Potential of aqueous ozone to control aflatoxigenic fungi in Brazil nuts

The Brazil nut (Bertholethia excelsa) is an important non timber forest product (NTFP) from the Amazonian forest. Despite their nutritious value, Brazil nuts are susceptible to contamination with Aspergillus section Flavi fungi and consequently with aflatoxins. Since aqueous ozone reduces microorganisms population and has oxidant effect on aflatoxins, the effect of ozone on. Both natural and artificially contaminated Brasil nuts were studied in the present work. The former were inoculated with either 1x106 or 1x107 conidia mL-1 of A. flavus (MUM 9201). Previous assays were carried out to determine optimal parameters of the treatment. Different aqueous ozone contact time was assayed. The duration was controlled by the addition of a sodium formate solution. Such assays evidenced that the effect of ozone is almost immediate. Also, different ozone concentrations were assayed. The optimum ozone concentration depended on the initial viable spores on the nutshell. Also, the effect of the ozonization on the shell nut color was assessed by measuring chromaticity values of the treated fruits in the L*a*b* space coordinates. High concentrations of ozone affected both the luminosity and the hue of the nutshell. Finally, a concentrated aqueous ozone solution was assayed on both natural and artificially contaminated nuts. The remaining viable spores in the ozone solution were recorded and the rate of inactivation for each treatment was determined by assessing the ratio between the CFU of each treatment and the control. Ozonized nuts were also plated on MEA to recover the fungi population. Aqueous ozone was effective in reducing the conidia of Aspergillus flavus and the natural fungal population associated to Brazil nuts, although it seems to affect external appearance of the shell. The aqueous ozone solution may be recommended to control other mycotoxin producer Aspergilli, since ozone is regarded a generally recognized as safe (GRAS) product and it has been already used in many agricultural products, including the organically labels one like Brazil nuts.Fundação para a Ciência e a Tecnologia (FCT

Detection and quantification of geosmin and other fungal metabolites in grape juice by HS-SPME coupled with GC/MS

(-)-Geosmin (GEO) (Figure 1) is an aromatic volatile metabolite the presence of which in grapes is associated with an earthy smell of crucial importance in grape products (e.g. grape juice). Two fungi are involved in the accumulation of geosmin in grapes: Botrytis cinerea and Penicillium expansum (La Guerche et al., 2005). Other compounds of fungal origin such as fenchone, fenchol, anisoles and methyl isoborneol (MIB) can also transmit undesirable aromas (Boutou and Chatonnet, 2007) According to literature (Boutou and Chatonnet, 2007; Baghery et al., 2007; Salto et al., 2008 and Prat et al., 2008), Head Space Solid-Phase Microextraction (HS-SPME) coupled with Gas Chromatography/Mass Spectrometry (GC/MS) methodology was used to detect and quantify GEO. The fiber used was divinilbenzene/carboxen/polidimetilsiloxane (DVB/CAR/PDMS). Grape juice spiked with standards of the analytes was analyzed to validate the method. Optimization of the method was performed by determining the efficiency of GEO extraction. The influence of the ionization of the analytes was tested by extracting at different pH (from ca. 3.5 to 7.0) and/or addition of NaCl (from none to saturation). Also, the optimal ethanol concentration in the sample was assessed (from none to 6% v/v). Time of extraction (from 20 to 65 min) and temperature of extraction (from 30 to 70 ºC) were assayed as was the position of the Head Space fiber. The results underwent ANOVA tests to evaluate the significance of the differences in geosmin extraction. Graphics of extraction of GEO as a function of the factors tested were plotted to choose those conditions that maximized the detection of GEO and permitted the detection of the other analytes. Finally, internal standards were tested (4- Nonanol; Methyl-4-pentan-2-ol and 2,3,4-Trichloroanisole) to obtain calibration curves. The calibration curves of GEO were linear with high correlation coefficients. Geosmin, MIB and most of the other metabolites were detected. Quantification of GEO was possible in the range of concentrations in naturally contaminated grape juice

Microextraction and gas chromatography/mass spectrometry for improved analysis of geosmin and other fungal "off" volatiles in grape juice

Geosmin is a volatile fungal metabolite with an earthy aroma produced in grape products from rotten grapes. The accumulation of geosmin in grapes is caused by the interaction of Botrytis cinerea and Penicillium expansum. Solid Phase Microextraction (SPME) has great utility for collecting volatile compounds in wine. However, contamination with earthy odours may have occurred previously in the must and novel methods are required for this commodity. In the present report, several parameters of the SPME were evaluated to optimize geosmin extraction. The method permitted quantification of geosmin and other fungal volatiles by Gas Chromatography–Mass Spectrometer (GC–MS) at very low concentrations. Limits of detection and quantification (LD and LQ) for geosmin were 4.7 ng L−1 and 15.6 ng L−1 respectively. The RSD was 4.1% and the recovery rates ranged from 115% to 134%. Uniquely, haloanisoles were analyzed by using only one internal standard (2,3,6-trichloroanisole) thus avoiding the synthesis of deuterated anisole analogues that are used as internal standard in other methods. The method was used for the analysis of grape juice samples inoculated with B. cinerea and P. expansum. Geosmin and methylisoborneol were the compounds that appeared to contribute most to earthy odours, although other fungal compounds which are claimed to cause earthy or mouldy off-odours were detected (e.g. 1-octen-3-ol and fenchol).Fundação para a Ciência e a Tenoplogia (FCT

Incidence of Botrytis cinerea and Penicillium spp. and other fungi in the regions of “Vinho Verde” (Portugal) and “Alvariño” (Spain)

Fundação para a Ciência e a Tecnologia (FCT

Micotoxinas detectadas en productos alimenticios en Portugal: revisión

Las micotoxinas son metabolitos tóxicos producidos por hongos filamentosos que aparecen de forma natural en productos agroalimentarios en todo el mundo. Las aflatoxinas, ocratoxina A, patulina, fumonisinas, zearalenona, tricotecenos y alcaloides del ergot son actualmente las más relevantes. Estas micotoxinas pueden ser producidas por especies que pertenecen a los géneros Aspergillus spp, Penicillium spp, Fusarium spp y Claviceps spp; y pueden ser carcinogénicas, mutagénicas, teratogénicas, citotóxicas, neurotóxicas, nefrotóxicas, estrogénicas e inmunosupresoras. La evaluación de la exposición de los seres humanos y animales a las micotoxinas se realiza sobre todo teniendo en cuenta los datos sobre su aparición en los productos alimenticios y sobre los hábitos de consumo. Esta evaluación es fundamental y sirve de soporte para la aplicación de medidas dirigidas a reducir la exposición de los consumidores a las micotoxinas. Este artículo intenta hacer una revisión sobre la aparición de micotoxinas y de los niveles de éstas encontrados en productos alimenticios portugueses para así contribuir a una visión global sobre esta problemática en Portugal

Diversity of Botrytis cinerea from vineyards in the north west Iberian peninsula

Botrytis cinerea is associated with a fungal gray rot in the concomitant regions of north west Spain and northern Portugal, where it is the most damaging pathogen and results in severe economic losses. Also, the physiological interactions of B. cinerea with Penicillium expansum are responsible for the production of geosmin, a volatile metabolite that transmit undesirable earthy odours to must and thus to wine. B. cinerea is not a homogeneous species and may be divided into several sub-species groupings. Some previous studies indicated two groups or cryptic species (I and II). Other work revealed that spore size and vegetative compatibility are characteristic features of these groups. In the present work a survey was taken of the population of B. cinerea from the above mentioned regions. The spore size and compatibility tests allowed characterisation of most isolates of B. cinerea into Group I or Group II taxa. Interestingly, some isolates could not be characterised according to their spore size and also presented ambiguous vegetative compatibility features. Furthermore, the influence of other factors on the spore size and grouping were studied. Grape variety, vineyard, country, sanitary state of the bunch and whether the isolates were obtained from the exterior or the interior of the bunch were compared with spore size and vegetative compatibility. Characterization of strains was affected by whether isolates were obtained from a particular grape variety. Also, isolates from Group II were obtained exclusively from a particular vineyard. The results suggest that there is considerable genetic diversity within the species which may explain patterns of gray rot within grapes.Fundação para a Ciência e a Tecnologia (FCT