Vincenzo Lippolis

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals.

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Rapid and non-invasive analysis of deoxynivalenol in durum and common wheat by Fourier-Transform Near Infrared (FT-NIR) spectroscopy

Fourier transform near-infrared spectroscopy (FT-NIR) was used for rapid and non-invasive analysis of deoxynivalenol (DON) in durum and common wheat. The relevance of using ground wheat samples with a homogeneous particle size distribution to minimize measurement variations and avoid DON segregation among particles of different sizes was established. Calibration models for durum wheat, common wheat and durum + common wheat samples, with particle size <500 µm, were obtained by using partial least squares (PLS) regression with an external validation technique. Values of root mean square error of prediction (RMSEP, 306–379 µg kg–1) were comparable and not too far from values of root mean square error of cross-validation (RMSECV, 470–555 µg kg–1). Coefficients of determination (r 2) indicated an “approximate to good” level of prediction of the DON content by FT-NIR spectroscopy in the PLS calibration models (r 2 = 0.71–0.83), and a “good” discrimination between low and high DON contents in the PLS validation models (r 2 = 0.58–0.63). A “limited to good” practical utility of the models was ascertained by range error ratio (RER) values higher than 6. A qualitative model, based on 197 calibration samples, was developed to discriminate between blank and naturally contaminated wheat samples by setting a cut-off at 300 µg kg–1 DON to separate the two classes. The model correctly classified 69% of the 65 validation samples with most misclassified samples (16 of 20) showing DON contamination levels quite close to the cut-off level. These findings suggest that FT-NIR analysis is suitable for the determination of DON in unprocessed wheat at levels far below the maximum permitted limits set by the European Commission.

Optimization of a Fluorescence Polarization Immunoassay for Rapid Quantification of Deoxynivalenol in Durum Wheat-Based Products

A fluorescence polarization immunoassay previously described for deoxynivalenol (DON) screening in wheat was optimized for the rapid quantification of DON in durum wheat kernels, semolina, and pasta. A background signal was observed in both spiked and naturally contaminated samples, strictly depending on the testing matrix. After subtracting the background DON level for durum wheat (0.27 microg of DON per g), semolina (0.08 microg of DON per g), and pasta (0.04 microg of DON per g), an accurate quantification of DON was possible at levels greater than 0.10 microg/g for all matrices. Average recoveries from spiked samples (0.25 to 1.75 microg/g) were 98, 102, and 101% for wheat, semolina, and pasta, respectively. Comparative analyses of 35 naturally contaminated durum wheat samples, 22 semolina samples, and 26 pasta samples performed by both the fluorescence polarization method and high-pressure liquid chromatography/immunoaffinity cleanup showed a good correlation (r > 0.995). The fluorescence polarization method showed better accuracy and precision with respect to the high-pressure liquid chromatography method and is suitable for the rapid and quantitative determination of DON in durum wheat-based products at levels foreseen by existing or coming international regulations.

Improvement of detection sensitivity of T-2 and HT-2 toxins using different fluorescent labeling reagents by high-performance liquid chromatography.

T-2 and HT-2 toxins are Fusarium mycotoxins that can occur in cereals and cereal-based products. Three fluorescent labeling reagents, i.e. 1-naphthoyl chloride (1-NC), 2-naphthoyl chloride (2-NC) and pyrene-1-carbonyl cyanide (PCC), were used for the determination of T-2 and HT-2 toxins by high-performance liquid chromatography (HPLC) with fluorescence detection (FD). Pre-column derivatization of T-2 and HT-2 toxins was carried out under mild conditions (50 degrees C, 10 min) in toluene with 4-dimethylaminopyridine (DMAP) as catalyst. All fluorescent derivatives were identified and characterized by HPLC-tandem mass spectrometry (HPLC-MS/MS). Optimal stoichiometric ratios (toxin:derivatizing reagent:catalyst), linear range and repeatability of the reaction, stability and sensitivity of the derivatives were determined. A wide linear range (10-1000 ng of either derivatized T-2 or HT-2 toxin), good stability (up to 2 weeks at -20 degrees C or 5 days at room temperature) of the fluorescent derivatives and good repeatability of the reaction (RSD</=8%) were observed. Detection limits (based on a signal-to-noise ratio of 3:1) were 10.0, 6.3 and 2.0 ng for derivatized T-2 toxin and 6.3, 2.3 and 2.8 ng for derivatized HT-2 toxin with 1-NC, 2-NC and PCC, respectively. In terms of sensitivity and repeatability, PCC and 2-NC reagents showed better performance than 1-anthroylnitrile (1-AN), a previously reported labeling reagent for T-2- and HT-2 toxins. Preliminary studies also showed the applicability of PCC and 2-NC as fluorescent labeling reagents for the simultaneous determination of T-2 and HT-2 toxins in cereal grains by HPLC/FD following immunoaffinity column clean-up.

Rapid prediction of ochratoxin A-producing strains of Penicillium on dry-cured meat by MOS-based electronic nose

The availability of rapid diagnostic methods for monitoring ochratoxigenic species during the seasoning processes for dry-cured meats is crucial and constitutes a key stage in order to prevent the risk of ochratoxin A (OTA) contamination. A rapid, easy-to-perform and non-invasive method using an electronic nose (e-nose) based on metal oxide semiconductors (MOS) was developed to discriminate dry-cured meat samples in two classes based on the fungal contamination: class P (samples contaminated by OTA-producing Penicillium strains) and class NP (samples contaminated by OTA non-producing Penicillium strains). Two OTA-producing strains of Penicillium nordicum and two OTA non-producing strains of Penicillium nalgiovense and Penicillium salamii, were tested. The feasibility of this approach was initially evaluated by e-nose analysis of 480 samples of both Yeast extract sucrose (YES) and meat-based agar media inoculated with the tested Penicillium strains and incubated up to 14 days. The high recognition percentages (higher than 82%) obtained by Discriminant Function Analysis (DFA), either in calibration and cross-validation (leave-more-out approach), for both YES and meat-based samples demonstrated the validity of the used approach. The e-nose method was subsequently developed and validated for the analysis of dry-cured meat samples. A total of 240 e-nose analyses were carried out using inoculated sausages, seasoned by a laboratory-scale process and sampled at 5, 7, 10 and 14 days. DFA provided calibration models that permitted discrimination of dry-cured meat samples after only 5 days of seasoning with mean recognition percentages in calibration and cross-validation of 98 and 88%, respectively. A further validation of the developed e-nose method was performed using 60 dry-cured meat samples produced by an industrial-scale seasoning process showing a total recognition percentage of 73%. The pattern of volatile compounds of dry-cured meat samples was identified and characterized by a developed HS-SPME/GC-MS method. Seven volatile compounds (2-methyl-1-butanol, octane, 1R-α-pinene, d-limonene, undecane, tetradecanal, 9-(Z)-octadecenoic acid methyl ester) allowed discrimination between dry-cured meat samples of classes P and NP. These results demonstrate that MOS-based electronic nose can be a useful tool for a rapid screening in preventing OTA contamination in the cured meat supply chain.

Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days.

Rapid screening of wheat bran contaminated by deoxynivalenol mycotoxin using Raman spectroscopy: a preliminary experiment

Deoxynivalenol (DON) is a mycotoxin frequently occurring in cereals and derived products, and regulated in many countries. Raman spectroscopy performed using optical fibers, with excitation at 1064 nm and a dispersive detection scheme, was utilized to analyze wheat bran samples naturally contaminated with DON. A multivariate processing of the spectroscopic data allowed to distinguish two classes of contamination, with DON below and above 400 μg/kg, respectively. Only one highly contaminated sample was misclassified. This preliminary result demonstrates the potential of Raman spectroscopy as a useful analytical tool for the non-destructive and rapid analysis of mycotoxins in food.

Interactions between cyclodextrins and fluorescent T-2 and HT-2 toxin derivatives: a physico-chemical study

T-2 and HT-2 toxins are mycotoxins produced by several Fusarium species that are commonly found in various cereal grains, including oats, barley, wheat and maize. Intake estimates indicate that the presence of these mycotoxins in the diet can be of concern for public health. In this work, the inclusion processes occurring between fluorescent anthracene-derivatives of T-2 and HT-2 toxins and different cyclodextrin (CD) molecules were investigated in aqueous solutions by means of UV–Vis absorption, fluorescence emission and dynamic light scattering. Binding constant values and chemico-physical parameters were calculated. It was found that β-CDs give stronger inclusion reactions with both T-2 and HT-2 derivatives, as stated by important emission intensity increments. Such interactions were found to be fundamentally enthalpy-driven. Among β-CDs, the effect of the methylation at hydroxyl groups was tested: as a result, the di-methyl form of β-CD was found to induce the best fluorescence intensity enhancements.

Rapid prediction of deoxynivalenol contamination in wheat bran by MOS-based electronic nose and characterization of the relevant pattern of volatile compounds: Prediction of DON contamination in wheat bran

BACKGROUND Deoxynivalenol (DON) is a mycotoxin, mainly produced by Fusarium sp., most frequently occurring in cereals and cereal-based products. Wheat bran refers to the outer layers of the kernel, which has a high risk of damage due to chemical hazards, including mycotoxins. Rapid methods for DON detection in wheat bran are required. RESULTS A rapid screening method using an electronic nose (e-nose), based on metal oxide semiconductor sensors, has been developed to distinguish wheat bran samples with different levels of DON contamination. A total of 470 naturally contaminated wheat bran samples were analyzed by e-nose analysis. Wheat bran samples were divided in two contamination classes: class A ([DON] ≤ 400 µg kg-1 , 225 samples) and class B ([DON] > 400 µg kg-1 , 245 samples). Discriminant function analysis (DFA) classified wheat bran samples with good mean recognizability in terms of both calibration (92%) and validation (89%). A pattern of 17 volatile compounds of wheat bran samples that were associated (positively or negatively) with DON content was also characterized by HS-SPME/GC-MS. CONCLUSIONS These results indicate that the e-nose method could be a useful tool for high-throughput screening of DON-contaminated wheat bran samples for their classification as acceptable / rejectable at contamination levels close to the EU maximum limit for DON, reducing the number of samples to be analyzed with a confirmatory method.

Discrimination of geographical origin of oranges (Citrus sinensis L. Osbeck) by mass spectrometry-based electronic nose and characterization of volatile compounds

An untargeted method using headspace solid-phase microextraction coupled to electronic nose based on mass spectrometry (HS-SPME/MS-eNose) in combination with chemometrics was developed for the discrimination of oranges of three geographical origins (Italy, South Africa and Spain). Three multivariate statistical models, i.e. PCA/LDA, SELECT/LDA and PLS-DA, were built and relevant performances were compared. Among the tested models, SELECT/LDA provided the highest prediction abilities in cross-validation and external validation with mean values of 97.8% and 95.7%, respectively. Moreover, HS-SPME/GC-MS analysis was used to identify potential markers to distinguish the geographical origin of oranges. Although 28 out of 65 identified VOCs showed a different content in samples belonging to different classes, a pattern of analytes able to discriminate simultaneously samples of three origins was not found. These results indicate that the proposed MS-eNose method in combination with multivariate statistical analysis provided an effective and rapid tool for authentication of the oranges geographical origin.