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 materials followed by a further clean-up in immunoaffinity column that was specific for the two toxins. The solvent was changed and the eluate was derivatized with pentafluoropropionic anhydride and injected into the GC system. The limits of detection (LOD) for T-2 and HT-2 toxins were 7 and 3 µg/kg, respectively, and the recovery rates for paprika spiked with 1000 µg toxin/kg were 71.1% and 80.1% for HT-2 and T-2 toxins, respectively. For DON determination, the optimized method consisted of extraction with acetonitrile-water (84:16, v/v) solution followed by a solid-phase extraction clean-up process in a cartridge made of different sorbent compounds. After solvent evaporation in N(2) stream, the residue was dissolved and DON was separated and determined by LC-MS/MS. The LOD for this method was 14 µg DON/kg paprika sample and the DON recovery rate was 86.8%.

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.

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.

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.

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.

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

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

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

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, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.

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.

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

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

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 characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B1 and B2 producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.

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

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-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC-FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C18 using methanol-acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1-1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.

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 were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.

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 grains regardless of incubation time. With regard to liquid cultures, OTA accumulation in YES supplemented with 5% bee pollen was significantly higher than in pollen-free liquid cultures. A positive correlation between the proportion of pollen added to YES medium and OTA level was observed. This is the first report concerning the use of bee pollen as a substrate to stimulate OTA production. On the basis of the preliminary results obtained in this study it can be hypothesized that bee pollen may constitute an important risk factor concerning the presence of OTA in the diet of consumers of that nutritious food.