Arnau Vidal

Determination of aflatoxins, deoxynivalenol, ochratoxin A and zearalenone in wheat and oat based bran supplements sold in the Spanish market.

The aflatoxins (AFs), deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based raw materials. Usually, the higher mycotoxins concentrations in cereals are found in the external layers of the grain (bran). Nowadays bran is increasingly consumed for its high fibre concentration. The objectives of this study were determining the concentration of these mycotoxins in bran samples intended for direct human consumption and to study the influence of some characteristics of the samples that may affect the mycotoxins content, there are not studies about fibre for direct human consumption. 67 bran samples from shops and supermarkets from two different Spanish cities were analyzed, being 37 samples of wheat bran and the remaining of oat bran. The results showed a major presence of DON in the analyzed samples, with levels above the EU legislation in some samples. Presence of DON was more frequent in wheat samples, compared to oats ones (p<0.05). Extruded or toasted samples, subjected to a heat treatment during processing, presented a significantly lower concentration of OTA, and differences between the organically and conventionally produced samples were also detected in OTA, which showed higher levels in the organic samples. Co-occurrence was frequently found between the Fusarium mycotoxins (ZEA and DON). Due to the high levels of DON in the analyzed samples, a calculation of DON intake has been made and it has been demonstrated that bran can account for an important percentage of DON exposure in the total diet.

The fate of deoxynivalenol and ochratoxin A during the breadmaking process, effects of sourdough use and bran content

Deoxynivalenol (DON) and ochratoxin A (OTA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based products such as bread. Due to the increasing interest in the beneficial effects of dietary bran, bran bread has attained high consumption. Usually, the higher mycotoxin concentrations in cereals are found in the external layers of the grain (bran), leading to higher concentration of DON and OTA in breads with added bran. Moreover, the use of sourdough in breadmaking is increasing, but no studies about its effect in the mycotoxins content exist. The objective of this study was to determine the variation of concentration of these mycotoxins during the breadmaking process including the following factors: two initial mycotoxin concentrations in the initial mix of ingredients, four different bran contents, and use of sourdough. OTA was confirmed to be quite stable during the breadmaking process, regardless of the assayed factors. DON concentration during breadmaking was not significantly affected by bran content of bread. However, it was significantly affected by kneading and fermentation steps in opposite way depending on sourdough use and flour contamination level: if DON reduction occurs during fermentation, this leads to a safer situation, but the possible increase in DON should be considered with care, as it can compensate the expected dilution effect by recipe. Finally, the results on deoxynivalenol-3-glucoside (DON-3-G), although preliminar, suggest an increase of this toxin during fermentation, but mainly during baking.

Thermal stability and kinetics of degradation of deoxynivalenol, deoxynivalenol conjugates and ochratoxin A during baking of wheat bakery products

The stability of deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-glucoside), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), de-epoxy-deoxynivalenol (DOM-1) and ochratoxin A (OTA) during thermal processing has been studied. Baking temperature, time and initial mycotoxin concentration in the raw materials were assayed as factors. An improved UPLC-MS/MS method to detect DON, DON-3-glucoside, 3-ADON, 15-ADON and DOM-1 in wheat baked products was developed in the present assay. The results highlighted the importance of temperature and time in mycotoxin stability in heat treatments. OTA is more stable than DON in a baking treatment. Interestingly, the DON-3-glucoside concentrations increased (>300%) under mild baking conditions. On the other hand, it was rapidly reduced under harsh conditions. The 3-ADON decreased during the heat treatment; while DOM-1 increased after the heating process. Finally, the data followed first order kinetics for analysed mycotoxins and thermal constant rates (k) were calculated. This parameter can be a useful tool for prediction of mycotoxin levels.

Enzyme bread improvers affect the stability of deoxynivalenol and deoxynivalenol-3-glucoside during breadmaking

The stability of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON-3-glucoside) during the breadmaking process was studied. Some enzymes used in the bakery industry were examined to evaluate their effects on DON and DON-3-glucoside. The level of DON in breads without added enzymes was reduced (17-21%). Similarly, the addition of cellulase, protease, lipase and glucose-oxidase did not modify this decreasing trend. The effect of xylanase and α-amylase on DON content depended on the fermentation temperature. These enzymes reduced the DON content by 10-14% at 45°C. In contrast, at 30°C, these enzymes increased the DON content by 13-23%. DON-3-glucoside levels decreased at the end of fermentation, with a final reduction of 19-48% when no enzymes were used. However, the presence of xylanase, α-amylase, cellulase and lipase resulted in bread with greater quantities of DON-3-glucoside when fermentation occurred at 30°C. The results showed that wheat bran and flour may contain hidden DON that may be enzymatically released during the breadmaking process when the fermentation temperature is close to 30°C.

Stability and kinetics of leaching of deoxynivalenol, deoxynivalenol-3-glucoside and ochratoxin A during boiling of wheat spaghettis

The stability of deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-glucoside) and ochratoxin A (OTA) during spaghetti production and cooking was investigated. Initial mycotoxin concentration, boiling time and use of egg as ingredient were assayed as factors. DON was stable during kneading and drying, but a consistent reduction of DON (>40%) was observed in boiled spaghettis. According to our results, DON was transferred to broth, where it was not degraded, and boiling time determined the extent of the transfer. A DON leaching model was fitted to data with a high goodness of fit (r2=0.99). This model can be used for prediction of final DON concentration in cooked pasta, and a useful tool in risk assessment models. DON-3-glucoside is totally stable through the pasta making process; moreover DON-3-glucoside is slightly released from pasta components and it is leached to broth. Similarly, OTA is also stable during pasta making; however, it is scarcely transferred to broth during boiling. The presence of egg as ingredient did not affect the final mycotoxin concentration in pasta in any case.

Multidetection of urinary ochratoxin A, deoxynivalenol and its metabolites: pilot time-course study and risk assessment in Catalonia, Spain

The presence of two main mycotoxins, ochratoxin A (OTA) and deoxynivalenol (DON), is widespread in cereal-based foodstuffs marketed in Europe. The objectives of this study were to develop and validate a multi-detection analytical methodology to simultaneously assess the urinary concentrations of OTA, DON and their metabolites, and to apply this methodology in a preliminary follow-up trial in Catalonia (Spain). Hence, an ultra-performance liquid chromatography with tandem mass spectrometry method was developed to simultaneously assess the urinary levels of OTA, DON, deoxynivalenol-3-glucoside (DON-3-glucoside), deoxynivalenol-3-glucuronide (DON-3-glucuronide), 3-acetyldeoxynivalenol (3-ADON) and de-epoxy-deoxynivalenol (DOM-1). Urine mycotoxins levels and food dietary intake were prospectively monitored in a group of volunteers throughout a restriction period followed by a free-diet period. The proposed multi-detection methodology for urinary OTA and DON metabolites was validated, providing suitable recove...

Genetic and toxigenic variability within Aspergillus flavus population isolated from maize in two diverse environments in Kenya

Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial β-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

Effect of xylanase and α-amylase on DON and its conjugates during the breadmaking process

Deoxynivalenol (DON) is one of the most frequently occurring mycotoxins in wheat crops worldwide and poses a risk to human and animal health due to its wide range of adverse effects. Deoxynivalenol-3-glucoside (DON-3-glucoside) is a DON plant conjugate that is widely found in cereal products. As DON accumulation in the field seems unavoidable, it is important to investigate all of the conditions that affect its stability during food processing. One of the most consumed cereal product around the world is bread, however the published information about DON stability in bread shows a large variability of results because a huge amount of factors affect DON and its modified forms. So, the aim of this research was to study the fate of DON and its modified forms through the breadmaking process with the addition of xylanase and α-amylase at different fermentation temperatures. Moreover, different α-amylase and xylanase concentrations were added to the dough to be fermented. To quantify DON and its derived forms in the samples, liquid chromatography with double mass spectrophotometer was used. DON was reduced during fermentation and baking; however, the reduction at each step was related to the fermentation temperature. The presence of α-amylase and xylanase caused increases in DON during fermentation and during early baking. DON-3-glucoside was slightly reduced after fermentation and was widely increased (>80%) after baking. Deepoxy-deoxynivalenol (DOM-1) increased during the breadmaking process. Breadmaking process can reduce DON concentration, however xylanase and α-amylase presence cause increases of DON.

Humans significantly metabolize and excrete the mycotoxin deoxynivalenol and its modified form deoxynivalenol-3-glucoside within 24 hours.

For the first time, a comprehensive human intervention study was conducted to unravel the urinary excretion profile and metabolism of the fungal metabolite deoxynivalenol (DON) and its modified form deoxynivalenol-3-glucoside (DON-3-glucoside). Twenty volunteers were restricted in consuming cereals and cereal-based foods for 4 days. At day 3, a single bolus of 1 µg/kg body weight of DON and a single bolus of 1 µg/kg body weight of DON-3-glucoside after a washing-out period of two months was administered, and a 24-h urine collection was performed. The urine was analysed for DON, DON-3-glucoside, 3-ADON, 15-ADON, deepoxy-deoxynivalenol (DOM-1), deoxynivalenol-3-glucuronide (DON-3-glucuronide) and deoxynivalenol-15-glucuronide (DON-15-glucuronide). The urinary biomarker-analysis revealed that DON and DON-3-glucoside were rapidly absorbed, distributed, metabolized and excreted. Sixty-four % of the administered DON and 58% of DON-3-glucoside was recovered in the urine collected within 24 h. DON-15-glucuronide was the most prominent urinary biomarker followed by free DON and DON-3-glucuronide. Moreover, correlations among the presence of DON-15-glucuronide and DON-3-glucuronide were observed (within 24 hours (r = 0.61)). The DOM-1 detected in the urine was higher after the DON-3-glucoside administration. The obtained results are imperative to construct a standardized method to estimate DON-intake by means of urinary biomarkers.

DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF CITRININ AND OCHRATOXIN A IN A VARIETY OF FEED AND FOODSTUFFS

An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI+/--MS/MS) method for the simultaneous analysis of citrinin (CIT) and ochratoxin A (OTA) in feed (chicken and pig) and food (cereal-based products, fruit, vegetable juices, nuts, seeds, herbs, spices, vegetarian and soy products, alcoholic beverages, baby food products and food supplements) was developed. The mycotoxins were extracted from these matrices using a QuEChERS-based extraction method without any further clean-up step. The samples were 5-fold concentrated. Final extracts were analyzed using a UPLC-MS/MS system and chromatographic separation was achieved by applying a gradient elution for a total run time of 10 min. Mycotoxins were quantified using an internal calibration via analyte/13C-labeled internal standard ratio. The developed method was validated according to the criteria described in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC. Specificity, linearity, apparent recovery, limit of detection and quantification, intraday and interday precision, measurement uncertainty, matrix effect, and extraction efficiency were the parameters studied. Finally, 90 Belgian chicken and pig feed samples were analyzed, revealing the simultaneous presence of CIT (