J. Diana Di Mavungu

Natural occurrence of mycotoxins and their masked forms in food and feed products

A total of 174 cereal-based food products, 67 compound feeds and 19 feed raw materials were analysed for the occurrence of deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, α-zearalenol, β-zearalenol, and their respective masked forms, including deoxynivalenol-3-glucoside, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate. Fibre-enriched bread, bran-enriched bread, cornflakes, popcorn and oatmeal were collected in Belgian supermarkets from April 2010 to October 2011. All food samples analysed were contaminated with an average of 2 to 6 mycotoxins, including 1 to 3 masked forms. Feed raw materials that were used in the analysed compound feeds were collected by the manufacturer. Feed raw materials included were beet pulp, sunflower seed meal, soy bean, soy peel, oats, barley, maize germs, maize gluten feed, maize, wheat gluten feed, wheat bran pellets, wheat bran and wheat. Beet pulp, sunflower seed meal, soy bean and soy peel w...

Screening, identification and quantification of glucosinolates in black radish (Raphanus sativus L. niger) based dietary supplements using liquid chromatography coupled with a photodiode array and liquid chromatography mass spectrometry

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48 mg/g), glucosisaustricin (0.37-0.91 mg/g), glucoraphenin (0.84-1.27 mg/g), glucoputrajivin (0.14-0.28 mg/g), glucosisymbrin (0.70-0.99 mg/g) and gluconasturtiin (0.06-0.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.

Developments in detection and determination of aflatoxins

Since the discovery of aflatoxins in the 1960s, much research has focused on detecting the toxins in contaminated food and feedstuffs in the interest of public safety. Most traditional detection methods involved lengthy culturing and/or separation techniques or analytical instrumentation and complex, multistep procedures that required destruction of samples for accurate toxin determination. With more regulations for acceptable levels of aflatoxins in place, modern analytical methods have become quite sophisticated, capable of achieving results with very high precision and accuracy, suitable for regulatory laboratories and for post-harvest sample testing in developed countries. Unfortunately, many countries around the world that are affected by the aflatoxin problem do not have ready access to high performance liquid chromatography and mass spectrometry instrumentation and require alternate, readily available and simple detection methods that may be used by small holdings farmers in developing countries. T...

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster

ABSTRACT The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

Pattern and distribution of ergot alkaloids in cereals and cereal products from European countries

This paper reports on the occurrence of ergot alkaloids in cereals and cereal products in Europe. It includes occurrence data our group previously submitted to the European Food Safety Authority and new data we gathered afterwards. A total of 1,065 samples of cereals and cereal products intended for human consumption and animal feeding were analysed by liquid chromatography-tandem mass spectrometry for the presence of ergot alkaloids. The sample set included rye-, wheat- and multigrain-based food as well as rye-, wheat- and triticale-based feed. The study revealed that 59% of the analysed food and feed samples were contaminated with ergot alkaloids to some extent. In 55% of the samples, the levels of the -ine isomers were above the limit of quantification (LOQ), while contamination with the -inine isomers was found in 51% of the samples. The median values for the main ergot alkaloids (-ine forms) and the epimers (-inine forms) were 1 and 2 μg/kg, respectively. Ergot alkaloids were present in 84% of rye fo...

Masked mycotoxins in food and feed : challenges and analytical approaches

Abstract: Conjugated mycotoxins, in which the toxin is usually bound to a more polar substance like glucose, are referred to as masked mycotoxins, as they escape commonly used methods of analysis but can release their toxic precursors after hydrolysis. Nowadays, there is a growing interest related to the determination of these hidden forms of mycotoxins in food and feed commodities, as they represent an additional potential risk for the consumer. This chapter reviews the current state of knowledge regarding masked mycotoxins and the current analytical strategies for their detection and quantification in food and feed. The main problems related to the extraction of these highly polar derivatives as well as the different alternatives employed for their simultaneous determination together with the precursor toxins are discussed.

Detached leaf in vitro model for masked mycotoxin biosynthesis and subsequent analysis of unknown conjugates

The manuscript details the development of an in vitro model plant system using detached leaves because there is a need for biosynthetic methods for the production and isolation of masked mycotoxins. This detached leaf in vitro model was firstly applied to deoxynivalenol with satisfying results. The biosynthesis of deoxynivalenol-3-glucoside was confirmed using its respective commercially available reference standard. Secondly, the detached leaf in vitro model was applied to T-2 toxin. Mono- and tri-glucoside derivatives of T-2 toxin and HT-2 toxin, T-2-(3)-glucoside, T-2-(3)-triglucoside and HT-2-(3)-glucoside were identified and characterised using Orbitrap high-resolution mass spectrometry. This is the first report on a triglucoside of T-2 toxin. The discovery of new masked forms implies the importance of the development of analytical methods for their detection, the constitution of toxicity studies, and proving the relevance of their presence in the food and feed chain.

Untargeted screening of secondary metabolites in fungal cultures and samples from mouldy indoor environments by time-of-flight mass spectrometry

Nowadays, complaints about poor indoor air quality have become common. The variety of indoor air health problems include chronic fatigue, allergy, skin and eye irritation, and can be caused by several factors including fungi and their metabolites present in a building. The objective of this study was to establish a method for untargeted analysis of secondary fungal metabolites in indoor environments. As a detection technique, time-of-flight mass spectrometry was chosen, as it provided mass accuracy and higher sensitivity in full scan acquisition mode compared to tandem mass spectrometers. The method was first applied to fungal cultures, namely Penicillium brevicompactum and Chaetomium murorum, which were isolated from mouldy houses and grown on building materials under laboratory conditions for 7-21 days. Following the proposed strategy based on accurate mass measurement and post-acquisition data processing using principal component analysis, roquefortine C, brevianamide A and mycophenolic acid were ident...

Collaborative investigation of matrix effects in mycotoxin determination by high performance liquid chromatography coupled to mass spectrometry

Liquid chromatography coupled to mass spectrometry (LC-MS) is a commonly used technique for mycotoxin determination in food and feed. However, accuracy and reliability of the obtained results can suffer from ion suppression/enhancement caused by co-eluting matrix components. Inter-laboratory study concerning relative and absolute matrix effects (MEs) in various food and feed matrices was carried out. The applicability of commonly used strategies in ME reduction were tested in the quantitative determination of nivalenol, deoxynivalenol, fumonisin B1 and B2, and zearalenone in complex feed matrices. ‘In house’ validated LC-MS methods were applied. The relative MEs were assessed on the different food/feed sample sets. Cereal based- and hay/silage feed were used as complex model matrices for absolute ME assessment. The relative MEs within the different sample sets were in range of 3-72%. The absolute MEs for all analytes were in the range of 60-100% and 7-187% for cereal based and hay silage feed, respectivel...

An integrated targeted and untargeted approach for the analysis of ergot alkaloids in cereals using UHPLC - hybrid quadrupole time-of-flight mass spectrometry

An ultra-high performance liquid chromatography hybrid quadrupole – time of flight (Q-TOF) mass spectrometry (MS) method is described for the simultaneous quantitative determination of common ergot alkaloids and the screening, detection and identification of unexpected (less studied or novel) members of this class of toxic fungal secondary metabolites. The employed analytical strategy involves an untargeted data acquisition (consisting of full scan TOF MS survey and information dependent acquisition MS/MS scans) and the processing of data using both targeted and untargeted approaches. Method performance characteristics for the quantitative analysis of 6 common ergot alkaloids i.e. ergometrine, ergosine, ergotamine, ergocornine, ergocristine, ergokryptine and their corresponding epimers in rye were comparable to those previously reported for triple-quadrupole (QqQ) MS/MS. The method limits of quantification (LOQ) were in the range from 3 to 19 μg/kg, and good linearity was observed for the different ergot ...