Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

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.

Silver Nanoparticle-Based Therapy: Can It Be Useful to Combat Multi-Drug Resistant Bacteria?

The present review focuses on the potential use of silver nanoparticles in the therapy of diseases caused by antibiotic-resistant bacteria. Such bacteria are known as "superbugs", and the most concerning species are Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus (methicillin and vancomycin-resistant), and some Enterobacteriaceae. According to the World Health Organization (WHO), there is an urgent need for new treatments against these "superbugs". One of the possible approaches in the treatment of these species is the use of antibacterial nanoparticles. After a short overview of nanoparticle usage, mechanisms of action, and methods of synthesis of nanoparticles, emphasis has been placed on the use of silver nanoparticles (AgNPs) to combat the most relevant emerging resistant bacteria. The toxicological aspects of the AgNPs, both in vitro using cell cultures and in vivo have been reviewed. It was found that toxic activity of AgNPs is dependent on dose, size, shape, and electrical charge. The mechanism of action of AgNPs involves interactions at various levels such as plasma membrane, DNA replication, inactivation of protein/enzymes necessary, and formation of reactive oxygen species (ROS) leading to cell death. Researchers do not always agree in their conclusions on the topic and more work is needed in this field before AgNPs can be effectively applied in clinical therapy to combat multi-drug resistant bacteria.

Lactic Acid Bacteria as Potential Agents for Biocontrol of Aflatoxigenic and Ochratoxigenic Fungi.

Aflatoxins (AF) and ochratoxin A (OTA) are fungal metabolites that have carcinogenic, teratogenic, embryotoxic, genotoxic, neurotoxic, and immunosuppressive effects in humans and animals. The increased consumption of plant-based foods and environmental conditions associated with climate change have intensified the risk of mycotoxin intoxication. This study aimed to investigate the abilities of eleven selected LAB strains to reduce/inhibit the growth of Aspergillus flavus, Aspergillus parasiticus, Aspergillus carbonarius, Aspergillus niger, Aspergillus welwitschiae, Aspergillus steynii, Aspergillus westerdijkiae, and Penicillium verrucosum and AF and OTA production under different temperature regiments. Data were treated by ANOVA, and machine learning (ML) models able to predict the growth inhibition percentage were built, and their performance was compared. All factors LAB strain, fungal species, and temperature significantly affected fungal growth and mycotoxin production. The fungal growth inhibition range was 0-100%. Overall, the most sensitive fungi to LAB treatments were P. verrucosum and A. steynii, while the least sensitive were A. niger and A. welwitschiae. The LAB strains with the highest antifungal activity were Pediococcus pentosaceus (strains S11sMM and M9MM5b). The reduction range for AF was 19.0% (aflatoxin B1)-60.8% (aflatoxin B2) and for OTA, 7.3-100%, depending on the bacterial and fungal strains and temperatures. The LAB strains with the highest anti-AF activity were the three strains of P. pentosaceus and Leuconostoc mesenteroides ssp. dextranicum (T2MM3), and those with the highest anti-OTA activity were Leuconostoc paracasei ssp. paracasei (3T3R1) and L. mesenteroides ssp. dextranicum (T2MM3). The best ML methods in predicting fungal growth inhibition were multilayer perceptron neural networks, followed by random forest. Due to anti-fungal and anti-mycotoxin capacity, the LABs strains used in this study could be good candidates as biocontrol agents against aflatoxigenic and ochratoxigenic fungi and AFL and OTA accumulation.

Potential Health Risk Associated with Mycotoxins in Oat Grains Consumed in Spain.

Spain is a relevant producer of oats (Avena sativa), but to date there has been no study on the occurrence/co-occurrence of mycotoxins in oats marketed in Spain. The present study is addressed to overcome this lack of knowledge. One hundred oat kernel samples were acquired across different Spanish geographic regions during the years 2015-2019 and analyzed for mycotoxin content using an ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method and matrix-matched calibration. The focus was on the regulated mycotoxins although other relevant mycotoxins were considered. The percentage of incidence (levels ≥ limit of detection), mean and range (ng/g) of mycotoxins were as follows: zearalenone (66%, mean 39.1, range 28.1-153), HT-2 toxin (47%, mean 37.1, range 4.98-439), deoxynivalenol, (34%, mean 81.4, range 19.1-736), fumonisin B1 (29%, mean 157.5, range 63.2-217.4), and T-2 toxin, (24%, mean 49.9, range 12.3-321). Fumonisin B2, 3-acetyldeoxynivalenol, aflatoxins B1, B2, and G2, and ochratoxin A were also detected at low levels, but aflatoxin G1 was not. The maximum limits established by the European Commission for unprocessed oats were not exceeded, except for zearalenone (in one sample), and the sum of aflatoxins (in two samples). Mycotoxin co-occurrence at quantifiable levels in the same sample (two to five combinations) was found in 31% of samples. The most common mixtures were those of HT-2 + T-2 toxins alone or together with deoxynivalenol and/or zearalenone.

Contamination of Wheat, Barley, and Maize Seeds with Toxigenic Fusarium Species and Their Mycotoxins in Tunisia.

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.

Machine learning approach for predicting Fusarium culmorum and F. proliferatum growth and mycotoxin production in treatments with ethylene-vinyl alcohol copolymer films containing pure components of essential oils.

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.

Antifungal effect of engineered silver nanoparticles on phytopathogenic and toxigenic Fusarium spp. and their impact on mycotoxin accumulation.

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.

Assessment of Toxic Effects of Ochratoxin A in Human Embryonic Stem Cells.

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.

Influence of nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains of Aspergillus spp. isolated from grapes.

This work studies the influence of nitrogen and carbon source on ochratoxin A production by three Aspergillus isolates A. ochraceus (Aso2), A. carbonarius (Ac25) and A. tubingensis (Bo66), all isolated from grapes. A basal medium (0.01 g/l FeSO4.7H2O, 0.5 g/l MgSO4.7H2O, 0.5 g/l Na2HPO4.2H2O, 1.0 g/l KCl) was prepared. This medium was supplemented with different nitrogen sources, both inorganic [(NH4)3PO(4), 0.3 g/l plus NH4NO3, 0.2 g/l] and organic (histidine, proline, arginine, phenylalanine, tryptophan or tyrosine) at two concentrations (0.05 g/l or 0.3 g/l), and different carbon sources (sucrose, glucose, maltose, arabinose or fructose) at three concentrations (10 g/l, 50 g/l or 150 g/l). A medium with sucrose (18 g/l) and glucose (1 g/l) was also tested. After a 10-day incubation period at 25 degrees C the highest levels of OTA (44.0 ng/ml, 13.5 ng/ml and 0.49 ng/ml for A. ochraceus, A. carbonarius and A. tubingensis, respectively) were obtained in the cultures containing 150 g/l of arabinose and 0.05 g/l of phenylalanine. Analysis of variance of the data showed that there were significant differences (p-value 0.05) among the OTA levels in the cultures with regard to carbon source and isolate. No significant differences were detected in OTA production regarding nitrogen source, although 0.05 g/l of phenylalanine generally favoured OTA production in the cultures of the three isolates. The dynamics of toxin production in the cultures of each isolate using the optimized basal medium supplemented with 0.05 g/l of phenylalanine and 150 g/l of arabinose for a period of 42 days at 25 degrees C was also studied. The maximum level of OTA was detected on the 3rd day of incubation in A. tubingensis cultures and on the 35th and 43(rd) days of incubation in A. ochraceus and A. carbonarius, respectively. This is the first study in which defined media have been used to assess the influence of carbon and nitrogen sources on OTA production by isolates of OTA-producing species isolated from grapes and to analyse the dynamics of toxin production in these species in a defined culture medium. This optimized medium for OTA production is being used in current studies aimed at elucidating its biosynthetic pathway.

Determination of multiple mycotoxins in feedstuffs by combined use of UPLC-MS/MS and UPLC-QTOF-MS.

In this report, a UPLC-ESI-MS/MS method for the simultaneous determination of aflatoxins, ochratoxin A, zearalenone, deoxynivalenol, fumonisins, T-2 and HT-2 toxins, fusarenone X, diacetoxyscirpenol, and 3- and 15-acetyldeoxynivalenol in feedstuffs was developed. A quadrupole-time-of-flight mass spectrometer detector (QTOF-MS) operating in full scan mode was combined with the UPLC-ESI-MS/MS system to confirm the identity of detected mycotoxins and to identify other possible microbial metabolites occurring in samples. Sixty-two feed samples from the Spanish market were analyzed. Extraction of metabolites was carried out with acetonitrile-water-formic acid (80:19:1, v/v/v). Method detection and quantification limits and performance criteria set by Commission Regulation (EC) No 401/2006 were fulfilled. Relatively high levels of the main regulated mycotoxins and presence of non-regulated mycotoxins in feed samples were found. This is the first study in which mycotoxins and other microbial metabolites occurring in feed are studied using a UPLC-QTOF-MS system being therefore a reference report.

Selected plant essential oils and their main active components, a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food.

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-1 were assayed and the effective doses for 50 (ED50) and 90% (ED90) growth inhibition were determined. The ED50 of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49-52.6, 98-145, 152-505, 295-560 mg l-1 and against A. parasiticus in the ranges 46-55.5, 101-175, 260-425 and 490-675 mg l-1, respectively, depending on environmental conditions. In A. flavus treatments ED90 were in the ranges 89.7-90.5, 770-860 and 820->1000 mg l-1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89-91, 855->1000 and 900->1000 mg l-1, respectively. ED90 values for oregano EO against both species were >1000 mg l-1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 aw. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.

Risk management of ochratoxigenic fungi and ochratoxin A in maize grains by bioactive EVOH films containing individual components of some essential oils.

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.

Electrochemical identification of toxigenic fungal species using solid-state voltammetry strategies.

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.

Real-Time RT-PCR assay to quantify the expression of fum1 and fum19 genes from the Fumonisin-producing Fusarium verticillioides.

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.

An overview of ochratoxin A in beer and wine.

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes.

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.

Assessment of azole fungicides as a tool to control growth of Aspergillus flavus and aflatoxin B1 and B2 production in maize.

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 the combination 37ºC/0.95 aw; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 aw, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l-1 prochloraz or 5.0 mg l-1 tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.

Impact of bioactive packaging systems based on EVOH films and essential oils in the control of aflatoxigenic fungi and aflatoxin production in maize.

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 0.25-4.0mg/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 0.22-0.23mg/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.

New method for determination of ochratoxin A in beer using zinc acetate and solid-phase extraction silica cartridges.

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 (r2 = 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).

Survey of the mycobiota of Spanish malting barley and evaluation of the mycotoxin producing potential of species of Alternaria, Aspergillus and Fusarium.

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.