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…

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) …

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…

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 …

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…

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…

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…

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…

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…

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

MycoKey, an EU-funded Horizon 2020 project, includes a series of “Roundtable Discussions” to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the…

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…

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…

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…

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

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. …

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

Influence of freezing temperatures prior to freeze-drying on viability of yeasts and lactic acid bacteria isolated from wine

Aims To determine the effect of three different freezing temperatures on post-freeze-drying survival rates of wine yeasts and lactic acid bacteria (LAB). To know if a similar freeze-drying protocol can be used for both micro-organisms. Methods and Results Cells from liquid culture media were recovered and concentrated in appropriate lyoprotectants. Aliquots of each strain were frozen at −20, −80 and −196°C before vacuum drying. Viable cell counts were done before freezing and after freeze-drying. Survival rates were calculated. Freezing temperatures differently affected yeast and bacteria survival. The highest survival rates were obtained at −20 and −80°C for yeasts, but at −196°C for LAB. …