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Background Fusarium is a worldwide distributed fungal genus. It includes different species pathogenic to cereals among others crops. Some of these species can also produce toxic compounds toward animals and humans. Objective In this work, occurrence of fumonisins B1+B2, zearalenone, type A trichothecenes (T-2 and HT-2 toxins), and type B trichothecenes (deoxynivalenol[DON] and nivalenol[NIV]) was studied in 65 samples of stored and freshly harvested wheat, barley, and maize collected in Tunisia. Methods Mycotoxins analyses were performed by using gas chromatography for type B trichothecenes and HPLC for other mycotoxins. Obtained results were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction (PCR). Results Fumonisins occurred in 20.83% of wheat, 40% of barley, and 57.14% of maize samples, at levels exceeding European limits and suggesting a risk in Tunisian cereals, especially maize. Zearalenone, DON, NIV, and T-2+HT-2 toxins were detected at lower values in only wheat and barley samples. PCR protocols showed the predominance of F. verticillioides especially in maize, and occurrence of F. equiseti and F. graminearum in wheat and barley, and F. proliferatum in only two maize samples. A very consistent correlation was found between the detection of F. verticillioides and the contamination by fumonisins, as well as between the presence of F. graminearum and the contamination by zearalenone, DON, and NIV in the analyzed cereals. Conclusions Consequently, the detection of Fusarium species with the current PCR assays strategy in wheat, barley, and maize grains may be considered predictive of their potential mycotoxin risk in these matrices. Highlights This work is the first to report information on the occurrence of fumonisins, trichothecene, and ZEN, together with their potentially producing Fusarium species in wheat, barley, and maize in Tunisia. The high level of fumonisins in cereals, especially maize, stresses the importance of the control and the regularization of these mycotoxins for food safety.

The potential use of nanotechnology in the control of toxigenic fungi and mycotoxin production has been little explored. In this report, engineered silver nanoparticles (AgNPs) were synthesized and characterized by single particle Inductively Coupled Plasma Mass Spectrometry. Then, their effectiveness in the control of the growth of the main aflatoxigenic and ochratoxigenic species affecting foods and aflatoxins (AFs) and ochratoxin A (OTA) production was studied. The target species and their associated mycotoxins were Aspergillus flavus (AFB1 and AFB2), A. parasiticus (AFB1, AFB2, AFG1 and AFG2), A. carbonarius, A. niger, A. ochraceus, A. steynii, A. westerdijkiae and Penicillium verrucosum (OTA). Spore suspensions supplemented with AgNPs (average diameter size 30 nm, range 14–100 nm) at doses 0–45 μg/mL were incubated for 2–30 h. At selected exposure times aliquots were removed and cultured on maize-based medium (MBM) for 10 days. In these cultures, percentage of viable spores, effective doses (EDs) to inhibit the number of viable spores to 50%, 90% and 100%, colony lag phases, colony growth rates (GR), EDs to inhibit the colony GR to 50%, 90% and 100% were estimated. AF and OTA levels were determined by UPLC-MS/MS. Under the assayed conditions, effective doses of the AgNPs against all the studied fungal species could be estimated. These doses generally decreased with increasing exposure time and were higher for A. flavus and A. parasiticus than for ochratoxigenic species. Colonies from spores treated at high exposure times (20–30 h) and variable AgNP doses showed long lag phases or even did not occur depending on the fungal species. Colony GR, as well as AF and OTA levels in MBM cultures decreased when AgNP dose and contact period increased. The factors species, AgNP dose, exposure time and their interactions significantly affect fungal growth and AF and OTA accumulation in MBM. The results suggest that AgNPs only or as active ingredient hosted in paints, films or other polymers could be a good strategy in the management of the main aflatoxigenic and ocratoxigenic species affecting food and AF and OTA contamination.

Fusarium culmorum and F. proliferatum can grow and produce, respectively, zearalenone (ZEA) and fumonisins (FUM) in different points of the food chain. Application of antifungal chemicals to control these fungi and mycotoxins increases the risk of toxic residues in foods and feeds, and induces fungal resistances. In this study, a new and multidisciplinary approach based on the use of bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing pure components of essential oils (EOCs) and machine learning (ML) methods is evaluated. Bioactive EVOH-EOC films were made incorporating cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG) or linalool (LIN). Several ML methods (neural networks, random forests and extreme gradient boosted trees) and multiple linear regression (MLR) were applied and compared for modeling fungal growth and toxin production under different water activity (aw) (0.96 and 0.99) and temperature (20 and 28 °C) regimes. The effective doses to reduce fungal growth rate (GR) by 50, 90 and 100% (ED50, ED90, and ED100) of EOCs in EVOH films were in the ranges 200 to >3330, 450 to >3330, and 660 to >3330 μg/fungal culture (25 g of partly milled maize kernels in Petri dish), respectively, depending on the EOC, aw and temperature. The type of EVOH-EOC film and EOC doses significantly affected GR in both species and ZEA and FUM production. Temperature also affected GR and aw only affected GR and FUM production of F. proliferatum. EVOH-CIT was the most effective film against both species and ZEA and FUM production. Usually, when the EOC levels increased, GR and mycotoxin levels in the medium decreased although some treatments in combination with certain aw and temperature values induced ZEA production. Random forest models predicted the GR of F. culmorum and F. proliferatum and ZEA and FUM production better than neural networks or extreme gradient boosted trees. The MLR mode provided the worst performance. This is the first approach on the ML potential in the study of the impact that bioactive EVOH films containing EOCs and environmental conditions have on F. culmorum and F. proliferatum growth and on ZEA and FUM production. The results suggest that these innovative packaging systems in combination with ML methods can be promising tools in the prediction and control of the risks associated with these toxigenic fungi and mycotoxins in food., Financial support from the European Regional Development Fund (ERDF) and Ministry of Economy and Competitiveness (MINECO, Spanish Government) (Project AGL2014-53928-C2-1-R) and ERDF and Ministry of Science, Innovation and Universities (Spanish Government) (Project RTI2018-097593-B-C22). J.V. Gómez is grateful to MINECO for a PhD contract (Ref. 543071242).

Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract–sucrose (YES) agar and in maize kernels under different water activities (aw) and temperatures. Aflatoxins B1 and B2 were determined by LC with fluorescence detection and post-column derivatisation of AFB1. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB1 and AFB2 production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at th...

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species, and it is considered a common contaminant in food and animal feed worldwide. On the other hand, human embryonic stem cells (hESCs) have been suggested as a valuable model for evaluating drug embryotoxicity. In this study, we have evaluated potentially toxic effects of OTA in hESCs. By using in vitro culture techniques, specific cellular markers, and molecular biology procedures, we found that OTA produces mild cytotoxic effects in hESCs by inhibiting cell attachment, survival, and proliferation in a dose-dependent manner. Thus, we suggest that hESCs provide a valuable human and cellular model for toxicological studies regarding preimplantation stage of human fetal development.

Cereal grains are essential ingredient in food, feed and industrial processing. One of the major causes of cereal spoilage and mycotoxin contamination is the presence of toxigenic Fusarium spp. Nanoparticles have immense applications in agriculture, nutrition, medicine or health but their possible impact on the management of toxigenic fungi and mycotoxins have been very little explored. In this report, the potential of silver nanoparticles (AgNPs) (size 14–100 nm) against the major toxigenic Fusarium spp. affecting crops and their effect on mycotoxin accumulation is evaluated for the first time. The studied Fusarium spp. (and associated mycotoxins) were F. graminearum and F. culmorum (deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone), F. sporotrichioides and F. langsethiae (T–2 and HT–2 toxins), F. poae (nivalenol), F. verticillioides and F. proliferatum (fumonisins B1 and B2) and F. oxysporum (mycotoxins no detected). The factors fungal species, AgNP dose (range 2–45 μg/mL), exposure time (range 2–30 h) and their interactions significantly influence spore viability, lag period and growth rate (GR) in subsequent cultures in maize-based medium (MBM) of all the studied species. The effective lethal doses (ED50, ED90 and ED100) to control spore viability and GR were in the range 1–>45 μg/mL depending on the remaining factors. At high exposure times (20−30 h), the three effective doses ranged 1–30 μg/mL for all the studied species. At the end of the incubation period (10 days) mycotoxin levels in MBM cultures inoculated with fungal spores from treatments were strongly related with the size reached by the colony at that time. None of the treatments produced stimulation in conidia germination, GR or mycotoxin biosynthesis with respect to controls. Thus, the antifungal effect of the assayed AgNPs against the tested Fusarium spp. suggests that AgNPs could be a new antifungal ingredient in bioactive polymers (paints, films or coating) likely to be implemented in the agro-food sector for controlling these important toxigenic Fusarium spp. and their main associated mycotoxins.

Mycotoxins are secondary metabolites produced mainly by fungal species belonging to the genera Fusarium, Aspergillus and Penicillium and belong to the most relevant contaminants of food and feed. Cereals are the main source of mycotoxins in the diet. The most prominent mycotoxins are aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), fumonisins B1 and B2 (FB1 and FB2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), 3- and 15-acetyl-deoxynivalenol (3- and 15-ADON), and T-2 and HT-2 toxins. Maximum levels allowed in food are very different depending on mycotoxin and food type, consumer susceptibility and current legislation in each country. Among cereals, maize and its derivatives deserve special attention. The present study was designed to investigate the occurrence of these mycotoxins in Spanish maize kernels collected in different regions for a five-year period (2015–2019) using a multi-mycotoxin UPLC-MS/MS method. Ninety-eight samples of maize kernels collected in 26 Spanish grain stores were ground to fine powder, extracted with acetonitrile-water-formic acid (80:19:1, v/v/v), centrifuged and analyzed by UPLC-ESI-MS/MS using matrix-matched calibration. The method was validated and mean recovery rates were 83–103.4% and mean relative standard deviations ranged from 4.1 to 8.5%. FB1, FB2, DON, 3-ADON, ZEA, AFB1, AFB2, AFG2, T-2 and HT-2 toxins were detected at levels higher than their respective limits of quantification in 71.4%, 56.2%, 31.6%, 5.02%, 24.5%, 9.2%, 9.2%, 2%, 5.1% and 5.1% of maize kernel samples, respectively. OTA was not detected. Among the positive samples, 20.4%, 5.1%, 3%, and 3% had levels exceeding the European Union permissible limits for FB1+FB2, ZEA, AFB1, and total aflatoxins, respectively. Co-occurrence of various mycotoxins (two to seven) in the same sample at quantifiable levels was observed in 33.5% of samples being the association of FB1, FB2 and DON the most frequent, followed by the presence of FB1, FB2, ZEA and DON.

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10-1000 mg l

Aspergillus steynii and Aspergillus tubingensis are possibly the main ochratoxin A (OTA) producing species in Aspergillus section Circumdati and section Nigri, respectively. OTA is a potent nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic and immunosuppressive compound being cereals the first source of OTA in the diet. In this study bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing cinnamaldehyde (CINHO), linalool (LIN), isoeugenol (IEG) or citral (CIT) which are major components of some plant essential oils (EOs) were produced and tested against A. steynii and A. tubingensis growth and OTA production in partly milled maize grains. Due to the favourable safety profile, these bioactive compounds are considered in the category “GRAS”. The study was carried out under different water activity (0.96 and 0.99 aw), and temperature (24 and 32 °C) conditions. ANOVA showed that class of film, fungal species, aw and temperature and their interactions significantly affected growth rates (GR), ED50 and ED90 and the doses for total fungal growth inhibition and OTA production. The most effective EVOH films against both species were those containing CINHO. ED50, ED90 and doses for total growth and OTA inhibition were 165–405, 297–614, 333–666 μg of EVOH-CINHO/plate (25 g of maize grains), respectively, depending on environmental conditions. The least efficient were EVOH-LIN films. ED50, ED90 and doses for total growth and OTA inhibition were 2800–>3330, >3330 and >3330 μg of EVOH-LIN/plate (25 g of maize grains), respectively. The effectiveness of the bioactive films increased with increasing doses. Overall, A. tubingensis was less sensitive to treatments than A. steynii. Depending on the species, aw and temperature affected GR and OTA production in a different way. In A. steynii cultures, optimal growth occurred at 0.96 aw and 32 °C while optimal OTA production happened at 0.99 aw and 32 °C. In A. tubingensis cultures optimal growth happened at 0.99 aw and 32 °C, although the best conditions for OTA production were 0.99 aw and 24 °C. Thus, these species can be very competitive in warm climates and storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study and applied in vapour phase, can be potent antifungal agents against A. steynii and A. tubingensis and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films containing individual components of EOs have on the growth of these ochratoxigenic fungi and on OTA production in maize grains.

Aspergillus flavus and A. parasiticus are the most common fungal species associated with aflatoxin (AF) contamination of cereals, especially maize, and other agricultural commodities. AFB1, the most frequent and toxic metabolite, is a powerful hepatotoxic, teratogenic and mutagenic compound. Effective strategies to control these fungal species and AFs in food and feed are required. Active packaging film containing essential oils (EO) is one of the most innovative food packaging concepts. In this study, ethylene-vinyl alcohol (EVOH) copolymer films incorporating EO from Origanum vulgare (ORE), Cinnamomum zeylanicum (CIN) or their major active constituents, carvacrol (CAR) and cinnamaldehyde (CINHO), respectively, were developed and assayed to control growth of A. flavus and A. parasiticus and AF production in maize grains under different aw and temperature regimens. EO doses assayed in cultures were in the range 0.25–4.0 mg/Petri dish. The factors aw, temperature, type of EVOH-EO film and fungal species significantly influenced the ED50 values of all assayed films. Growth rate (GR) of both species was usually higher at 0.99 than at 0.96 aw and at 37 °C than at 25 °C. However, the contrary was found with regard to AF production. The order of efficacy of EVOH-EO films to control growth of both species and AF production was EVOH-CINHO > EVOH-CAR > EVOH-ORE > EVOH-CIN. The effective dose (ED50) (mg EO/plate) for EVOH-CINHO and EVOH-CIN films against A. flavus were in the ranges of 0.125 and 2.475–3.500 and against A. parasiticus in the ranges of 0.121–0.133 and 2.275–3.625, respectively. Under the assayed conditions, the ED90 for EVOH-CINHO film were 0.22–0.23 mg/plate for both species. It was the most effective bioactive film to control fungal growth (vapour phase) and AF production, regardless of aw and temperature. This is the first study about the impact that interacting environmental conditions and bioactive EVOH-CINHO, EVOH-ORE, EVOH-CIN EVOH-CAR films have on the growth of aflatoxigenic fungi and on AF production in maize grains., The authors acknowledge financial support from FEDER and Ministry of Economy and Competitiveness (MINECO, Spanish Government) (Project AGL2014-53928-C2-1-R). E.M. Mateo is grateful to “Generalitat Valenciana” for financial support (APOSTD/2016/102) and J.V. Gómez is grateful to FEDER and MINECO for a PhD contract (BES-2015-071242).

Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner.

An electrochemical methodology for the characterization of mycotoxin-producing fungal species from the genera Aspergillus and Fusarium using solid-state voltammetry is described. Upon attachment of fungal colony microsamples to glassy carbon electrodes in contact with aqueous acetate buffer, characteristic voltammetric signals mainly associated to the oxidation of polyphenolic metabolites are recorded. The possibility of fungi-localized electrochemical processes was assessed by means of electron microscopy and field emission scanning electrochemical microscopy coupled to the application of oxidative potential inputs. Using pattern recognition methods, the determined voltammetric profiles were able to discriminate between mycotoxin-producing fungi from different sections and to identify selected toxigenic species of the Aspergillus and Fusarium genera isolated from grapes and cereals.

The health-beneficial properties of oats have led to an increase in the consumption of oats and oat-based food products in recent years. Fusarium langsethiae grows on small grain cereals, especially oats and can result in contamination with type A trichothecenes (T-2 toxin (T-2) and HT-2 toxin (HT-2)) in crops pre- and post-harvest. The aim of this work was to assess the efficacy of three fungicides (fenpropimorph, prochloraz and tebuconazole) on temporal growth of two F. langsethiae strains and the accumulation of T-2 and HT-2 in oat grains, under different water activities (aw; 0.95 and 0.98) and temperatures (15 and 25 °C). All the antifungal agents reduced growth rates when compared to controls, and this increased with increasing fungicide dose. The ranges of ED50 values (mg/kg) were 65–270 for fenpropimorph, 0.25–4.2 for prochloraz, and 0.3–14 for tebuconazole. The ED90 values (mg/kg) ranged from 170 to > 800, 0.5 to > 10 and 0.5 to > 15 for these three fungicides respectively. The ED50 values were not statistically significantly affected by the factors aw, temperature or strain. However, there were significant differences among the fungicides. Fenpropimorph proved less efficient that the two azoles, which do not differ significantly. In general, levels of HT-2 were higher than T-2 in all cultures regardless of environmental conditions. Overall, HT-2 concentration was always higher at 25 than 15 °C and increased from day 14 to day 21. Levels of both toxins generally decreased with increasing fungicide dose regardless of fungicide type, strain and incubation time. No toxins were detected in cultures at 0.95 aw in the presence of any of the three fungicides. Also under wetter conditions at 0.98 aw neither mycotoxin was found in cultures treated with prochloraz at doses >1 mg/kg (15 °C) or >3 mg/kg (25 °C) or tebuconazole at doses >6 mg/kg (15 or 25 °C). ANOVA showed that in treatments with each fungicide the factors dose, time and temperature significantly affected toxin production while there was no difference between the strains.

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED₅₀ and ED₉₀ values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED₅₀ and ED₉₀ values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 a(w). Mancozeb at 40 mg l(-1 )inhibited fungal growth and provided lag phases24 days at 10-20 mg l(-1) and 15°C. OTA was observed only at 25°C and doses10 mg l(-1). At 15°C, copper oxychloride proved inhibitory at 800 mg l(-1), while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5-8 g l(-1) (at 15°C) while at 25°C growth took place even at 8 g l(-1), although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozebcopper oxychloridesulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.

Patulin (PAT) is a mycotoxin produced in fruits, mainly in apples, by several fungal species that can be carried into industrial apple juice by-products during factory processing. An analytical method for determination of PAT in apple juice and another one for determination of this compound in apple purees and apple compotes by liquid chromatography are proposed in the present paper. These methods have better precision and sensitivity than previously reported methods and focus mainly on extraction and clean-up. To accomplish analytical methods with higher accuracy, lower limits of detection and simpler procedures for application in quality control of the goods, different extraction and clean-up procedures for PAT were comparatively studied. PAT recoveries in apple juice spiked with 1.0mg PAT/kg varied between 52.3% and 81.0%. The highest PAT recovery in apple puree spiked with 0.1mg PAT/kg was 82.9%. Addition of NaH(2)PO(4) during the extraction phase here reported for the first time has the advantage of keeping the pH slightly acidic, thus avoiding PAT degradation.

Fusarium graminearum is a mould that causes serious diseases in cereals worldwide and that synthesises mycotoxins such as deoxynivalenol (DON), which can seriously affect human and animal health. Predicting the level of mycotoxin accumulation in food is very difficult, because of the complexity of the influencing parameters. In this work, we have studied the possibility of using artificial neural networks (NN) to predict DON level attained in F. graminearum wheat cultures taking as inputs the fungal contamination level of the cereal, the water activity as a measure of the available water for fungal growth in the cereal, the temperature and time. DON analysis was performed by gas chromatography with electron capture detection. The data matrix was used to train and validate various types of NN using MATLAB 7.0. The aim was to obtain a network that provided the best possible fit between predicted and target DON levels by minimising the mean-square error of test. Radial basis function-NNs attained lower errors and better generalisation than multi-layer perceptron networks to predict DON accumulation in wheat. This is the first time that NNs have been used to predict DON accumulation in wheat based on the studied factors.

Aims: To examine the efficacy of natamycin produced by Streptomyces natalensis against strains of Aspergillus carbonarius growth and ochratoxin A (OTA) production under different environmental factors on a grape juice-based medium. Methods and Results: Detailed studies in the range 0–20 ng ml−1 for control of growth and ochratoxin production by strains of A. carbonarius at 0·98, 0·96 and 0·94 water availabilities (aw) and 15–25°C on a fresh red grape extract medium were examined. Inhibition of growth was depending on temperature and aw level. At 15°C, 5–10 ng ml−1 natamycin was effective in reducing growth almost completely. However, at 20–25°C and all the three aw levels, growth was only slightly inhibited by 5–10 ng ml−1 natamycin. There were strain differences with regard to inhibition of OTA production. At 15°C and 0·98 aw, 10 ng ml−1 was required to inhibit production by >90%. However, at 0·96 and 0·94 aw, almost complete inhibition occurred. At 20°C, OTA production was only significantly inhibited by 10 ng ml−1 natamycin at 0·94 aw. At 0·96 and 0·98 aw, some inhibition occurred with 5–10 ng ml−1, but greater concentrations would be required for effective inhibition. At 25°C, 5 ng ml−1 was effective at all aw levels. However, at 15°C and 25°C and a wide range of aw levels, natamycin effectively controlled OTA production. Conclusions: Natamycin appears to be a very effective for controlling growth and OTA production by strains of A. carbonarius over a range of aw and temperature conditions on grape-based media. Significance and Impact of the Study: This is the first detailed study to demonstrate the impact of natamycin against A. carbonarius. This study suggests that use of natamycin at 50–100 ng ml−1 can give complete inhibition of growth of A. carbonarius and OTA production over a range of environmental conditions. Natamycin could be an important component of a system to prevent OTA contamination of wine as well during the drying and production of vine fruits.

Fumonisins are a group of mycotoxins produced by Fusarium species of the Gibberella fujikuroi species complex that contaminate food and feed products, and represent a risk for human and animal health. In this work, we have developed a specific real-time reverse transcription-PCR (RT-PCR) assay to quantify the level of expression of two genes of the fumonisin biosynthetic cluster in F. verticillioides: fum1 (that encodes a polyketide synthase enzyme) and the ABC transporter encoding gene fum19. The level of expression of both genes was compared with the amount of fumonisin B(1) (FB(1)), measured by HPLC, produced by several strains of F. verticillioides in liquid culture. The results indicated a good correspondence between the levels of fum1 and fum19 expression and the production of fumonisin B(1). The analysis described provides a good approach for the rapid and specific detection and characterization of the potential ability of F. verticillioides strains to produce fumonisins.

A new method for the determination of ochratoxin A (OTA) in beer has been developed. The new method has been compared with a reference method currently accepted as AOAC official first action. The limits of detection and quantification of the proposed method were 0.0008 and 0.0025 ng/ml, respectively, while they were 0.0025 and 0.0075 ng/ml, respectively, in the AOAC method used as reference. The recovery levels in the 0.025–0.40 ng OTA/ml spiking range for the proposed and the reference methods were 80.6–87.6% and 78.2–83.8%, respectively. The relative standard deviations of recoveries were 2.6–7.5% for the proposed method and 0.7–6.1% for the reference method. Passing and Bablok regression analysis of recovery data obtained by the proposed method versus data obtained by the reference method on an OTA-spiked beer sample showed good correlation ( r 2 = 0.9993), while the slope and intercept were 1.049 and −0.0013, respectively. The advantage of the proposed method is the low cost of the materials used in sample preparation because expensive immunoaffinity columns are not needed to clean-up samples while it maintains or even increases the good performance of the reference method. The proposed method was applied to 69 beer samples from different geographic origins (national and imported) but purchased in the Spanish market. They were found to be contaminated with OTA in the range from 0.008 to 0.498 ng/ml (average: 0.070 ng/ml). Five samples surpassed the limit recommended by the European Union (0.2 ng OTA/g).

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB1, FB2, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.

The present work deals with the toxigenic mycobiota occurring in Spanish malting barley and the capability for producing mycotoxins by several important toxigenic fungi. One hundred and eighty seven samples of malting barley were gathered from Spanish breweries before processing. One hundred and fifty kernels per sample were surface-sanitized with a 2% sodium hypochlorite solution and incubated on three culture media. The most abundant fungi were species of Alternaria, Aspergillus, Penicillium and Fusarium, which were present in 93%, 82.3%, 57.8% and 27.8% of the samples, respectively. To evaluate their mycotoxin producing potential a number of isolates belonging to each genus, except Penicillium, were randomly selected and incubated on culture media known to be appropriate for production of mycotoxins. Alternariol and alternariol monomethyl ether were produced by 26.7% of Alternaria spp. isolates (all belonged to Alternaria alternata). All tested isolates of F. verticillioides produced fumonisin B(1) (FB(1)) and 61.3% of them produced fumonisin B(2) (FB(2)), whereas FB(1) was synthesized by 83.3% and FB(2) by 77.8% of F. proliferatum isolates. Twenty percent of the isolates of the Aspergillus flavus/A. parasiticus group had the capability to produce aflatoxin B(1) and aflatoxin B(2). Thirty out of 34 isolates of F. graminearum produced deoxynivalenol and zearalenone whereas the other 4 isolates produced nivalenol. Ochratoxin A was detected in 75% and 15% of isolates of Aspergillus section Nigri and A. ochraceus, respectively. This is the first survey carried out in Spain on the toxigenic mycobiota contaminating malting barley in breweries and the mycotoxin producing capacity of several species. The information obtained is useful for assessing the risk of mycotoxins in beer.

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.

The natural mycobiota occurring in bee pollen is studied in the present report with special attention to analyze the incidence of fungal species that are potential producers of mycotoxins. A total of 90 ready-to-eat bee pollen samples were analyzed. Eighty-seven samples were collected in stores placed in different Spanish areas and three were from Buenos Aires (Argentina). The statistical results (ANOVA) showed that yeasts and Penicillium spp. were the predominant fungi. With regard to the potential mycotoxin producing species, Penicillium verrucosum, Aspergillus niger aggregate, Aspergillus carbonarius, Aspergillus ochraceus, Aspergillus flavus, Aspergillus parasiticus and Alternaria spp. were found. The last genus was isolated very frequently. The potential ability for producing ochratoxin A (OTA) and aflatoxins B(1), B(2), G(1) and G(2) was studied by culturing in vitro the isolates followed by analysis of these mycotoxins in culture extracts by HPLC with fluorescent detection. It was found that 100%, 53.3%, 33.3% and 25% of the isolates of A. carbonarius, A. ochraceus, P. verrucosum and A. niger aggregate, respectively, produced OTA. Moreover, 28.6% of the isolates from the A. flavus plus A. parasiticus group were able to produce aflatoxin B(1). Aflatoxin B(2) was detected in only 10% of the cultures. Aflatoxins G(1) and G(2) were not detected in cultures under the assayed conditions. This is the first report carried out on the natural mycobiota occurring in bee pollen in general and on the toxigenic capability of these isolates in particular.

A new sample treatment for liquid chromatographic analysis of ochratoxin A (OTA) in beer is proposed. Degassed beer is mixed with lead hydroxyacetate, which precipitates some bulk components but does not remove OTA. The precipitate is separated and the acidified liquid is extracted with chloroform. The solvent is evaporated and the residue is dissolved in mobile phase (acetonitrile–water, 40:60, v/v; acidified at pH 3.0 with phosphoric acid) and separated by liquid chromatography using fluorescence detection. The limit of detection was 0.005 ng/ml. The average recovery rate and the average RSD of recovery in the spiking level range 0.01–0.5 ng/ml were 95.5% and about 5%, respectively. The method is cheaper that other alternative ones using immunoaffinity columns or other solid-phase extraction cleanup. The separation was optimised with regard to composition and flow of the mobile phase and no interference from the matrix was found. The method was applied to 88 samples of beer (domestic and imported) marketed in Spain. OTA was detected in 82.9% of them. The range for positive samples was 0.007–0.204 ng of OTA/ml.

Various solid-phase extraction (SPE) procedures for clean-up, two perfluoroacylation reagents (pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA)) and two chromatographic columns (HP-1701 and HP-5) have been assessed comparatively to achieve the determination of type B trichothecenes (deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol (3- and 15-ADON)) in wheat grain by gas chromatography (GC)–electron-capture detection (ECD). Spiked wheat samples were extracted with acetonitrile–water (84:16, v/v). Tested SPE procedures were MycoSep 225 column, Florisil and different cartridges prepared in the laboratory with mixtures of various sorbents like alumina, Celite 545, C18, silica and charcoal. We propose MycoSep 225 column, and cartridges made with alumina–charcoal–silica and alumina–charcoal–C18 silica mixtures as clean-up procedures on the basis of recovery values (89.6, 87.3 and 86.1% for deoxynivalenol, respectively, at 1.0 mg/kg spiking level). The two last procedures are less expensive. Pentafluoropropionic anhydride was more stable against moisture and less expensive, while recoveries were similar to those obtained with heptafluorobutyric anhydride. HP-1701 column can separate 3- and 15-acetyldeoxynivalenol derivatives while HP-5 cannot, although this last column provided lower bleed and better sensitivity.

Thirty-three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B1 and B2 except those isolated from banana. Analysis of the IGS region by PCR-RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin-producing group associated with cereals and the fumonisin non-producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.

Fumonisins are important mycotoxins basically produced by strains from the Gibberella fujikuroi species complex (with anamorphs in Fusarium genus) which contaminate food and feed products representing a risk to human and animal health. In this work, we report for the first time the fumonisin production of Fusarium moniliforme Sheldon strains associated to edible pine nuts of Pinus pinea. P. pinea is an important and widely distributed Pinus species in the Mediterranean area where their pine nuts are consumed raw or slightly processed in diverse food products. In this work, characterization and further identification of those strains were performed by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the intergenic spacer region of the rDNA (IGS) with the aid of the eight mating populations (A–H) described for G. fujikuroi species complex. The method was powerful to detect polymorphism, allowing discrimination between individuals and could be used to study the genetic relationships among them and within the G. fujikuroi species complex. Fusarium strains associated to Pinus radiata were also included in the present study. These strains did not produce fumonisins and showed no close relation with the strains isolated from P. pinea. The approach used in this work was rapid and proved to be efficient to assist identification and to characterize and analyse relatedness of new isolates within the G. fujikuroi species complex.

The capacity of four isolates belonging to the Gibberella fujikuroi complex to produce fumonisin B1 and fumonisin B2 when grown in liquid medium supplemented with one sugar and one amino acid at various concentration levels has been investigated. The sugars used for supplementing the medium were glucose, fructose, rhamnose, sucrose, maltose, and trehalose at 5, 10 or 20 g/l. The amino acids used were serine, threonine, glutamic acid, aspartic acid, valine, isoleucine, methionine, glycine, alanine, and cystine at 1 or 10 g/l. Fumonisins were extracted from culture filtrates, purified by SAX column and determined by reversed-phase C18 HPLC with fluorescence detection of the o-phthaldialdehyde derivatives. Two isolates produced very low concentrations of fumonisins with all sugars. The remaining isolates provided increased contents of fumonisins when sugar level increased. Concerning the amino acids, production of fumonisins was also dependent on the isolate, although at 1 g/l, the production of fumonisins was greater than at 10 g/l. The results indicate that the sugar–amino acid-isolate combination is basic in fumonisin biosynthesis and that the particular behaviour of each isolate in the different nutritional conditions may constitute a piece of interesting information in the fields of the Taxonomy, Physiology and Toxicology of these fungi. This is the first report on the influence of the carbon and nitrogen sources on fumonisin production by isolates of the G. fujikuroi complex.

This article is a revision of the previous edition article by Imad Ali Ahmed, volume 2, pp. 1526–1532, © 1999, Elsevier Ltd.

Las fumonisinas B1 (FB1) y B2 (FB2) son las principales micotoxinas producidas por algunas cepas del complejo Gibberella fujikuroi, hongos ampliamente presentes en cereales. La contaminacion de alimentos y piensos por estos hongos se ha asociado con la aparicion de graves enfermedades en animales y humanos. En este trabajo se analizan las etapas del analisis de FB1 y FB2 mediante cromatograna liquida (HPLC) con la finalidad de reducir el tiempo de analisis, costes y pureza de los extractos. Las fumonisinas se extrajeron con acetonitrilo/agua (1:1) y los extractos se purificaron mediante cartuchos de extraccion en fase solida C18. Estos cartuchos presentan ciertas ventajas respecto a los cartuchos SPE-SAX, como la reduccion del tiempo de analisis y la obtencion de extractos con menos impurezas. Las fumonisinas se eluyeron de los cartuchos con metanol/agua (7:3) y con oftalaldialdehido y 2-mercaptoetanol se obtuvieron los derivados fluorescentes. Se empleo una columna de fase inversa y un detector de fluorescencia para la separacion final y la determinacion de las micotoxinas por HPLC. El metodo analitico fue lineal para cantidades de micotoxinas comprendidas entre 10 y 4000 ng. El limite de deteccion en muestras de maiz y arroz (con una razon senal/ruido de fondo de 3:1) fue de 5 ng/g para las dos fumonisinas.

Various analytical methods used in the analysis of type B trichothecenes (deoxynivalenol, nivalenol, 3- and 15-acetyldeoxynivalenol) in cereals were compared and optimised in this work. These methods use either GC-electron-capture detection (ECD) of trimethylsilyl, trifluoroacetyl and heptafluorobutyryl derivatives or HPLC with UV or photodiode array detection of analytes. A new HPLC procedure using fluorescence detection prior derivatisation with coumarin-3-carbonyl chloride has been also tested. Five extraction solvents and two solid-phase extraction cartridges (silica, Florisil) plus a especial clean-up column (MycoSep 225) were compared in order to obtain the best recovery of the mycotoxins with minimal presence of coextractives in the chromatograms. The chosen extraction solvent was a mixture of acetonitrile-water (84:16, v/v). The MycoSep 225 column was chosen as the best alternative for clean-up of grain samples. For GC-ECD analysis, derivatisation of analytes with heptafluorobutyric anhydride prior the final determination was chosen as the most suitable procedure. HPLC-photodiode array (at 221 nm) analysis was more suitable for determination of type B trichothecenes than HPLC of the fluorescent coumarin-3-carbonyl derivatives. Recoveries obtained in spiked corn, rice and wheat are reported. The utility of the proposed methodology was assayed in cereal cultures of various Fusarium strains.

This work describes for the first time the production of trichothecenes and fumonisins in autoclaved tiger nuts by strains of Fusarium sporotrichioides and F. moniliforme respectively. The study was carried out using a high performance liquid chromatograph with fluorescence detector. The results obtained show that the strains studied are capable of producing these mycotoxins in tiger nuts and that the concentrations of toxin found depend on the variety of tiger nut used as substrate. The amount of type A trichothecenes produced in tiger nuts by the different strains of F. sporotrichioides is smaller than that produced by the same strains in other substrates. However, the strains of F. moniliforme have shown that their ability to produce fumonisin B1 is similar to that which they have in rice or wheat, although we have not found any detectable amounts of fumonisin B2 in tiger nuts.

The addition of inocula of a commercially available strain of Saccharomyces cerevisiae to musts with a high initial sugar content at various fermentation stages induces variable changes in the different components of the volatile fraction of wine and, potentially, its sensory properties as well. Inoculation alters the concentration ratio between the 2,3-butanediol isomers and causes a decrease in acetates and ethyl ester content. We propose an analytical ratio to evaluate the ‘aromatic’ quality of the wine.

The authors studied the changes in yeast and mould counts, pH, free and lactone acidity, colour and sugar composition undergone by honeys with water contents of 169, 188 and 220 g kg-1 on storage at refrigeration temperature (4–7°C), room temperature and 28°C for two years. Of the solid culture media assayed, honey agar and osmophilic medium fostered yeast growth while malt-extract agar and yeast-extract-malt-extract agar allowed for better development of filamentous fungi. The yeasts isolated belonged to the genera Saccharomyces, Schizosaccharomyces and Zygosaccharomyces, while filamentous moulds were of the genera Aspergillus, Penicillium, Fusarium and Alternaria. The honeys studied underwent no visible microbial alternations under any of the conditions tested, although yeast population increased significantly with time. Free and lactone acidity increased slightly but the pH was practically constant. The honeys darkened gradually with time, the effect increasing with the storage temperature. The analysis of various sugars carried out by capillary column gas chromatography of their trimethylsilyloxime derivatives indicated that all of them changed significantly after 2 years. Levels of fructose, glucose, sucrose, maltose, kojibiose, isomaltose and trisaccharides decreased with time whereas those of maltulose and turanose increased over the same period.

The most important trichothecenes are HT-2 toxin (HT2) and T-2 toxin (T2) from type A and deoxynivalenol (DON) from type B. Thus, the aim of the current study was to assess the occurrence of these trichothecenes in the Catalonian market. 479 food samples were taken from the most susceptible to trichothecenes contamination and most commonly consumed in Catalonia commodities. DON, T2 and HT2 toxin were determined in breakfast cereals, snacks and pasta samples following extraction, clean-up, derivatization and finally analysis by GC–ECD. Moreover, these mycotoxins were determined in sliced bread, sweet corn and beer by LC–DAD. Our results showed that DON was the main trichothecene present in the cereal-based food from Catalonian market with percentages of positive samples ranging from 1.4 to 100.0%. Despite the high incidence of DON, only five samples were above EU limits. Concerning HT2 toxin, it was present in a low percentage of samples of sliced bread (15.3%), wheat flakes (14.8%), pasta (10.0%), corn snacks (8.5%), sweet corn (6.9%) and corn flakes (6.2%), while the T2 toxin only was quantified in 5 samples out of the total 479.

The capacity of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict deoxynivalenol (DON) accumulation in barley seeds contaminated with Fusarium culmorum under different conditions has been assessed. Temperature (20-28 °C), water activity (0.94-0.98), inoculum size (7-15 mm diameter), and time were the inputs while DON concentration was the output. The dataset was used to train, validate and test many ANNs. Minimizing the mean-square error (MSE) was used to choose the optimal network. Single-layer perceptrons with low number of hidden nodes proved better than double-layer perceptrons, but the performance depended on the training algorithm. The RBFN reached lower errors and better generalization than MLP-ANN but they required a high number of hidden nodes. Accurate prediction of DON accumulation in barley seeds by F. culmorum was possible using MLP-ANNs or RBFNs. © 2010 Elsevier Ltd., This work was supported by the Spanish "Ministerio de Educacion y Ciencia" (projects AGL-2004-07549-C05-02, AGL2007-66416-C05-01/ALI, and a research grant) and the Valencian Government (Conselleria de Empresa, Universitat i Ciencia) (project GV04B-111, action ACOMP/2007/155, and a research grant).

In the present work, a phylogenetic study based on protein electrophoretic profiles of Saccharomyces strains isolated from different Spanish wine regions has been carried out. Qualitative differences between the protein electrophoregrams were found at inter- and intraspecific level, but not between electrophoregrams of strains isolated at the same ecosystem. The numerical analysis of these results allowed us to conclude that intraspecific relationships are determined by ecological factors, as well as human influences (dispersion and artificial selection). A correlation between ecological and/or geographical origin and the relationships among strains was observed.

Banana fruits were studied over a six-month period in order to determine the incidence of species of the Fusarium genus and assess their potential pathogenicity. The 72 samples studied were commercially available in Italy and Spain, where they were brought from Panama, Ecuador and Canary Islands. Among the species detected in the fruits, Fusarium semitectum var. majus Wollenw. was predominant, followed by F. moniliforme Sheld., F. solani (Mart.) Appel & WoUenw., F. oxysporum Schlecht., F. proliferatum (Matsushima) Nirenberg, F. graminearum Schw., F. camptoceras WoUenw. &C Reinking, F. subglutinans (WoUenw. & Reinking) Nelson et al., F. dimerum Penzig in Sacc, F. acuminatum EU. & Ev., and F. equiseti (Corda) Sacc. Fusarium proliferatum had never been reported to occur as contaminating fungi in banana fruits to date. Fusarium subglutinans, F. acuminatum and F. graminearum were found to be the most markedly pathogenic of all. The lack of noticeable differences in relation to the incidence of the different species isolated from the samples indicates that the mycoflora found is typical of this fruit and does not depend on its origin.

There is a need to develop sensitive and accurate analytical methods for determining deoxynivalenol (DON), HT-2 toxin and T-2 toxin in paprika to properly assess the relevant risk of human exposure. An optimized analytical method for determination of HT-2 toxin and T-2 toxin using capillary gas chromatography with electron capture detection and another method for determination of DON by liquid chromatography-mass spectrometry in paprika was developed. The method for determination of HT-2 toxin and T-2 toxin that gave the best recoveries involved extraction of the sample with acetonitrile-water (84:16, v/v), clean-up by solid-phase extraction on a cartridge made of different sorbent materials followed by a further clean-up in immunoaffinity column that was specific for the two toxins. The solvent was changed and the eluate was derivatized with pentafluoropropionic anhydride and injected into the GC system. The limits of detection (LOD) for T-2 and HT-2 toxins were 7 and 3 μg/kg, respectively, and the recovery rates for paprika spiked with 1000 μg toxin/kg were 71.1% and 80.1% for HT-2 and T-2 toxins, respectively. For DON determination, the optimized method consisted of extraction with acetonitrile-water (84:16, v/v) solution followed by a solid-phase extraction clean-up process in a cartridge made of different sorbent compounds. After solvent evaporation in N(2) stream, the residue was dissolved and DON was separated and determined by LC-MS/MS. The LOD for this method was 14 μg DON/kg paprika sample and the DON recovery rate was 86.8%.