. The isolates from the region of Baddar produced the highest OTA yields. A 24 h light cycle generally enhanced the growth of A. carbonarius. Growth rates cycles of 11 h/13 h light/darkness and 24 h darkness were often similar for individual isolates, such conditions enhanced OTA production in two of the six isolates tested., (2010 Elsevier B.V. All rights reserved.)

This study investigated the effects of temperature (15 to 37 degrees C) and water activity (0.90 to 0.99) on the growth and production of ochratoxin A (OTA) by Aspergillus carbonarius cultured on synthetic nutrient medium (SNM) after 5 and 10 d of incubation. Total of 8 ochratoxigenic A. carbonarius, isolated from vineyards located in different regions of Tunisia, were used. Growth data were modeled by the flexible model of Baranyi and growth rates at each set of conditions were obtained. For both growth and OTA production, optimal water activity was 0.99; however, optimal temperature varied. The optimal temperature for growth was 30 degrees C. At 37 degrees C, the growth rate decreased significantly (P < 0.05). Maximum toxin production occurred at temperatures in the range of 15 to 25 degrees C with the optimum one depending on the isolate tested. Significant amounts of OTA were produced after only 5 d of incubation. Our results showed that A. carbonarius isolated from Tunisian grapes behave as those from European and Australian grapes, as reported in the literature, although some differences in trends for growth and OTA production were observed.

The aim of this study was to assess the colonizing capability and OTA production of different populations of Aspergillus section Nigri spp. in grapes, as affected by the interactions with other fungi, during a simulated in vitro sun-drying. Mature white grapes were divided into two lots of healthy and artificially injured grapes and inoculated with A. section Nigri spp. (A. carbonarius OTA producer, A. niger aggregate OTA producer, A. niger aggregate OTA non-producer), Eurotium amstelodami and Penicillium janthinellum, in different combinations. The drying process was simulated adjusting water activity firstly at 0.98 a(w) and gradually decreasing it to 0.76 a(w) for a total of 20 days. Colonizing grape percentages were recorded after 5, 10, 15 and 20 days of dehydration and OTA content was measured after 5, 7, 10, 12, 15, 17 and 20 days. Colonization of grapes increased with time in all treatments. A. niger aggregate OTA-positive showed the highest colonization percentage, followed by A. carbonarius, and finally their mixed inoculum. When the two OTA-producing strains were combined, addition of any other microorganism increased the percentage of infection by A. section Nigri. A. carbonarius was the highest OTA producer in pure culture, followed by A. niger aggregate OTA-positive. In general, when competing fungi were added to A. carbonarius inoculum, the OTA content was reduced. E. amstelodami was the only competing fungus which increased OTA accumulation. The sun-drying process may be conducive to OTA accumulation in dried grapes. The complex fungal interactions which may take place during this process, may act as an additional control factor, given that the higher presence of A. niger aggregate OTA-negative inhibits OTA accumulation by OTA producing species.

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.

The effect of the application of two fungicides (cyprodinil alone and a mixture of cyprodinil and fludioxonil) on the mycoflora of grapes, especially ochratoxigenic fungi, was studied. Different doses and application times were analyzed. Grape mycobiota was isolated and identified, and the classification of black aspergilli was carried out. We found that 81.7% of the isolates belonged to Aspergillus niger aggregate and 18.3% to Aspergillus carbonarius. The ability to produce ochratoxin A (OTA) was studied on Czapek yeast extract agar (CYA) medium in 238 isolates. Most A. carbonarius (97.2%) produced detectable amounts of OTA, while only 2.9% of the A. niger aggregate were OTA producers. Most of the isolates (58%) produced less than 2.5 microg OTA per g of CYA. That, together with the highest levels of black aspergilli detected near harvest, proved the reported theory that they are primarily responsible for OTA in grapes. The fungicides studied had a significant effect on black aspergilli in three of the four vineyards sampled, as the natural increase of black aspergilli when approaching harvest was in general lower in all the fields treated with fungicides as compared to the control treatment. A mixture of cyprodinil (37.5%) and fludioxonil (25%) applied at veraison and 21 days before harvest was the most effective treatment to prevent black aspergilli in grapes, together with a single application of this mixture at veraison followed by an application of cyprodinil (50%) 21 days before harvest. No OTA was detected in musts (n=112) produced from either the control treatment or the treated grapes.

The aim of this study was to characterize Ochratoxin A (OTA) production by Aspergillus carbonarius under different environmental conditions, and to elucidate the diffusion capacity of OTA throughout synthetic medium. One strain belonging to the species A. carbonarius isolated from vine dried fruit was single-point inoculated onto triplicate synthetic nutrient medium plates at two water activities (0.92 and 0.97) and two temperatures (20 and 30 degrees C). Daily radii were measured and OTA production was tested after 4, 7, 10, 14 and 18 days of incubation at four distances from the centre of colony (1-4 cm). OTA production was detected mainly at 0.97 a(w). Earlier production was detected at 30 degrees C (optimum for growth), whereas maximum OTA concentrations were found at 20 degrees C. OTA production was detected from mycelium that was only a few days old and attained its optimum when mycelium was 4-7 days old at 0.97 a(w). OTA diffusion was observed at 0.92 a(w) and 20 degrees C. Thus OTA production is discernable in young A. carbonarius mycelium and diffusion of the toxin has been shown to occur in a solid substrate.

Ochratoxin A is a well-known mycotoxin produced by species of the genera Penicillium and Aspergillus. OTA-producing species from A. section Nigri are considered the source of OTA detected in grapes, dried vine fruits and wines. Other fungi present in grapes during their maturation can grow and interact with OTA-producing Aspergillus species and affect OTA production. In this study seven fungi (Alternaria alternata, Cladosporium herbarum, Eurotium amstelodami, Trichoderma harzianum, Penicillium decumbens, P. janthinellum and Candida sp.) disolated from grapes and dried vine fruits were grown in SNM medium paired with OTA-positive A. carbonarius at two temperatures (20 and 30 degrees C) and at two water activities (0.92 and 0.97). OTA production was tested after 5, 7, 10, 14 and 18 days of incubation, at four distances (1, 2, 3 and 4 cm) from A. carbonarius inoculation point in the inter-colony axis. At 0.92 a(w) OTA production was almost negligible. At 0.97 a(w) and 30 degrees C OTA accumulation was reduced when A. carbonarius was grown in paired cultures, particularly with A. alternata, C. herbarum, P. decumbens and P. janthinellum. At 0.97 a(w) and 20 degrees C, there was no clear effect of the interacting species on OTA accumulation; in general E. amstelodami and Candida sp. seemed to stimulate OTA production, whereas T. harzianum and P. decumbens reduced it. Competing mycoflora acted as an additional control factor against OTA accumulation at 30 degrees C; but at 20 degrees C, where OTA production is optimal, this did not happen. Thus maintaining the temperature of grapes at or above 30 degrees C during dehydration may provide some control against OTA accumulation in grapes.

The objective of this work was to determine the effects of extrusion cooking on the stability of ochratoxin A (OTA) in an artificially contaminated hulled barley meal (0.73-mm grain diameter) using a single screw extruder. The extrusion cooking parameters were temperature (140, 160, and 180 degrees C), initial moisture content of barley meal (24, 27 and 30%), and residence time (30, 40, 50, 60, and 70 s). Both unextruded and extruded samples were analyzed for OTA by high-performance liquid chromatography. Extrusion cooking variables significantly affected the stability of OTA (P < 0.05). Greater OTA reductions were achieved at higher residence time (70 s), medium temperature level (160 degrees C), and either high (30%) or low moisture (24%) content of samples. The amount of OTA destroyed during the extrusion process ranged from 17 to 86% depending on the studied parameters. The decrease in the amount of OTA after extrusion cooking followed first-order kinetics, showing that the fastest treatment in OTA reduction was that at 140 degrees C and 24% of moisture content.

Ochatoxin A (OTA) is one of the most important mycotoxins based on its toxicity. The oral route is the main gateway of entry of OTA into the human body, and specialized epithelial cells constitute the first barrier. The present study investigated the in vitro cytotoxic effect of OTA (5, 15 and 45 μM) and production of OTA metabolities in Caco-2 and HepG2 cells using a co-culture Transwell System to mimic the passage through the intestinal epithelium and hepatic metabolism. The results derived from MTS cell viability assays and transepithelial electrical resistance measurements showed that OTA was slightly cytotoxic at the lowest concentration at 3 h, but significant toxicity was observed at all concentrations at 24 h. OTA metabolites generated in this co-culture were ochratoxin B (OTB), OTA methyl ester, OTA ethyl ester and the OTA glutathione conjugate (OTA-GSH). OTA methyl ester was the major metabolite found in both Caco-2 and HepG2 cells after all treatments. Our results showed that OTA can cause cell damage through several mechanisms and that the OTA exposure time is more important that the dosage in in vitro studies. OTA methyl ester is proposed as an OTA exposure biomarker, although future studies should be conducted. [ABSTRACT FROM AUTHOR]

The stability of deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-glucoside) and ochratoxin A (OTA) during spaghetti production and cooking was investigated. Initial mycotoxin concentration, boiling time and use of egg as ingredient were assayed as factors. DON was stable during kneading and drying, but a consistent reduction of DON (> 40%) was observed in boiled spaghettis. According to our results, DON was transferred to broth, where it was not degraded, and boiling time determined the extent of the transfer. A DON leaching model was fitted to data with a high goodness of fit (r 2 = 0.99). This model can be used for prediction of final DON concentration in cooked pasta, and a useful tool in risk assessment models. DON-3-glucoside is totally stable through the pasta making process; moreover DON-3-glucoside is slightly released from pasta components and it is leached to broth. Similarly, OTA is also stable during pasta making; however, it is scarcely transferred to broth during boiling. The presence of egg as ingredient did not affect the final mycotoxin concentration in pasta in any case. [ABSTRACT FROM AUTHOR]

Abstract: The fate of deoxynivalenol (DON) and ochratoxin A (OTA) during the breadmaking process was studied. In particular, toxin content was analysed in mixed baking ingredients before kneading, after fermentation and proofing, and finally after baking. Fermentation and proofing were carried out at 30 °C for 1 h, while baking was performed at different temperature levels (from 170 to 210 °C) and baking times from 45 to 135 min, in a full factorial design. DON increased from unkneaded mix to fermented dough, and decreased due to baking; this trend depended on the initial concentration of DON in the flour. The level in the bread was significantly lower than in the initial mix of ingredients. In contrast, deoxynivalenol-3-glucoside (DON-3-G) content increased both during kneading and fermentation, and also during baking. Moreover, the results confirmed the high stability of OTA as no significant change in its content could be observed as a result of the breadmaking process. As conclusion, the design of bakery product processes may help to control DON in final products, because although quite stable, its levels can be reduced to some extent. However, high levels of DON-3-G were released during baking, and this point should be further investigated. Mycotoxins have been always considered as stable compounds; however, in depth knowledge of the processing steps that may lead to some reduction (although limited) and those which can stimulate their release from conjugated forms, will definitely help in their control in finished foodstuffs. [Copyright &y& Elsevier]

Abstract: The occurrence of Ochratoxin A (OTA) was examined in 121 special wines made using different winemaking techniques and from many European origins. The wine groups with the highest OTA content and occurrence, above 90%, were those were the must was fortified before fermentation (mean: 4.48μg/l) and those made from grapes dried by means of sun exposure (mean: 2.77μg/l). Fortified wines with long aging in wooden casks were about 50% contaminated, with OTA levels below 1.00μg/l. Wines affected by noble rot, late harvest wines and ice wines did not contain OTA. Overall, 19.8% of the wines studied contained OTA levels above the maximum permissible limit for the European Union (2μg/kg) in wine (excluding liqueur wines). [Copyright &y& Elsevier]