Antifungal activity of Saccharomyces cerevisiae and assessment of ochratoxigenic load on currants by means of Real Time PCR.

Currants are prone to contamination by ochratoxin during cultivation, processing and storage conditions. Saccharomyces cerevisiae is considered to be among the main species of grape yeast flora able to control antagonistic fungi. In this study, the potential of S. cerevisiae Y33 was investigated to inhibit the growth of several fungal species indigenous to the microbiota of grapes. Moreover, the efficacy of this yeast species was investigated to inhibit OTA by toxin producing fungi both in vitro and in situ. For this purpose thirty-five different fungal species, belonging to the genera Aspergillus, Penicillium, Cladosporium, Fusarium and Alternaria interacted in vitro with S. cerevisiae on Malt Extract agar plates, stored at 25 °C for 14 days. Results showed that the highest OTA producer A. carbonarius F71 was inhibited more than 99% from day 7, in contrast to A. niger strains that presented enhanced OTA production at day 14 due to interaction with S. cerevisiae Y33. Additionally, the antifungal potential of the selected yeast was also studied in situ on currants subjected to different treatments and stored at 25 °C for 28 days. Microbiological analysis was undertaken for the enumeration of the bacterial and fungal flora, together with OTA determination at 7 and 21 days. To quantify A. carbonarius on all treated currant samples, molecular analysis with Real Time PCR was employed. A standard curve was prepared with A. carbonarius DNA. The efficiency of the curve was estimated to 10.416, the slope to -3.312 and the range of haploid genome that could be estimated was from 1.05 to 105∙105. The amount of A. carbonarius DNA in all treated currants samples, where the fungus was positively detected, ranged from as low as 0.08 to 562 ng DNA/g currants. The antifungal activity of S. cerevisiae Y33 was observed in all studied cases, causing inhibition of fungal growth and OTA production.

Effects of environmental conditions and substrate on growth and ochratoxin A production by Penicillium verrucosum and Penicillium nordicum: Relative risk assessment of OTA in dry-cured meat products.

Ecological conditions during ripening of dry-cured meat products favour the growth of an uncontrolled mould population that could suppose a risk of ochratoxin A (OTA) production. In this work the influence of water activity (aw), temperature and substrate composition on fungal growth and OTA production by Penicillium nordicum and Penicillium verrucosum isolated from dry-cured meat products have been studied. In addition, the relative risk of OTA presence on dry-cured meat products has been evaluated using the Risk Ranger software. Fungal growth was observed in the range of 0.99-0.90 aw and 15-25 °C being mainly temperature-dependent. P. nordicum and P. verrucosum were able to produce OTA in every substrate in these ranges of aw and temperature. The production of OTA by P. verrucosum was mainly influenced by temperature and media composition. However, P. nordicum it is affected mainly by substrate or temperature depending on the strain studied. Both species produce a large amount of OTA on dry-cured ham and on dry-cured fermented sausage "salchichón" in environmental conditions usually found throughout the ripening of these products. The Risk Ranger software reveals that the relative risk of OTA on dry-cured meat products is 75%. Thus, control measures during dry-cured meat products processing to prevent OTA risk should be established.

Assessment of the ochratoxin A production ability of Aspergillus tubingensis.

Aspergillus tubingensis is a black Aspergillus frequently isolated from different agricultural products, including grapes. Conflicting results have been published in recent years about its ability to produce ochratoxin A (OTA), a potent nephrotoxic and carcinogenic mycotoxin. This study re-examined six A. tubingensis strains deposited in international culture collections for OTA production. OTA could not be detected in any A. tubingensis extract using HPLC coupled with a fluorescence detector (FLD), whereas it was easily detected in ochratoxigenic A. niger extracts used as positive control. The same outcome was obtained using LC-MS. The presence of other metabolites with retention times similar to the OTA signal in the A. tubingensis extracts or background noise of the growth media may be reasons for the misinterpretation of the chromatograms obtained by HPLC-FLD.

Natural occurrence of ochratoxin A in wolfberry fruit wine marketed in China.

Wolfberry fruit wine (WFW) is widely used as a global functional food to improve the immune system and prevent human disease. A total of 36 bottled WFWs were randomly collected in China between 2005 and 2010. Samples were analysed for the presence of ochratoxin A (OTA) using immunoaffinity column (IAC) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Positive results were confirmed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The limit of detection (LOD), based on a signal-to-noise ratio of 3, was 0.05 ng mL⁻¹. Recoveries ranged from 78.3% to 94.7% and relative standard deviations from 1.1% to 4.3% within the spiking range of 0.2-20 ng mL⁻¹. OTA was detected in one sample, below the maximum allowable limit as established by the European community.

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks.

AIMS: To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity (a(w)) and sub-inhibitory doses of the fungicide carbendazim. METHODS AND RESULTS: A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20-28 degrees C), a(w) (0.94-0.98), carbendazim level (0-450 ng ml(-1)) and time (3-15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed. CONCLUSIONS: ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a(w), temperature and carbendazim doses. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.

Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR.

Aspergillus carbonarius is an ochratoxin producing fungus that has been considered to be responsible of the ochratoxin A (OTA) contamination in grapes and wine. In order to monitor and quantify A. carbonarius, a specific primer pair Ac12RL_OTAF/Ac12RL_OTAR has been designed from the acyltransferase (AT) domain of the polyketide synthase sequence Ac12RL3 to amplify 141 bp PCR product. Among the mycotoxigenic fungi tested, only A. carbonarius gave a positive result. This specific primer pair was also successfully employed in real-time PCR conjugated with SYBR Green I dye for the direct quantification of this fungus in grape samples. A positive correlation (R(2)=0.81) was found between A. carbonarius DNA content and OTA concentration in 72 grape samples, allowing for the estimation of the potential risk from OTA contamination. Consequently, this work offers a quick alternative to conventional methods of OTA quantification and mycological detection and quantification of A. carbonarius in grapes.