Rapid profiling of antimicrobial compounds characterising B. subtilis TR50 cell-free filtrate by high-performance liquid chromatography coupled to high-resolution Orbitrap™ mass spectrometry.

RATIONALE: Several Bacillus strains, typically isolated from different food sources, represent renowned producers of a multitude of low and high molecular weight compounds, including lipopeptides and macrolactones, with an importance for their antimicrobial activity. The high homology shared by many of these compounds also occurring as closely related isoforms poses a challenge in their prompt detection. METHODS: Identification and structural elucidation is generally achieved by matrix-assisted laser desorption/ionization (MALDI) or liquid chromatography (LC) coupled to mass spectrometry (MS) after a pre-fractionation and/or purification step of the extract. In this paper we report the application of a method based on LC separation and high-resolution Orbitrap™-based MS for the rapid screening of raw filtrate of the strain Bacillus subtilis TR50 endowed with antimicrobial activity, without requiring any sample pre-treatment. RESULTS: Upon direct analysis of the cell-free filtrate of Bacillus subtilis TR50 by high-resolution mass spectrometry (HRMS), different compounds families, that proved to exert a remarked antimicrobial activity against several foodborne pathogens, can be readily displayed along the chromatographic run. Among them, three different classes were identified and characterized belonging to the iturin, fengycin and surfactin groups. The high resolving power and accurate mass accuracy provided by the HRMS system in use ensured an enhanced selectivity compared to other mass spectrometers. In addition, after activation of the HCD cell, the HR-MS/MS spectra can provide insights in the structural elucidation of several compounds. CONCLUSIONS: The acquisition of HRMS spectra of raw filtrates of subtilis strains allows untargeted analysis of the major classes of compounds produced to be performed, thus facilitating identification of other unknown bioactive molecules after retrospective analysis. These features make this approach a fast tool applicable to the rapid screening and further identification of antimicrobial compounds released by Bacillus strains in raw filtrates.

Rapid and label-free detection of egg allergen traces in wines by surface plasmon resonance biosensor.

The development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg-related fining allergens in wines is herein described. Ovalbumin (OVA) was chosen as the target protein to be monitored due to its highest abundance in the egg white powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. With the aim of optimizing the assay conditions, different parameters able to influence the final biosensor response were carefully investigated (i.e., pH, ionic strength, and additional surfactant concentration). After the fine tuning of these parameters, the assay was tested in the direct analysis of OVA in commercial wines artificially contaminated with egg white powder at different concentration levels in order to assess the reliability of the biosensor in detecting traces of OVA in complex matrices. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment required, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 µg/mL. Finally, the response provided by the developed biosensor was correlated with an established liquid chromatography mass spectrometry (LC-MS) method developed in our laboratories, and performances of both approaches were assessed for the fast monitoring of egg allergen contamination in fined wines.

Assessment of toxic potential of mycotoxin contaminated bread during in vitro human digestion on human B lymphoid cell line.

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.

Pepsin-digested bovine lactoferrin prevents Mozzarella cheese blue discoloration caused by Pseudomonas fluorescens.

The aim of this work was to check the efficacy of bovine lactoferrin hydrolyzed by pepsin (LFH) to prevent blue discoloration of Mozzarella cheese delaying the growth of the related spoilage bacteria. Among 64 Pseudomonas fluorescens strains, isolated from 105 Mozzarella samples, only ten developed blue discoloration in cold-stored Mozzarella cheese slices. When Mozzarella cheese samples from dairy were treated with LFH and inoculated with a selected P. fluorescens strain, no pigmentation and changes in casein profiles were found up to 14 days of cold storage. In addition, starting from day 5, the count of P. fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. ESI-Orbitrap-based mass spectrometry analyses allowed to reveal the pigment leucoindigoidine only in the blue LFH-free cheese samples indicating that this compound could be considered a chemical marker of this alteration. For the first time, an innovative mild approach, based on the antimicrobial activity of milk protein hydrolysates, for counteracting blue Mozzarella event and controlling psychrotrophic pigmenting pseudomonads, is here reported.

Orbitrap™ monostage MS versus hybrid linear ion trap MS: application to multi-allergen screening in wine.

Food allergen research has made giant steps in the last years thanks to the features offered by the latest technology of mass analyzers placed on the market allowing multiplex sensitive detection of proteins. Potentials and features of two mass analyzers namely a linear ion trap capable of performing a data dependent or selected reaction monitoring analysis and an Orbitrap(TM) stand-alone MS enabling a broadband fragmentation without mass selection at highest mass resolving power are herein described and applied to the multiplex screening of allergens in a type of wine chosen as a reference matrix. Quantitative and confirmative capabilities of both platforms were assessed on the specific case study, the multiple detection of egg and milk -related proteins, typically employed in white wines as fining agents. Commercial bioinformatic tools used for a quick allergen identification will be also discussed.

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.

Multi-allergen detection in food by micro high-performance liquid chromatography coupled to a dual cell linear ion trap mass spectrometry.

There is a raising demand for sensitive and high throughput MS based methods for screening purposes especially tailored to the detection of allergen contaminants in different food commodities. A challenging issue is represented by complex food matrices where the antibody-based kits commercially available might encounter objective limitations consequently to epitope masking phenomena due to a multitude of interfering compounds arising from the matrix. The performance of a method duly optimized for the extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multi-target SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent™ Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1µg/g for milk to 0.3µg/g for egg and 2µg/g for soy.

Improved method for the simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in cereals and derived products by liquid chromatography-tandem mass spectrometry after multi-toxin immunoaffinity clean up.

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1µg/kg to 30µg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins.

Use of liquid chromatography-high-resolution mass spectrometry for isolation and characterization of hydrolyzed fumonisins and relevant analysis in maize-based products.

The synthesis of partially hydrolyzed fumonisins (PHFB1 and PHFB2) and hydrolyzed fumonisins (HFB1 and HFB2) by chemical hydrolysis of pure fumonisins (FB1 and FB2) is reported together with the isolation and characterization by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Two structural isomers of partially hydrolyzed forms of FB1 and FB2 were identified, namely PHFB(1a) and PHFB(1b) and PHFB(2a) and PHFB(2b). Reaction yields were 21% for PHFB1 (sum of the two isomers), 52% for HFB1, 31% for PHFB2 (sum of the two isomers) and 30% for HFB2. Purity of each isolated compound was >98%. An LC-HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzed derivatives was applied to 24 naturally contaminated samples of maize and maize-based products. The majority of samples (18 out of 24) were contaminated with fumonisins B1 and B2. Fumonisins co-occurred with both partially hydrolyzed and hydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co-occurrence of fumonisins with partially hydrolyzed fumonisins was also recorded in one sample of maize kernels and four samples of maize-based products (i.e. maize meal, cous-cous, corn-cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 µg/kg for fumonisins (sum of FB1 and FB2), from 10 to 210 µg/kg for partially hydrolyzed fumonisins (sum of PHFB1 and PHFB2) and from 30 to 200 µg/kg for hydrolyzed fumonisins (sum of HFB1 and HFB2). This is the first report of the isolation of PHFB2 and the co-occurrence of FB1, FB2, PHFB1, PHFB2, HFB1 and HFB2 in maize products. Considering the growing use of nixtamalized and maize-based products, the monitoring of fumonisins and their partially and totally hydrolyzed forms in these products may represent an important contributing factor in evaluating the relevant human risk exposure.

Toxic mechanisms induced by fumonisin b1 mycotoxin on human intestinal cell line.

The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 µM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 µM, followed by inhibition of cell proliferation (up to 8.6 µM), the immunomodulatory effect (up to 17.2 µM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 µM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.

Assessment of multi-mycotoxin exposure in southern Italy by urinary multi-biomarker determination.

Human exposure assessment to deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) can be performed by measuring their urinary biomarkers. Suitable biomarkers of exposure for these mycotoxins are DON + de-epoxydeoxynivalenol (DOM-1), aflatoxin M1 (AFM1), FB1, ZEA + α-zearalenol (α-ZOL) + ß-zearalenol (ß-ZOL) and OTA, respectively. An UPLC-MS/MS multi-biomarker method was used to detect and measure incidence and levels of these biomarkers in urine samples of 52 volunteers resident in Apulia region in Southern Italy. The presence of ZEA + ZOLs, OTA, DON, FB1 and AFM1 were detected in 100%, 100%, 96%, 56% and 6%, of samples, respectively. All samples contained biomarkers of two or more mycotoxins. The mean concentrations of biomarkers ranged from 0.055 ng/mL (FB1) to 11.89 ng/mL (DON). Urinary biomarker concentrations were used to estimate human exposure to multiple mycotoxin. For OTA and DON, 94% and 40% of volunteers, respectively exceeded the tolerable daily intake (TDI) for these mycotoxins. The estimated human exposure to FB1 and ZEA was largely below the TDI for these mycotoxins for all volunteers.

Assessment of multi-mycotoxin adsorption efficacy of grape pomace.

Grape pomace (pulp and skins) was investigated as a new biosorbent for removing mycotoxins from liquid media. In vitro adsorption experiments showed that the pomace obtained from Primitivo grapes is able to sequester rapidly and simultaneously different mycotoxins. Aflatoxin B1 (AFB1) was the most adsorbed mycotoxin followed by zearalenone (ZEA), ochratoxin A (OTA), and fumonisin B1 (FB1), whereas the adsorption of deoxynivalenol (DON) was negligible. AFB1 and ZEA adsorptions were not affected by changing pH values in the pH 3-8 range, whereas OTA and FB1 adsorptions were significantly affected by pH. Equilibrium adsorption isotherms obtained at different temperatures (5-70 °C) and pH values (3 and 7) were modeled and evaluated using the Freundlich, Langmuir, Sips, and Hill models. The goodness of the fits and the parameters involved in the adsorption mechanism were calculated by the nonlinear regression analysis method. The best-fitting models to describe AFB1, ZEA, and OTA adsorption by grape pomace were the Sips, Langmuir, and Freundlich models, respectively. The Langmuir and Sips models were the best models for FB1 adsorption at pH 7 and 3, respectively. The theoretical maximum adsorption capacities (mmol/kg dried pomace) calculated at pH 7 and 3 decreased in the following order: AFB1 (15.0 and 15.1) > ZEA (8.6 and 8.3) > OTA (6.3-6.9) > FB1 (2.2 and 0.4). Single- and multi-mycotoxin adsorption isotherms showed that toxin adsorption is not affected by the simultaneous presence of different mycotoxins in the liquid medium. The profiles of adsorption isotherms obtained at different temperatures and pH and the thermodynamic parameters (ΔG°, ΔH°, ΔS°) suggest that mycotoxin adsorption is an exothermic and spontaneous process, which involves physisorption weak associations. Hydrophobic interactions may be associated with AFB1 and ZEA adsorption, whereas polar noncovalent interactions may be associated with OTA and FB1 adsorption. In conclusion, this study suggests that biosorption of mycotoxins onto grape pomace may be a reasonably low-cost decontamination method.

Assessment of Mycotoxin Exposure in Côte d'ivoire (Ivory Coast) Through Multi-Biomarker Analysis and Possible Correlation with Food Consumption Patterns.

SCOPE: The aim of the presented study was to investigate the mycotoxin exposure of Ivorian population related to the consumption patterns of maize, peanuts, millet, and cassava product (attiéké). MATERIALS AND METHODS: Maize flour samples (n = 51) were purchased from all Abidjan local markets, in the south of Ivory Coast, and urine (n = 99) was collected during the same reference period (July-September 2011) from volunteers living in Abidjan and Daloa cities. Reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) was used to analyze aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), fumonisins (FB1, FB2), deoxynivalenol (DON), zearalenone (ZEA), and T-2 and HT-2 toxins in maize flour samples, and their relevant biomarkers (AFM1, DON, DON + de-epoxydeoxynivalenol (DOM-1), FB1, α-zearalenol (ZOL), ß-ZOL, and OTA) in urine samples. RESULTS: Critical maize contamination was observed by AFs occurrence (total AFs 4.5 - 330.0 µg/kg) while OTA was found in 13% of samples analyzed. AFM1 was detected in 40% of urines samples (0.06 - 14.11 ng/ml), OTA in 37% (0.01 - 0.42 ng/ml), FB1 in 27% (0.07 to 15.31 ng/ml) and, DON was found in 21% of samples at levels up to 10.0 ng/ml. The correlation coefficients (R(2)) obtained by plotting the percentage of biomarker occurrence (positive samples) versus the frequency of food consumption revealed maize, peanuts, millet and attiéké were strongly linked to AFB1 and OTA exposure with values of R(2) ranged from 0.462 to 0.956. CONCLUSION: The present study provided data on mycotoxin risk in Ivory Coast, revealing a frequent co-exposure to the major mycotoxins such as AFs, OTA, and fumonisins, which appeared to be related to the frequency of peanuts, maize, millet and attiéké consumption.

Mycotoxin profile of Fusarium langsethiae isolated from wheat in Italy: production of type-A trichothecenes and relevant glucosyl derivatives.

Fusarium langsethiae, formally described as a new species over a decade ago, has been identified as the main producer of HT-2 (HT2) and T-2 (T2) toxins in Europe in small cereal grains. Mycotoxin contamination caused by this Fusarium species can represent a food safety hazard that deserves further attention. In the present work, the mycotoxin profile in wheat cultures of F. langsethiae is presented with particular reference to the production of major type-A trichothecenes and their glucosyl derivatives. F. langsethiae isolates, representative of the major Italian wheat cultivation areas, were tested for the production of T2, HT2, diacetoxyscirpenol (DAS) and neosolaniol (NEO), and relevant glucosyl derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiae isolates under investigation resulted to be potent producers of T2, HT2 and NEO. Furthermore, a well-defined set of isolates, all originating from Central Italy, produced also DAS. All isolates were found to be able to produce HT2 glucosyl derivatives, whereas only traces of T2 glucoside were detected in one sample. Furthermore, two mono-glucosyl derivatives of NEO and one mono-glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylated trichothecenes in analyzed fungal extracts revealed a general co-occurrence of these derivatives with the parent toxin at levels that could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the production of glucosylated trichothecenes by F. langsethiae cultured on small grains.

Multi-allergen quantification of fining-related egg and milk proteins in white wines by high-resolution mass spectrometry.

RATIONALE: A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin. METHODS: The method was based on the combination of ultrafiltration (UF) of protein-containing wines, tryptic digestion of the dialyzed wine extracts and liquid chromatography/high resolution mass spectrometry (LC/HRMS) analysis of tryptic digests. Peptides providing the most intense electrospray ionization (ESI)-MS response were chosen as quantitative markers of the proteins under investigation. RESULTS: Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL. CONCLUSIONS: The developed method is suited for assessing the contemporary presence of allergenic milk and egg proteins characterizing egg white and caseinate, fining agents typically employed for wine clarification. The LODs of the method enable the detection of sub-ppm concentrations of residual fining agents, that could represent a potential risk for allergic consumers.

Mycological analysis and multimycotoxins in maize from rural subsistence farmers in the former Transkei, South Africa.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 µg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 µg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 µg/kg in good maize, respectively, and of 14 and 5.8 µg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 µg/kg in good maize, respectively, and of 111 and 135 µg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

Multiple mycotoxin exposure determined by urinary biomarkers in rural subsistence farmers in the former Transkei, South Africa.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean±standard deviation 0.342±0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4±49.4 ng/mg creatinine) after hydrolysis with ß-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with ß-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529±1.60 ng/mg creatinine), FB1 (96%; 1.52±2.17 ng/mg creatinine), α-zearalenol (92%; 0.614±1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3±27.1 ng/mg creatinine), ß-zearalenol (75%; 0.702±2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041±0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

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 × 10(5) CFU g(-1) homogenate) than those of A. carbonarius population (mean 4.9 × 10(3) 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.

Experimental design for in-house validation of a screening immunoassay kit. The case of a multiplex dipstick for Fusarium mycotoxins in cereals.

The benefits of using rapid qualitative methods to verify compliance of food and feed with legislation requirements include user-friendly format, the possibility of detection without expensive instrumentation, rapid response and affordable price. Prior to their use, however, the methods have to pass validation experiments, in order to assess their performance profile. An experimental protocol for in-house validation of a screening immunoassay has been designed and applied to evaluate performance characteristics of a multiplex dipstick kit for the determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat and maize. The test is intended for screening of cereals on the presence/absence of these mycotoxins at maximum permitted levels established by European legislation or target levels. The response of the measurement is determined with a reader device. Samples classified as negative are considered as compliant, whereas positive samples need to be re-analysed with confirmatory methods. The in-house validation design consisted of three steps, namely (1) estimating the precision of the method including "between day" effects and influences from different varieties of the matrices, (2) establishing robust cutoff values for the dipstick response at target mycotoxin levels assuming an acceptable rate of false negative results of 5% and (3) assessment of the rate of false positive results of blank samples and samples containing the target analytes below the legal limits. The total precision expressed as relative standard deviation and determined individually for each analyte/concentration/matrix combination varied from 9 to 30% and was considered as acceptable. In 17 out of 28 cases, the repeatability standard deviation was the most important factor. The predominance of the repeatability compared to the other factors (matrix, days) was an indicator for the ruggedness of the assay. The validation study demonstrated that the test was able to differentiate blank samples from samples contaminated at target mycotoxin levels with a false positive rate lower than 6%. Considering realistic mycotoxin occurrence in European samples, significant economical benefits can be expected when using the test under real-world conditions.

Fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside conjugates from flour to bread: an investigation by high-performance liquid chromatography high-resolution mass spectrometry.

Deoxynivalenol, T-2 and HT-2 toxins are mycotoxins frequently occurring in cereals and cereal-based products along with their conjugated forms. In this paper, we provide insights into the fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside derivatives during bread making, using naturally contaminated wheat flour. High-resolution mass spectrometry was used to assess the extent of degradation of the three mycotoxins during bread baking and to identify some glucoside conjugates, namely deoxynivalenol, T-2 and HT-2 mono-glucosides, detected both in the flour and in the respective breads. Our findings show deoxynivalenol's levels markedly increased upon baking, whereas those of HT-2 and T-2 toxins were decreased in the final bread with special regard to the T-2 toxin.