Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium.

Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus. Temperature and water activity are the two key determinants in pre and post-harvest environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental to know which combinations can control or be conducive to aflatoxin contamination. Little information is available about the influence of these parameters on aflatoxin production on almonds. The objective of this study was to determine the influence of different combinations of temperature (20 °C, 28 °C, and 37 °C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth, aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28 °C and 0.96 aw; no fungal growth and AFB1 production were observed at 20 °C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37 °C AFB1 production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28 °C) and minimum (20 °C and 37 °C) AFB1 production. Conversely the two structural genes (aflD and aflO) were highly expressed only at maximum AFB1 production (28 °C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which is strictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO), but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are most likely subordinated to other regulatory processes acting at post-translational level. The results of this study are useful to select conditions that could be used in the almond processing chain to suppress aflatoxin production in this important product.

Systemic growth of F. graminearum in wheat plants and related accumulation of deoxynivalenol.

Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock.

First evidence of placental transfer of ochratoxin A in horses.

Ochratoxin A (OTA) is a renal mycotoxin and transplacental genotoxic carcinogen. The aim of this study was to evaluate the natural occurrence of OTA in equine blood samples and its placental transfer. For the assessment of OTA levels, serum samples were collected from 12 stallions, 7 cycling mares and 17 pregnant mares. OTA was found in 83% of serum samples (median value = 121.4 pg/mL). For the assessment of placental transfer, serum samples were collected from the 17 mares after delivery and from the umbilical cords of their foals, after foaling. Fourteen serum samples from pregnant mares contained OTA (median value = 106.5 pg/mL), but only 50% of their foals were exposed (median values = 96.6 pg/mL). HPLC analysis carried out on four serum samples (collected from two mares and their respective foals) supported the ELISA results on OTA placental transfer. This is the first report on the natural occurrence of OTA in horse serum samples and placental transfer in horses.

Determination of ochratoxin A in wine by means of immunoaffinity and aminopropyl solid-phase column cleanup and fluorometric detection.

A new analytical method for the determination of ochratoxin A (OTA) in red wine has been developed by using a double-extract cleanup and a fluorometric measurement after spectral deconvolution. Wine samples were diluted with a solution containing 1% polyethylene glycol and 5% sodium hydrogencarbonate, filtered, and purified by immunoaffinity and aminopropyl solid-phase column. OTA contents in the purified extract were determined by a spectrofluorometer (excitation wavelength, 330 nm; emission wavelength, 470 nm) after deconvolution of fluorescence spectra. Average recoveries from wine samples spiked with OTA at levels ranging from 0.5 to 3.0 ng/mL were 94.5-105.4% with relative standard deviations (RSD) of <15% (n = 4). The limit of detection (LOD) was 0.2 ng/mL, and the total time of analysis was 30 min. The developed method was tested on 18 red wine samples (naturally contaminated and spiked with OTA at levels ranging from 0.4 to 3.0 ng/mL) and compared with AOAC Official Method 2001.01, based on immunoaffinity column cleanup and HPLC with fluorescence detector. A good correlation (r(2) = 0.9765) was observed between OTA levels obtained with the two methods, highlighting the reliability of the proposed method, the main advantage of which is the simple OTA determination by a benchtop fluorometer with evident reductions of cost and time of analysis.

Determination of HT-2 and T-2 toxins in oats and wheat by ultra-performance liquid chromatography with photodiode array detection.

European intake estimates indicate that the presence of HT-2 and T-2 toxins in cereals, mainly in oats, can be of concern for human health. Therefore, the development of sensitive, rapid and reliable methods for determining these mycotoxins in cereals, in particular oats, has high priority. A rapid ultra-performance liquid chromatographic (UPLC) method has been developed for the simultaneous determination of HT-2 and T-2 toxins in oats and wheat at µg kg(-1) level. Ground samples were extracted with methanol/water (90:10, v/v) and the diluted extracts were cleaned up through immunoaffinity columns. HT-2 and T-2 toxins were separated and quantified by UPLC with photodiode array (PDA) detector (λ=202 nm) in less than 5 min. Mean recoveries from blank oats samples spiked with HT-2 and T-2 toxins at levels of 50-1000 µg kg(-1) ranged from 87 to 96%, with relative standard deviations (RSDs) lower than 7%; mean recoveries from wheat spiked with HT-2 and T-2 toxins at levels of 25-100 µg kg(-1) ranged from 91 to 103%, with RSDs lower than 5%. The limit of detection of the method was 8 µg kg(-1) for both toxins (signal-to-noise ratio 3:1). The method was successfully applied to the analysis of HT-2 and T-2 toxins in naturally contaminated oats and wheat samples. A good correlation was found by comparative analysis of naturally contaminated samples of oats (r=0.9985) and wheat (r=0.9058) using the proposed method or a reliable HPLC method with fluorescence detection after pre-column derivatization with 1-anthroylnitrile.

Removal of ochratoxin A from contaminated red wines by repassage over grape pomaces.

Ochratoxin A contamination of red wines might be quite severe in certain high-risk regions and vintages, thus requiring corrective measures to fulfill acceptable standards for human consumption. This work proposes an innovative and environmentally friendly corrective measure to reduce ochratoxin A levels by repassage of contaminated musts or wines over grape pomaces having no or little ochratoxin A contamination. Grape pomaces have a high affinity for ochratoxin A and have been shown to remove ochratoxin A from must and wine during vinification. Time course experiments showed that ochratoxin A adsorption by pomaces is a rapid process, reaching equilibrium in less than 10 h, and is not affected by the tested toxin concentrations. Repassage of wine from Primitivo grapes spiked with 2-10 microg/kg ochratoxin A over pomaces obtained from the same grapes removed up to 65% ochratoxin A within 24 h. Similar results (50-65% ochratoxin A reduction) were obtained with Primitivo or Negroamaro wines repassed over pomaces from different grape varieties including white grapes (Malvasia, Greco di Tufo) and red grapes (Sangiovese, Aglianico). Grape pomaces maintained a good efficacy in removing ochratoxin A after being reused four times. Unlike other enological fining agents, the use of grape pomaces to adsorb ochratoxin A from red wines of the same grape variety (Primitivo) did not affect relevant wine quality parameters, including color intensity and health-promoting phenolic content (trans-resveratrol, quercetin, total polyphenols). These quality parameters were instead positively or negatively affected when contaminated wines were repassed over grape pomaces from other grape varieties, the effect being related to the intrinsic characteristics of the pomace variety. The proposed decontamination procedure can be applied in a modern winery provided that contaminated grapes are identified early and processed separately from uncontaminated grapes.

Determination of ochratoxin A in grapes, dried vine fruits, and winery byproducts by high-performance liquid chromatography with fluorometric detection (HPLC-FLD) and immunoaffinity cleanup.

A liquid chromatographic method for the determination of ochratoxin A in grapes, dried vine fruits, and winery byproducts was developed. A mixture of either acetonitrile/water or acetonitrile/water/methanol was used as an extraction solvent mixture. After immunoaffinity column cleanup, the final extract was analyzed by high-performance liquid chromatography (HPLC) with a fluorometric detector (FLD). Mean recoveries from grapes, grape pomace, and lees samples spiked in the range of 1-200 microg/kg were 78, 86, and 88%, respectively, with a detection limit of 0.1 microg/kg and within-laboratory repeatability ranging from 6 to 15%. Tested on naturally contaminated samples of grapes, grape pomace, and sultanas, the method showed better performances as compared to two other methods also based on immunoaffinity cleanup and HPLC/FLD determination. Ochratoxin A was detected in samples of grape pomace (levels ranging from 34.2 to 456.8 microg/kg) and lees (levels ranging from 48.3 to 602.5 microg/kg) derived from the wine making of red grapes of 2004 and 2005 vintages in southern Italy. After distillation of contaminated grape pomace in a pilot-scale equipment to produce grappa, the toxin remained unchanged in the exhausted pomace and was not detected in any of the distilled fractions (detection limit of 0.02 microg/L).