Lucia Gambacorta

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) + β-zearalenol (β-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.

Biomonitoring of concurrent mycotoxin exposure among adults in Sweden through urinary multi-biomarker analysis.

Mycotoxin producing moulds may contaminate numerous agricultural commodities either before harvest or during storage. A varied diet consisting of different foods may therefore be contaminated with a range of mycotoxins. The aim of the present study was to study concurrent exposure to mycotoxins through urinary multi-biomarker analysis, as well as its possible associations with the diet. Urinary samples from 252 adults, participating in the Swedish national dietary survey Riksmaten 2010-11, were collected together with a 4-day diet record. Concurrent mycotoxin exposure was studied using a multi-biomarker LC-MS/MS method. The results revealed that exposure to mycotoxins is common and concurrent exposure to more than one toxin was found in 69% of the study population. However, when comparing the number of toxins detected with the reported consumption data it was difficult to distinguish food patterns which would indicate an increased risk of exposure to many mycotoxins simultaneously. This is the first study to investigate concurrent mycotoxin exposure and urinary levels of fumonisin B1 (FB1), fumonisin B2 (FB2), nivalenol (NIV), ochratoxin A (OTA), zearalenone (ZEA), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and de-epoxydeoxynivalenol (DOM-1) among adults in Sweden.

Food coloring agents and plant food supplements derived from Vitis vinifera: a new source of human exposure to ochratoxin A.

Grape pomaces are increasingly being used as starting material in the industrial production of plant food supplements (PFS), food coloring, and tartrates, but they are at risk of ochratoxin A (OTA) contamination, a mycotoxin with nephrotoxic and carcinogenic effects. We analyzed 24 commercial PFS and 13 food coloring samples derived from Vitis vinifera, mainly pomaces, using a HPLC-FLD method for OTA determination. OTA was found in 75% of PFS samples and 69% of food coloring samples at levels of <1.16-20.23 μg/kg and <1.16-32.00 μg/kg, respectively. The four commercial leavening agents containing tartrates were found to be negative for OTA. All eight samples collected in two distilleries that use grape pomaces and wine lees to produce tartrates and other byproducts contained OTA at levels of <1.16-240.93 μg/kg. The high incidence of OTA contamination in PFS and food coloring agents derived from V. vinifera suggests that maximum permitted level(s) should be established for this mycotoxin in these products.

Comparison of single and multi-analyte methods based on LC-MS/MS for mycotoxin biomarker determination in human urine

The performances of four LC-MS/MS methodologies for determination of up to eight mycotoxin biomarkers in human urines were compared by involving three laboratories that analysed common urine samples spiked at two levels of each biomarker. Each laboratory received a calibration solution, spiked urines and the corresponding unspiked urine. The two spiking levels for each biomarker were chosen by considering the levels naturally occurring in human urines and the limits of quantification of the LC-MS/MS methodologies used by the participating laboratories. The results of each laboratory were evaluated for their z-score values. The percentage of satisfactory z-scores (| z | < 2) were: 100% for deoxynivalenol, de-epoxy deoxynivalenol, aflatoxin M1, β-zearalenol and zearalenone, 87% for α-zearalenol, 50% for ochratoxin A and 42% for fumonisin B1. Good method performances were obtained for most biomarkers at the levels tested in this study, as demonstrated by the overall percentage of satisfactory z-scores for al...

Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

ABSTRACT Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTA cluster of A. niger. The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius. IMPORTANCE Ochratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius. The encoding gene, AcOTAhal, is contiguous to biosynthetic genes that we have already described (nrps and pks), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius, which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.

Grape Pomace, an Agricultural Byproduct Reducing Mycotoxin Absorption: In Vivo Assessment in Pig Using Urinary Biomarkers

The efficacy of four agricultural byproducts (ABPs) and two commercial binders (CBs) to reduce the gastrointestinal absorption of a mixture of mycotoxins was tested in piglets using urinary mycotoxin biomarkers as indicator of the absorbed mycotoxins. Twenty-eight piglets were administered a bolus contaminated with the mycotoxin mixture containing or not ABP or CB. Twenty-four hour urine was collected and analyzed for mycotoxin biomarkers by using a multiantibody immunoaffinity-based LC-MS/MS method. Each bolus contained 769 μg of fumonisin B1 (FB1), 275 μg of deoxynivalenol (DON), 29 μg of zearalenone (ZEN), 6.5 μg of aflatoxin B1 (AFB1) and 6.6 μg of ochratoxin A (OTA) corresponding to 2.2, 0.8, 0.08, 0.02, and 0.02 μg/g in the daily diet, respectively. The percentage of ABP in each bolus was 50%, whereas for the two CBs the percentages were 5.2 and 17%, corresponding to 2.8, 0.3, and 0.9% in the daily diet, respectively. The reduction of mycotoxin absorption was up to 69 and 54% for ABPs and CBs, respectively. White grape pomace of Malvasia was the most effective material as it reduced significantly (p < 0.05) urinary mycotoxin biomarker of AFB1 (67%) and ZEN (69%), whereas reductions statistically not significant were observed for FB1 (57%), DON (40%), and OTA (27%). This study demonstrates that grape pomace reduces the gastrointestinal absorption of mycotoxins. This agricultural byproduct can be considered an alternative to commercial products and used in the feed industries as an effective, cheap, and natural binder for multiple mycotoxins.

Effect of almond processing on levels and distribution of aflatoxins in finished products and byproducts.

The fate of aflatoxins during processing of contaminated almonds into nougat, pastries, and almond syrup was evaluated by testing the effect of each processing step (blanching, peeling, roasting, caramelization, cooking, and water infusion) on the distribution and levels of aflatoxins. Blanching and peeling did not reduce total aflatoxins that were distributed between peeled almonds (90-93%) and skins (7-10%). Roasting of peeled almonds reduced up to 50% of aflatoxins. Up to 70% reduction of aflatoxins was observed during preparation and cooking of almond nougat in caramelized sugar. Aflatoxins were substantially stable during preparation and cooking of almond pastries. The whole process of almond syrup preparation produced a marked increase of total aflatoxins (up to 270%) that were distributed between syrup (18-25%) and spent almonds (75-82%). The increase of total aflatoxins was probably due to the activation of almond enzymes during the infusion step that released free aflatoxins from masked aflatoxins.

Comparison of Data from a Single-Analyte and a Multianalyte Method for Determination of Urinary Total Deoxynivalenol in Human Samples

Deoxynivalenol (DON) exposure is estimated by the combined measures of urinary DON and DON-glucuronides. In this study, data from single-mycotoxin (SM) and a multimycotoxin (MM) methods were compared for 256 Swedish adult urine samples. Both methods included β-glucuronidase predigestion, immunoaffinity enrichment, and LC-MS/MS. However, the specific reagents, apparatus, and conditions were not identical in part because the MM method measures additional mycotoxins. DON was detected in 88 and 63% of samples using the SM and MM methods, respectively, with the following mean and median concentrations: SM, mean = 5.0 ng/mL, SD = 7.4, range of positives = 0.5-60.2 ng/mL, median = 2.5 ng/mL, IQR = 1.0-5.5 ng/mL; MM, mean = 4.4 ng/mL, SD = 12.9, range of positives = 0.5-135.2 ng/mL, median = 0.8 ng/mL, IQR = 0.3-3.5. Linear regression showed a significant, albeit modest, correlation between the two measures (p = 0.0001, r = 0.591). The differences observed may reflect subtle handling differences in DON extraction and quantitation between the methods.

Co-occurrence of toxigenic moulds, aflatoxins, ochratoxin A, Fusarium and Alternaria mycotoxins in fresh sweet peppers (Capsicum annuum) and their processed products.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) were screened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were used for the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1 and FB2), nivalenol (NIV), deoxynivalenol (DON), T-2 and HT-2 toxins and Alternaria mycotoxins altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TTX) and tenuazonic acid (TeA). Frequency of potential toxigenic fungal species occurrence was: 87% Aspergillus Sect. Nigri; 58% Aspergillus Sect. Flavi; 38% Aspergillus Sect. Circumdati; 42% Alternaria spp.; 33% Penicillium spp. and 20% Fusarium spp. Frequency of mycotoxin occurrence and mean of positives were: 51% OTA, 29.5 µg/kg, 5 samples above the EU limit of 20 µg/kg; 31% aflatoxins, 12.8 µg/kg, two samples above the EU limit of 5 µg/kg for aflatoxin B1; 91% ZEA, 1.4 µg/kg; 78% FB2, 7.6 µg/kg; 58% FB...

Reduction of aflatoxins in apricot kernels by electronic and manual color sorting

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB1 + AFB2, whereas AFG1 and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%–99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB1 + AFB2 measured in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB1 and from 0.06 to 1.79 μg/kg for total aflatoxins.