Determination of type A and type B trichothecenes in paprika and chili pepper using LC-triple quadrupole-MS and GC-ECD.

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 material…

Effect of ethanol on the ability of Oenococcus oeni to remove ochratoxin A in synthetic wine-like media

The study focused on the changes in bacterial population, malolactic fermentation and ochratoxin A (OTA) levels in cultures of Oenococcus oeni performed in synthetic medium supplemented with ethanol and OTA. Growth was better in cultures containing 5% ethanol and was not observed in cultures containing 15% ethanol. The OTA removal ability of O. oeni depended on ethanol and initial OTA levels. The highest toxin removal percentage was observed in cultures containing 5% ethanol and 2 μg OTA/l. In ethanol-containing medium part of OTA was not sorbed by O. oeni and remained in the liquid medium. Thus, it cannot efficiently eliminate OTA in acidic ethanol-containing beverages, such as wine. The e…

Bee pollen, a substrate that stimulates ochratoxin A production by Aspergillus ochraceus Wilh.

The capacity of bee pollen as a substrate for production of ochratoxin A (OTA) by a strain of Aspergillus ochraceus was studied. For control purposes corn, wheat and rice grains, and eleven liquid media were assayed. They were Yeast Extract Sucrose broth (YES), YES supplemented with 0.05, 0.1, 0.5, 1 and 5% bee pollen, YES supplemented with 0.5% peptone, 50% must, Wickerham medium, Aflatoxin Production medium and Coconut Broth Medium. Cultures were maintained at 28 degrees C for 4 weeks and were analyzed every seven days for OTA by liquid chromatography with fluorescence detection. OTA production in bee pollen was significantly (P < 0.01) higher than production in corn, wheat and rice grain…

Microbiological and Enological Parameters during Fermentation of Musts from Poor and Normal Grape-Harvests in the Region of Alicante (Spain)

Must and wine from grapes harvested in two vintages (1986 and 1987) were anlyzed during vinification for physicochemical and microbiological characteristics. The 1986 vintage would be considered abnormal or poor vintage because of higher rainfall at harvest, and the 1987 one a normal vintage. Low reducing sugars and high volatile acidity at the beginning of the poor fermentation was observed as compared to normal vinification. The yeast population showed atypical evolution through the process since oxidative yeasts were isolated in the first stages of the poor vinification.

Contamination of barley seeds with Fusarium species and their toxins in Spain: an integrated approach.

Fusarium is a globally distributed fungal genus that includes different species pathogenic to cereals among others crops. Some of these Fusarium species can also produce toxic compounds towards animals and humans. In this work, the presence of the most important Fusarium toxins was determined in barley seeds from Spain, sampled according to European Union requirements. The results obtained were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction protocols. Fumonisins B(1) and B(2), zearalenone, trichothecenes type A (T-2 and HT-2) and trichothecenes type B (deoxynivalenol and nivalenol) were analy…

Critical study of and improvements in chromatographic methods for the analysis of type B trichothecenes

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 mycoto…

Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media.

A LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol water), extraction times (30', 60' or 90') and drying methods were investigated. Results showed that extraction with methanol: water (80:20, v/v) for 90 min, drying with N-2 and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0…

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) …

Conventional and Real-Time PCR Assays for Detection and Quantification of the Expression of Fumonisin Biosynthetic Genefum5 inFusarium verticillioides

Determination of ochratoxin A in beer marketed in Spain by liquid chromatography with fluorescence detection using lead hydroxyacetate as a clean-up agent

Abstract 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%, respective…

Influence of the Interactions among Ecological Variables in the Characterization of Zearalenone Producing Isolates of Fusarium spp.

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 …

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri.

ABSTRACT The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria , Cladosporium , and Aspergillus . The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respecti…

Changes in ochratoxin A and type B trichothecenes contained in wheat flour during dough fermentation and bread baking processes

Ochratoxin A (OTA) and type B trichothecenes are mycotoxins that occur frequently in cereals and thus can be found in cereal by-products such as bread. The aim of this work was to study the variation of the levels of OTA, deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) during the bread-making process. This was done by using wheat flour spiked with different levels of toxins. Mycotoxin levels were controlled after fermentation of the dough with yeasts (Saccharomyces cerevisiae) and after further baking at different temperature-time combinations. Analysis of variance (ANOVA) of the results showed a significant reduction in OTA level (p < 0.05) during fermentation of …

Bioproduction of an extract from Penicillium funiculosum Thom with activity against Ceratitis capitata and Tetranychus urticae

The bioproduction conditions of a crude extract from a strain of Penicillium funiculosum Thom, which shows activity against Ceratitis capitata and Tetranychus urticae, have been optimized. The efficacy degree was 95% (C. capitata) and 100% (T. urticae), according to Abbot. This is the first report on the pesticide activity of an extract from P. funiculosum against these species. The best yields were obtained when P. funiculosum was grown in Raulin-Thom medium at pH 6.5 with sucrose candy as the carbon source and ammonium sulphate and phosphate as the nitrogen source. Other favourable conditions were static cultures, at 28 degrees C, and in the dark.

Comparison of extraction and clean-up procedures for analysis of zearalenone in corn, rice and wheat grains by high-performance liquid chromatography with photodiode array and fluorescence detection.

The aim of this work was the optimization of some procedures usually used in the analysis of zearalenone (ZEA) in corn and other cereals by high-performance liquid chromatography (HPLC) with photodiode array and/or fluorescence detection. The comparison of five extraction solvents is presented. Three solid-phase extraction cartridges (C-18, silica, Florisil) and immuno-affinity columns were also compared to obtain the best recovery of the mycotoxin with the minimal presence of co-extractives in the chromatograms. Mixtures of methanol-1% aqueous NaCl (80.20 or 60:40 v/v) were the best extraction solvents. Florisil provided higher recovery of ZEA than C-18, and silica proved unsuitable. The i…

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp.

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. O…

Study on mycotoxin contamination of maize kernels in Spain

Abstract 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 a…

Trichothecenes and fumonisins produced in autoclaved tiger nuts by strains of Fusarium sporotrichioides andFusarium moniliforme

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. monil…

Optimization of clean-up procedure for patulin determination in apple juice and apple purees by liquid chromatography

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 clea…

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

Abstract 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 mycotoxig…

Monoterpenes in grape juice and wines.

The importance of monoterpenes on varietal flavour of wines has been reviewed. These compounds were mainly found linked to sugar moieties in the grape juice and wines, showing no olfactive characteristics. In this way, mechanisms to liberate terpenes were studied, making a comparative study between acidic and enzymic hydrolysis of terpene glycosides. Finally, analytical techniques developed to study these compounds, in both free or glycosidically forms, and also to fractionate glycosidic precursors, have been discussed.

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims: To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity (a(w)) and sub-inhibitory doses of the fungicide carbendazim. Methods and Results: A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20-28 degrees C), a(w) (0 center dot 94-0 center dot 98), carbendazim level (0-450 ng ml(-1)) and time (3-15 days after the lag phase). The output of the ANNs was OTA level determined by liqui…

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography.

Abstract Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC–FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC–FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-u…

Impact of non-selective fungicides on the growth and production of ochratoxin A byAspergillus ochraceusandA. carbonariusin barley-based medium

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. …

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 a…

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

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 tri…

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 sa…

Differentiation of Penicillium griseofulvum Dierckx isolates by enzyme assays and by patulin and griseofulvin analyses

The production of patulin and griseofulvin by 49 different isolates of Penicillium griseofulvum Dierckx was analyzed by high-performance liquid chromatography. Eleven isolates were obtained from pistachio nuts, 37 were obtained from wheat seeds, and 1 was obtained from the American Type Culture Collection. Activities of 19 enzymes were also assayed by the API ZYM system. From these results it may be deduced that there are two different groups among the strains tested which cannot be distinguished by morphological and cultural characteristics. One group of isolates did not produce detectable amounts of patulin and griseofulvin when grown in sucrose-yeast extract and Wickerham media, while en…

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

Abstract 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 …

Comparative assessment of solid-phase extraction clean-up procedures, GC columns and perfluoroacylation reagents for determination of type B trichothecenes in wheat by GC–ECD

Abstract 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 sorben…

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

Abstract 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. culmor…

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 …

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B1 and 15 ppb aflaxtoxin B2) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to pro…

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 to…

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 Penic…

Lactic acid bacteria: a potential tool to reduce ochratoxin A in wine

E. M. Mateo, A. Medina, F. Mateo, F.M. Valle Algarra, R. Mateo, and M. Jimenez 1 Dpto. de Microbiologia i Ecologia. Universitat de Valencia. Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain. 2 Dpto. de Quimica Analitica. Universitat de Valencia. Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain. 3 Dpto. de Ingenieria Electronica, Universitat Politecnica de Valencia, Camino de Vera, 14. E-46022, Valencia, Spain.

Ochratoxin A removal in synthetic media by living and heat-inactivated cells of Oenococcus oeni isolated from wines

The capacity of Oenococcus oeni to eliminate ochratoxin A (OTA) from synthetic media in different conditions was studied. Ten tested O. oeni strains removed OTA from the medium but with significant differences depending on the strain, incubation period, and initial OTA level in the medium. Mycotoxin reductions higher than 60% were recorded in 14-day cultures spiked with 2 mu g OTA/l. Toxin removal was independent of bacterial viability and culture medium composition. This is the first study carried out to study OTA removal dynamics by living and heat-inactivated cells of O. oeni. The results aim that this bacterium may be a very useful tool to control OTA in food and beverages. (C) 2009 Els…

Multilayer perceptron neural networks and radial-basis function networks as tools to forecast accumulation of deoxynivalenol in barley seeds contaminated with Fusarium culmorum

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 …

Fumonisin production by Gibberella fujikuroi strains fromPinus species

Abstract 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-restr…

Liquid chromatographic determination of toxigenic secondary metabolites produced by Fusarium strains.

Various liquid chromatographic methods used in the analysis of mycotoxins (zearalenone, trichothecenes and fumonisins) produced by Fusarium species were compared in this work. The results demonstrate the suitability of modern clean-up procedures employing multifunctional MycoSep and immunoaffinity columns although these methods are more expensive than conventional methodologies for clean-up. HPLC with both fluorescence and photodiode array detection is a suitable technique for the analysis of toxic secondary metabolites produced by Fusarium species; different derivatisation strategies have been studied to improve the sensitivity of the technique because of the low concentration of these met…

Determination of mycotoxins produced by Fusarium isolates from banana fruits by capillary gas chromatography and high-performance liquid chromatography.

A method of analysis for trichothecenes (nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, T-2 tetraol, T-2 and HT-2 toxins), zearalenone and zearalenols, and another method for determination of fumonisin B1 are described and applied to cultures of Fusarium isolated from bananas. Both methods were adapted from different techniques of extraction, clean-up and determination of these mycotoxins. The first method involves extraction with methanol-1% aqueous sodium chloride, clean-up of extracts by partition with hexane and dichloromethane, additional solid reversed-phase clean-up and analysis of two eluates by both high-performance liquid chromatography…

Detection and quantification of patulin and griseofulvin by high pressure liquid chromatography in different strains of Penicillium griseofulvum Dierckx.

Patulin and griseofulvin production by twelve strains ofPenicillium griseofulvum Dierckx, eleven of which were isolated from pistachio (Pistacia vera) nuts and the other was supplied by the Spanish Collection of Type Culture, was investigated. Six strains of the eleven isolated had ability to produce patulin and griseofulvin in Yes medium. All the strains studied had no ability to produce patulin in Wickerham medium. Griseofulvin production was significant in both media but higher in Wickerham.These metabolites were separated and determined in the chloroform extracts of cultures by high performance liquid chromatography with ultraviolet detection. The best conditions were: acetonitrile - wa…

Presence of trichothecenes and co-occurrence in cereal-based food from Catalonia (Spain)

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 t…

Aroma compounds in wine as influenced by apiculate yeasts

Aroma compounds of wines resulting from fermentation of sterile grape musts from Monastrell variety inoculated with pure and mixed cultures of apiculate and Saccharomyces yeasts, were isolated and analyzed by gas chromatography with flame ionization and mass spectrometry. Samples fermented with mixed cultures produced a higher concentration of selected compounds and higher total amounts of alcohols and acids, in contrast with wines produced with pure cultures of Saccharomyces spp. Apiculate yeasts are important in the chemical composition and quality of wine.

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 networ…

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…

Influence of the storage conditions on some physicochemical and mycological parameters of honey

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 under…

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

ABSTRACTAspergillus 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…

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

Abstract 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 r…

Potential impact of engineered silver nanoparticles in the control of aflatoxins, ochratoxin A and the main aflatoxigenic and ochratoxigenic species affecting foods

Abstract 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 …

Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops.

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity (a(w)) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 degrees C. The a(w)-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect signific…

MYCOTOXINS | Detection and Analysis by Classical Techniques

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

Characterization ofFusarium verticillioides strains by PCR-RFLP analysis of the intergenic spacer region of the rDNA

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 …

Neural network models for prediction of trichothecene content in wheat

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 chromatogr…

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 indicat…

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 an…

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates.

Summary In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B1 and fumonisin B2 by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers. The molecular char…

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

Ochratoxin A levels in the plasma of healthy blood donors from Valencia and estimation of exposure degree: comparison with previous national Spanish data.

Blood plasma levels of ochratoxin A, a toxic secondary metabolite of several fungal species belonging to the genera Aspergillus and Penicillium, were determined in 168 blood donors from the population of Valencia (Spain) using LC-FLD. In conjunction with blood collection, detailed information on diet was obtained by using a questionnaire that encompassed a wide range of products potentially contaminated with the toxin. The investigation revealed a detection frequency of 100%. Mean level was 1.09 microg OTA/l of plasma and concentrations ranged between 0.15 and 5.71 microg OTA/l of plasma. Men's levels were slightly higher than levels observed in women. Results were analysed by Spearman rank…

Fumonisin production in rice cultures of Fusarium verticillioides under different incubation conditions using an optimized analytical method.

Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the main members of a family of mycotoxins produced by various fungal species belonging to the Gibberella fujikuroi complex. The present work shows the results of a comparative study of various clean-up and derivatization procedures for analysis of fumonisins in rice cultures. Fumonisins were extracted from rice with acetonitrile/water (50/50, v/v). For clean-up, three solid-phase extraction procedures were assayed (C18 cartridge, SAX cartridge, and a combination of both). Two reagents (o-phthaldialdehyde and 4-fluoro-7-nitro-benzofurazan) were studied comparatively for formation of fluorescent derivatives. The separation was carried out by LC u…

Characterization of Gibberella fujikuroi complex isolates by fumonisin B-1 and B-2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Summary Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B 1 and B 2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which wer…

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 …

Efficacy of natamycin for control of growth and ochratoxin A production by Aspergillus carbonarius strains under different environmental conditions

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…

Comparison of Different Analytical Methods for Determination of Type B Trichothecenes in Wheat and Ochratoxin A in Beer

Capillary column gas chromatographic identification of sugars in honey as trimethylsilyl derivatives.

A method for identifying carbohydrates (mono-, di- and trisaccharides) in honey is presented. It is based on the separate preparation of both trimethylsilyl ethers and oxime trimethylsilyl ethers of the sugars followed by their gas chromatographic separation on a fused-silica capillary column coated with OV-101 using temperature programming. From the two chromatograms, the number of peaks given by each derivatized sugar, their relative retention times and peak-area ratios are used for identification. The identities of two unidentified trisaccharide peaks are considered. Quantitative applications to honey sugar analysis are discussed.

Occurrence of mycotoxin producing fungi in bee pollen

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. …

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 we…

An Overview on the Status of Toxigenic Fungi and Mycotoxins in Spain

Mycotoxins are a group of secondary metabolites which are produced by various filamentous fungi, and which can produce a toxic response in animals or humans after ingestion of contaminated food. The main toxigenic fungi isolated from foods and feeds belong to the genera Penicillium, Aspergillus, Fusarium and Alternaria. Spores of these moulds are almost universally present everywhere, they fall on plants and, under appropriate conditions, they can germinate and generate mycelia causing crop spoilage and/or production of mycotoxins.

Comparison of different analytical processes for patulin determination in apple juice

F. M. Valle-Algarra, R. Mateo, A. Medina, F. Mateo, E. M. Mateo, E. Sanchis Blanco, J.V. Gimeno-Adelantado, J. Peris-Vicente and M. Jimenez 1 Dpto. de Quimica Analitica, Facultad de Quimica, Universidad de Valencia, Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain 2 Dpto. de Microbiologia y Ecologia, Facultad de Biologia, Universidad de Valencia, Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain 3 Dpto. de Ingenieria Electronica, Universidad Politecnica de Valencia, Camino de Vera 14, E-46022, Valencia, Spain

Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection.

A method for the analysis of type A trichothecenes T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol by high-performance liquid chromatography with fluorescence detection using coumarin-3-carbonyl chloride has been developed. Different parameters concerning the analytical procedure such as stability of both the reagent and derivatised analytes, time and temperature of the derivatisation reaction, were studied and optimised. Three different clean-up procedures (solid-phase extraction with silica gel or C-18 cartridges, and liquid-liquid partition between toluene and dihydrogen phosphate buffer) were tested in order to remove the excess reagent peaks. The last procedure gave the best …