Biodiversity of Aspergillus section Flavi in Europe in relation to the management of aflatoxin risk
Aflatoxins and the producing fungi Aspergillus section Flavi are widely known as the most serious and dangerous mycotoxin issue in agricultural products. In Europe, before the outbreak of aflatoxins on maize (2003–2004) due to new climatic conditions, their contamination was confined to imported foods. Little information is available on molecular biodiversity and population structure of Aspergillus section Flavi in Europe. Preliminary reports evidenced the massive presence of Aspergillus flavus L -morphotype as the predominant species in maize field, no evidence of the highly toxigenic S-morphotype and of other aflatoxigenic species are reported. The risk of a shift in traditional occurrence areas for aflatoxins is expected in the world and in particular in South East of Europe due to the increasing average temperatures. Biological control of aflatoxin risk in the field by atoxigenic strains of A. flavus starts to be widely used in Africa and USA. Studies are necessary on the variation of aflatoxin production in populations of A. flavus to characterize stable atoxigenic A. flavus strains. The aim of present article is to give an overview on biodiversity and genetic variation of Aspergillus section Flavi in Europe in relation to the management of aflatoxins risk in the field.
An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1
A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn.
Mathematical approach for sampling plan performance assessment for aflatoxin B1 in pistachios
According to the EU Regulation 165/2010, the level of aflatoxin B1 and total aflatoxins in pistachios must not exceed 8 μg/kg and 10 μg/kg, respectively. Due to the heterogeneous distribution of this contaminant, the performance of sampling plans highly depends on the way they are designed. So, the sampling plan to be used must be assessed thanks to an OC (Operating Characteristic) curve showing both consumer and producer risks. The first risk is the risk of authorizing pistachios for sale while the contamination level is above the threshold; whereas the second risk is the risk of rejecting a lot having a contamination level under the threshold. The method developed the use of early split contamination levels and incidence in individual pistachios to derive OC curves for various sampling plan designs for aflatoxin B1. Contamination levels in individuals were calculated from small sample contamination levels, thus having a negligible probability of containing more than one contaminated individual. Incidence levels of early split pistachios were derived for each harvest day. Indeed, early split nuts are dubious nuts that were considered to contain all the aflatoxin content found in a global sample. As their percentage increased along the harvesting period, it led to various contamination distributions in 10 kg samples. The EU sampling plan (Regulation 178/2010) was found to give a consumer risk with a probability of acceptance at 5% for a lot mean aflatoxin B1 concentration of 75.34 μg/kg and a producer risk with a probability of acceptance at 95% for a lot mean concentration of 1.62 μg/kg. Finally, the performance of several sampling plan designs was evaluated to demonstrate how to manipulate the number of samples to reduce misclassification of pistachio lots.
Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts
Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 105 conidia per reaction with the primer set ID9 for A. nomius and 104 conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 101 and 102 conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61.5% and 66.7% for A. nomius and A. flavus, respectively. When LAMP results were compared with the presence of aflatoxins in corresponding samples, the Negative Predictive Values were 22.2% and 44.4% and the Positive Predictive Values were 52.2% and 78.3% for aflatoxins produced by A. nomius and A. flavus, respectively. The LAMP assays described in this study have been demonstrated to be a specific, sensitive and easy to use tool for the survey of Brazil nuts for contaminations with potential aflatoxin-producing A. nomius and A. flavus in low tech environments where resources may be limited.
Patulin & ergosterol: New quality parameters together with aflatoxins in hazelnuts
Hazelnuts of three different categories, mouldy, hidden mould and sound (undamaged), were investigated for their contents of aflatoxins (B1, B2, G1 and G2), patulin, and ergosterol. Samples were obtained from five hazelnut processing plants located in a major hazelnut producing area in the Black Sea region in Turkey. All aflatoxins, patulin and ergosterol were determined by high performance liquid chromatography (HPLC). Sound hazelnuts were contaminated with trace or zero amounts of aflatoxins, patulin and ergosterol, so they posed no risk for the consumer when national and/or international regulatory limits were considered. Mouldy and hidden mould hazelnuts were contaminated with high (246–510 ppb; 141–422 ppb) aflatoxin levels, respectively. Aflatoxin B1 content was significantly correlated with the patulin and ergosterol contents in mouldy and hidden mould hazelnuts. However, there was no significant correlation between patulin and ergosterol contents of mouldy and hidden mould hazelnuts.
Monoclonal antibody-quantum dots CdTe conjugate-based fluoroimmunoassay for the determination of aflatoxin B1 in peanuts
A fluoroimmunoassay towards aflatoxin B1 (AFB1) was presented using quantum dots as the fluorescent label. The CdTe QDs were successfully linked to the monoclonal antibody against AFB1. Based on the conjugated complexes, a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) was developed for AFB1 detection. The 50% inhibition value (IC50) of the cFLISA was 0.149 ng/mL in peanuts matrix. The method performance included the limit of detection (LOD) of 0.016 ng/mL and considerable recoveries of 85-117% at three fortification levels (0.075, 0.15, and 0.3 ng/g) from spiked AFB1 blank peanuts samples, along with coefficients of variation (CVs) below 10%. The cFLISA provided an alternative of rapid and sensitive detection for AFB1 and, moreover provided great potential for multiplexed mycotoxins determination simultaneously.
Effect of ozone on aflatoxins detoxification and nutritional quality of peanuts
Aflatoxins are a group of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus with carcinogenicity, teratogenicity, and mutagenicity. Aflatoxins may be found in a wide range of agri-products, especially in grains, oilseeds, corns, and peanuts. In this study, the conditions for detoxifying peanuts by ozonation were optimized. Aflatoxins in peanuts at moisture content of 5% (w/w) were sensitive to ozone and easily degraded when reacted with 6.0 mg/l of ozone for 30 minutes at room temperature. The detoxification rates of the total aflatoxins and aflatoxin B1 (AFB1) were 65.8% and 65.9%, respectively. The quality of peanut samples was also evaluated in this research. No significant differences (P > 0.05) were found in the polyphenols, resveratrol, acid value (AV), and peroxide value (PV) between treated and untreated samples. The results suggested that ozonation was a promising method for aflatoxin detoxification in peanuts.