Annalisa De Girolamo

Screening and Initial Binding Assessment of Fumonisin B1 Aptamers

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column.

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300μl oligosorbent (24nmol DNA) showed a linear (r=0.999) behaviour in the range of 0.4-500ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5-50ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r=0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.

Effect of processing on fumonisin concentration in corn flakes.

The stability of fumonisin B1 and fumonisin B2 during processing of corn flakes was investigated with three different methods for analysis of the naturally contaminated raw material (corn flour), intermediate product (extruded, but not roasted corn flakes), and final product (roasted corn flakes). Only one method, using immunoaffinity column clean-up, provided reliable results in the determination of fumonisins in corn flake samples at the intermediate and final steps of processing. About 60 to 70% of the initial amount of fumonisins were lost during the entire cycle of corn flake processing, with less than 30% losses occurring during the intermediate extrusion step (70 to 170 degrees C for 2 to 5 min). The effect of different additives commonly present in commercial products (sodium chloride, sucrose, and ferrous sulfate heptahydrate) on the reliability of fumonisin analysis has also been investigated. The presence of sodium chloride strongly reduced fumonisin recovery when strong anion-exchange (SAX) columns were used for the clean-up step, whereas the other additives appeared to have little or no effect on the accuracy of fumonisin analysis. The use of reliable analytical methods that are effective for both raw materials and processed products is of paramount relevance for studying the effect of food processing on mycotoxin-contaminated commodities. Despite the fact that some effective fumonisin decontamination occurring during corn flake processing has been shown, more work is needed to identify the thermal breakdown products of fumonisins and their relevant toxicity.

Determination of fumonisins B1 and B2 in cornflakes by high performance liquid chromatography and immunoaffinity clean-up.

The determination of fumonisins in cornflakes is a challenging matter as the actually available methods for the analysis of corn do not perform well when applied to this more complex matrix. After testing several factors that may affect the analytical performance, an accurate method for the determination of fumonisin B1 (FB1) and B2 (FB2) in cornflakes has been developed. The method uses immunoaffinity chromatography for clean-up and high performance liquid chromatography (HPLC) for quantification of the toxins. Samples were extracted twice with acetonitrile-methanol-water (25:25:50) and the combined extracts were diluted with phosphate buffered saline (PBS) and applied to a FumoniTest™ immunoaffinity column. After washing with PBS, fumonisins were eluted from the column with methanol and reacted with o-phthaldialdehyde/2-mercaptoethanol to form fluorescent derivatives. Fumonisin derivatives were analysed by reversed phase HPLC with fluorometric detection using methanol-0.1 M phosphate buffer (77:23; pH adjusted at 3.35) as mobile phase. The average recoveries for FB1 and FB2 spiked in the ranges of 0.33–2.80 μg/g and 0.17–1.40 μg/g were 102.6% and 95.1%, respectively, with average relative standard deviations of 9% and 8%. The limit of quantiWcation for FB1 and FB2 was 0.005 μg/g based on a signal-to-noise ratio of 6:1 by using a sensitive Xuorescence detector. The method was used to analyse 18 cornflakes and cornflake cereals samples for FB1 and FB2 contamination. All but one sample were found to be contaminated, with maximum FB1 and FB2 concentrations of 1.092 μg/g and 0.235 μg/g, respectively. Mean FB1 and FB2 concentrations were 0.157 μg/g and 0.036 μg/g, respectively.

LC–MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.

Comparison of different extraction and clean-up procedures for the determination of fumonisins in maize and maize-based food products.

In order to optimize the analytical method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in different maize products, five materials (maize flour, cornflakes, extruded maize, muffins and infant formula) were investigated under a variety of experimental conditions organized in a ruggedness test according to a factorial design. The influence of five factors (extraction solvent, extraction mode, volume of extraction solvent, test sample size and clean-up) on method performances was tested by four laboratories using spiked materials (0.5 μg/g and 1.5 μg/g FB1 + FB2) and naturally contaminated materials (ca 1.5 μg/g with FB1 + FB2). The end determination step was performed by high-performance liquid chromatography analysis of the o-phthaldialdehyde derivatized extracts. The ruggedness test permitted identification of two critical factors in the analysis of fumonisins in the above products, namely ‘extraction solvent’ and ‘cleanup procedure’. In particular, the use of acetonitrile (ACN)-water (1 + 1, v + v) as extraction solvent and immunoaffinity column for clean-up provided better recovery of fumonisins and chromatographic resolution as compared with methanol (MeOH)-water (3 + 1, v + v) and strong anion exchange (SAX), respectively. However, phase separation occurring after extraction with ACN-water may have given incorrect results. Based on the information obtained with the present study it was possible to develop a new method horizontally applicable to all the above mentioned maize-based food matrices.

Comprehensive Analytical Comparison of Strategies Used for Small Molecule Aptamer Evaluation

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Effect of quercetin and umbelliferone on the transcript level of Penicillium expansum genes involved in patulin biosynthesis

Penicillium expansum is commonly associated with patulin accumulation in pome fruits. In in vitro studies, two phenolic compounds (quercetin and umbelliferone) proved to be effective in reducing patulin accumulation, particularly when applied in combination, without consistently affecting mycelial growth. To investigate the mode of action of quercetin and umbelliferone, the expression of five genes likely involved in patulin biosynthesis was evaluated using real-time PCR in the presence and absence of the tested phenolic compounds. The relative expression of genes coding isoepoxydon dehydrogenase (IDH), 6-methylsalicylic acid synthase (msas) and an ATP-binding cassette transporter (peab1) proved to be down-regulated when quercetin and umbelliferone were added in combination. Furthermore, the relative expression of two putative cytochrome P450 monooxygenases (p450-1 and p450-2) was reduced by all treatments, although the combination of the two substances was the most effective. These results provide evidence that quercetin and umbelliferone reduce patulin accumulation by acting on the transcription level of the tested genes.

Occurrence of ochratoxin A, fumonisin B2 and black aspergilli in raisins from Western Greece regions in relation to environmental and geographical factors

The presence of ochratoxin A (OTA), fumonisin B2 (FB2) and black aspergilli in raisins from Western Greece regions (Messinia, Corinthia, Achaia, Ilia and Zante Island) was investigated in relation to the different geographic and climatic conditions in the 2011 growing season. The biseriate species Aspergillus niger “aggregate” and A. carbonarius were mainly identified. The population of A. niger “aggregate” species occurred in all raisin samples at colony-forming units (CFU) concentrations significantly higher (mean 2.2 × 105 CFU g−1 homogenate) than those of A. carbonarius population (mean 4.9 × 103 CFU g−1 homogenate), which occurred in 80% of the raisin samples. OTA was found in 73% of the samples at levels ranging from 0.1 µg kg−1 to 98.2 µg kg−1, with the highest level occurring in a raisin sample from Ilia that also contained the highest level of A. carbonarius. The European Union legal limit for OTA was exceeded in 15% of the raisin samples. FB2 was found in 29% of the raisin samples at levels ranging from 7.1 µg kg−1 to 25.5 µg kg−1, with 20% of the samples co-occurring with OTA. Principal-component analysis was applied to levels of mycotoxins, fungal contamination, geographical data and environmental conditions recorded in the harvesting (August) or drying (September) period. Principal-component analysis clearly indicated a good direct correlation of rainfall and relative humidity with OTA and A. carbonarius contamination. A lack of clustering was observed when A. niger and FB2 contamination were considered. This is the first report on the co-occurrence of the mycotoxins OTA and FB2 in dried vine fruits from Greece.

Rapid Analysis of Deoxynivalenol in Durum Wheat by FT-NIR Spectroscopy

Fourier-transform-near infrared (FT-NIR) spectroscopy has been used to develop quantitative and classification models for the prediction of deoxynivalenol (DON) levels in durum wheat samples. Partial least-squares (PLS) regression analysis was used to determine DON in wheat samples in the range of <50–16,000 µg/kg DON. The model displayed a large root mean square error of prediction value (1,977 µg/kg) as compared to the EU maximum limit for DON in unprocessed durum wheat (i.e., 1,750 µg/kg), thus making the PLS approach unsuitable for quantitative prediction of DON in durum wheat. Linear discriminant analysis (LDA) was successfully used to differentiate wheat samples based on their DON content. A first approach used LDA to group wheat samples into three classes: A (DON ≤ 1,000 µg/kg), B (1,000 < DON ≤ 2,500 µg/kg), and C (DON > 2,500 µg/kg) (LDA I). A second approach was used to discriminate highly contaminated wheat samples based on three different cut-off limits, namely 1,000 (LDA II), 1,200 (LDA III) and 1,400 µg/kg DON (LDA IV). The overall classification and false compliant rates for the three models were 75%–90% and 3%–7%, respectively, with model LDA IV using a cut-off of 1,400 µg/kg fulfilling the requirement of the European official guidelines for screening methods. These findings confirmed the suitability of FT-NIR to screen a large number of wheat samples for DON contamination and to verify the compliance with EU regulation.