Francisco M. Valle-Algarra

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, 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.

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

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 the dough. The reduction ranged between 29.8% and 33.5%, depending on the initial concentration of toxin in the flour. During this period, the level of the other mycotoxins studied was not modified. By contrast, in the baking phase there were significant changes in the levels of the four mycotoxins, although the reduction was similar under all the baking conditions. Considering all the temperature–time conditions tested, it can be concluded that during the baking period the average reduction of OTA, NIV, 3-ADON, and DON was 32.9%, 76.9%, 65.6%, and 47.9%, respectively.

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.

Mycobiota and co-occurrence of mycotoxins in Capsicum powder

This study aimed to: (1) determine the mycobiota of Capsicum powder samples, paying a special attention to the mycotoxigenic moulds; (2) evaluate the contamination levels of aflatoxins (AF), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T2 and HT2 toxins in those samples. Thirty-two samples were obtained through the methods of sampling established by the European Union legislation. Aspergillus and Eurotium were the most frequently found genera. Aspergillus section Nigri had the higher relative frequency in the samples, A. niger aggregate being the most representative group of this section. Other potentially mycotoxigenic Aspergillus, Fusarium and Penicillium species were found, but in a lower frequency. Co-occurrence of mycotoxins was confirmed in the 32 Capsicum powder samples. All samples were contaminated with AF and OTA, 27% with ZEA (36% of chilli and 18% of paprika samples), 9% with DON (18% of chilli and 6% of paprika samples), 6% with T2 (18% of chilli samples) and none of the samples contained HT2. Although in the present study the most common genera found (Aspergillus and Eurotium) belong to storage moulds, some field fungi such as Fusarium spp. were also found, and their toxins were sometimes detected. This fact supports the hypothesis that mycotoxin contamination of Capsicum products may occur both in the field and/or during storage.

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%.

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium

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

On the dehydroindigo contribution to Maya Blue

A series of data from voltammetric, spectral, and UPLC–MS and Py–GC–MS analyses of extracts from synthetic Maya Blue-type specimens provides evidence on the presence of a significant amount of dehydroindigo, identified on the basis of its MS, FTIR, and UV–Vis signatures, accompanying indigo and other minority compounds, supporting the view of this material as a complex polyfunctional organic–inorganic hybrid material. Estimates of dehydroindigo/indigo in-depth distribution and thermochemical data for the dye association to the clay from chromatographic and voltammetric data are provided.

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 clean-up procedures for PAT were comparatively studied. PAT recoveries in apple juice spiked with 1.0mg PAT/kg varied between 52.3% and 81.0%. The highest PAT recovery in apple puree spiked with 0.1mg PAT/kg was 82.9%. Addition of NaH(2)PO(4) during the extraction phase here reported for the first time has the advantage of keeping the pH slightly acidic, thus avoiding PAT degradation.

‘Maya chemistry’ of organic–inorganic hybrid materials: isomerization, cyclicization and redox tuning of organic dyes attached to porous silicates

Association of indigo and lapachol dyes to aluminosilicate clays yields polyfunctional organic–inorganic hybrid materials forming Maya Blue-like systems. Upon partial removing of clays zeolitic water by moderate thermal treatment, abundant isomerization, cyclicization and oxidation reactions occur defining a ‘Maya chemistry’ whose complexity could explain the versatile use of such materials in the pre-Columbian cultures and permits the preparation of polyfunctional materials potentially usable for therapeutic and catalytic purposes.