Characterization of an ethanol-tolerant 1,4-ß-xylosidase produced by Pichia membranifaciens.

AIMS: The purification and biochemical properties of the 1,4-ß-xylosidase of an oenological yeast were investigated. METHODS AND RESULTS: An ethanol-tolerant 1,4-ß-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50kDa by SDS-PAGE. The activity of 1,4-ß-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48±0·06mmol l(-1) and a Vmax of 7·4±0·1µmol min(-1)mg(-1) protein for p-nitrophenyl-ß-d-xylopyranoside. CONCLUSIONS: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may be useful for assessing the ability of the 1,4-ß-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Characterization of glycolytic activities from non-Saccharomyces yeasts isolated from Bobal musts.

A large number of non-Saccharomyces yeasts were isolated from grapes of Bobal variety and identified according to their physiological and molecular characteristics. The yeasts were tested to determine the presence of ß-glucosidase, ß-xylosidase, α-arabinosidase, and α-rhamnosidase activities and five isolates were selected. All enzymatic activities were induced by the presence of glycosides as the only carbon source in the medium, which seems to be a characteristic of the yeast isolate, and were characterized according to different parameters of enological interest.

Sugars and amino acids as factors affecting the synthesis of fumonisins in liquid cultures by isolates of the Gibberella fujikuroi complex.

The capacity of four isolates belonging to the Gibberella fujikuroi complex to produce fumonisin B1 and fumonisin B2 when grown in liquid medium supplemented with one sugar and one amino acid at various concentration levels has been investigated. The sugars used for supplementing the medium were glucose, fructose, rhamnose, sucrose, maltose, and trehalose at 5, 10 or 20 g/l. The amino acids used were serine, threonine, glutamic acid, aspartic acid, valine, isoleucine, methionine, glycine, alanine, and cystine at 1 or 10 g/l. Fumonisins were extracted from culture filtrates, purified by SAX column and determined by reversed-phase C18 HPLC with fluorescence detection of the o-phthaldialdehyde derivatives. Two isolates produced very low concentrations of fumonisins with all sugars. The remaining isolates provided increased contents of fumonisins when sugar level increased. Concerning the amino acids, production of fumonisins was also dependent on the isolate, although at 1 g/l, the production of fumonisins was greater than at 10 g/l. The results indicate that the sugar-amino acid-isolate combination is basic in fumonisin biosynthesis and that the particular behaviour of each isolate in the different nutritional conditions may constitute a piece of interesting information in the fields of the Taxonomy, Physiology and Toxicology of these fungi. This is the first report on the influence of the carbon and nitrogen sources on fumonisin production by isolates of the G. fujikuroi complex.

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 metabolites in contaminated food. The utility of the proposed methodology was assessed in cereal cultures of various Fusarium strains.

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 immuno-affinity column was very efficient in cleaning the extracts, but its sample capacity was lower than that of SPE columns due to saturation. The mobile phase (methanol-water 80:20 v/v) gave a low retention time for ZEA (approximately 5 min), high sensitivity and acceptable separation between this mycotoxin and alpha-zearalenol. The optimized protocol is straightforward, provides high ZEA recoveries in spiked corn (mean 102.4%), has an acceptable sensitivity and has a lack of interference with fluorescence detection (detection limit 4 ng ZEA g(-1) corn). The photodiode array detector was useful, except at very low ZEA levels, to confirm the identity of the mycotoxin. The method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and F. culmorum.

Accumulation of type A trichothecenes in maize, wheat and rice by Fusarium sporotrichioides isolates under diverse culture conditions.

Toxigenic isolates of Fusarium sporotrichioides were tested for the production of type A trichothecenes (T-2 toxin, HT-2 toxin, diacetoxyscirpenol and neosolaniol) when grown on three substrates (maize, rice and wheat) under various conditions of temperature and water activity in the laboratory for 3 weeks. Trichothecenes were determined by high-performance liquid chromatography with fluorescence detection, after derivatisation with coumarin-3-carbonyl chloride. This is the first time this analytical method has been applied to an extensive study of trichothecene accumulation. With minor exceptions, greater trichothecene production occurred when samples were incubated at 20 degrees C and moistened with 35% water (water activity 0.990) although incubation conditions affected the substrates studied in different ways. No correlation between the different pairs of trichothecenes was found except for neosolaniol and diacetoxyscirpenol (r=0.56). Principal component analysis results show that the data points can be grouped in three rough clusters related to cereal type, which points out that the composition of these cereals can influence the production of type A trichothecenes.

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 mycotoxins with minimal presence of coextractives in the chromatograms. The chosen extraction solvent was a mixture of acetonitrile-water (84:16, v/v). The MycoSep 225 column was chosen as the best alternative for clean-up of grain samples. For GC-ECD analysis, derivatisation of analytes with heptafluorobutyric anhydride prior the final determination was chosen as the most suitable procedure. HPLC-photodiode array (at 221 nm) analysis was more suitable for determination of type B trichothecenes than HPLC of the fluorescent coumarin-3-carbonyl derivatives. Recoveries obtained in spiked corn, rice and wheat are reported. The utility of the proposed methodology was assayed in cereal cultures of various Fusarium strains.

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.

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 results when the buffer pH was 3-5.5, and is therefore recommended. Separations were performed on a stainless steel LiChrospher 100 C-18 reversed-phase column with pre-column of the same phase. The mobile phase was acetonitrile/water (65:35, v/v) containing 0.75% acetic acid at a flow-rate of 1.0 ml/min. The proposed method provides good separation between the four trichothecenes and good reproducibility (RSD of calibration standards <5%). The limits of detection of the studied trichothecenes at a signal-to-noise ratio of 3:1, with an injection volume of 20 microl were 10 ng/g sample for T-2 toxin and about 15 ng/g sample for the remaining mycotoxins. The calibration curve was linear between 10 and 2000 ng for the four trichothecenes assayed. The method was applied to the analysis of these mycotoxins in fungal cultures (corn and rice) of Fusarium sporotrichioides, and is also perfectly suitable for the quantification of type A trichothecenes in contaminated cereals.

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA.

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 B1 and B2 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 were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.