Marika Jestoi

Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples.

Fusarium mycotoxins beauvericin, enniatins (A, A1, B, B1) and moniliformin were analysed in 38 Finnish grain samples (14 wheat, 22 barley, one rye, one oats) harvested in 2001–02. The contaminating Fusarium species were identified with the primer-specific polymerase chain reaction as well as with morphological studies. All the studied mycotoxins were found in the samples. Enniatins B and B1 were detected in all samples, and enniatin A, enniatin A1, beauvericin and moniliformin in 74, 95, 95 and 74% of the samples, respectively. There were higher concentrations of the mycotoxins analysed in 2001 compared with 2002. The highest levels of mycotoxins were detected in samples harvested late in the autumn after a long rainy period. Fusarium avenaceum was the most abundant Fusarium species in Finland during both years (0–29.5%) measured as infected kernels. A significant correlation was found between F. avenaceum contamination level and the concentration levels of enniatins B and B1, as well as moniliformin.

Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry.

A simple and feasible method is described for analysing nine mycotoxins in cheese matrix. The method involves liquid extraction followed by high performance liquid chromatographic separation and mass spectrometric detection of the analytes, and allows the determination of aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, mycophenolic acid, penicillic acid and roquefortine C simultaneously. Average recoveries of the mycotoxins from spiked samples at concentration levels of 5–200u2009µgu2009kg−1 ranged from 96–143%. Within-day relative standard deviations at these concentration levels varied from 2.3–12.1%. The limit of quantification for aflatoxin M1 was 0.6u2009µgu2009kg−1 and for the other compounds 5u2009µgu2009kg−1. The method developed was applied for analysing these mycotoxins in blue and white mould cheeses purchased from Finnish supermarkets. Roquefortine C was detected in all of the blue mould cheese samples in concentrations of 0.8–12u2009mgu2009kg−1. One blue cheese contained also 0.3u2009mgu2009kg−1 mycophenolic acid. The other investigated mycotoxins were absent in the samples.

Mycotoxin production of selected Fusarium species at different culture conditions.

The toxin producing capacity of seven Fusarium species (F. langsethiae, F. sporotrichioides, F. poae, F. avenaceum, F. tricinctum, F. graminearum and F. culmorum) and the effect of culture conditions on the toxin production were studied. The strains were isolated from Finnish grains and cultivated on a grain mixture at three different water activity/temperature combinations (i.e. 0.994/15 degrees C; 0.994/25 degrees C; 0.960/25 degrees C). The mycotoxins produced were analyzed with a multi-toxin method based on liquid chromatography-tandem mass spectrometry enabling the simultaneous determination of 18 different Fusarium toxins. The general toxin profiles revealed F. langsethiae and F. sporotrichioides as producers of diacetoxyscirpenol, neosolaniol, HT-2 and T-2-toxins. F. sporotrichioides produced additionally beauvericin. In the F. poae cultures, only beauvericin was detected. F. avenaceum and F. tricinctum were capable of producing enniatins, moniliformin and antibiotic Y, and F. graminearum and F. culmorum produced zearalenone, deoxynivalenol and 3-acetyl deoxynivalenol. Differences existed in the quantitative toxin production between the individual strains representing the same species. Additionally, the culture conditions affected the range and amounts of toxins produced. In general, a(w) 0.994 and temperature of 15 degrees C favoured the type-A trichothecene production of F. langsethiae and F. sporotrichioides. The beauvericin production of F. sporotrichioides occurred more favourably at a(w) 0.960 and 25 degrees C. F. poae produced the highest concentrations of beauvericin under two different conditions, namely at a(w) 0.994/15 degrees C and a(w) 0.960/25 degrees C. None of the combinations particularly favoured toxin production of F. avenaceum, with all three toxins being produced extensively at all culture conditions. F. tricinctum produced enniatins most efficiently at a(w) 0.994/25 degrees C. The moniliformin production of both these two species occurred readily at a(w) 0.960/25 degrees C. F. culmorum and F. graminearum produced the highest concentrations and variety of mycotoxins at a(w) 0.960/25 degrees C. The results give valuable information on the toxigenicity of some important Fusarium species. Additionally, this is the first in-depth study to investigate the influence of environmental conditions on the toxin production by F. langsethiae, F. poae, F. avenaceum and F. tricinctum.

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4–5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.

Simultaneous determination of major type A and B trichothecenes, zearalenone and certain modified metabolites in Finnish cereal grains with a novel liquid chromatography-tandem mass spectrometric method

A reliable and sensitive liquid chromatography-tandem mass spectrometric method was developed for the simultaneous quantitative determination in cereals of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol and zearalenone, as well as the modified metabolites 3-acetyl-deoxynivalenol, α-zearalenol, β-zearalenol, deoxynivalenol-3-glucoside, HT-2-3-glucoside, nivalenol-3-glucoside, zearalenone-14-glucoside, zearalenone-14-sulphate, zearalenone-16-glucoside, α-zearalenol-14-glucoside and β-zearalenol-14-glucoside. The ‘dilute and shoot’ approach was used for sample preparation after extraction with acetonitrile:water:acetic acid (79:20:1, v/v/v). Separation was carried out using reversed-phase liquid chromatography, and detection was performed using tandem mass spectrometry in the selected reaction monitoring mode. The method was in-house validated according to performance characteristics, established in Commission Regulation EC No 401/2006 and Commission Decision EC No 657/2002, prior to its application in a nationwide survey for the analysis of barley, oat and wheat samples (nu2009=u200995) harvested in Finland during 2013. Deoxynivalenol and its glucosylated form were the most abundant of the analytes, being detected in 93 and 81xa0% of the samples, respectively. Concentrations of deoxynivalenol were unusually high in 2013, especially in oats, with some cases exceeding the maximum legislative limits for unprocessed oats placed on the market for first-stage processing. All modified mycotoxins analysed were detected, and the natural occurrence of some of these compounds (e.g. zearalenone-16-glucoside and nivalenol-3-glucoside) in barley, oats and/or wheat was documented for the first time.

Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat

To investigate the metabolic fate of HT-2 toxin (HT2) and T-2 toxin (T2) in wheat (Triticum aestivum L.), an untargeted metabolomics study utilizing stable isotopic labeling and liquid chromatography–high resolution mass spectrometry was performed. In total, 11 HT2 and 12 T2 derived in planta biotransformation products were annotated putatively. In addition to previously reported mono- and diglucosylated forms of HT2, evidence for the formation of HT2-malonyl-glucoside and feruloyl-T2, as well as acetylation and deacetylation products in wheat was obtained for the first time. To monitor the kinetics of metabolite formation, a time course experiment was conducted involving the Fusarium head blight susceptible variety Remus and the resistant cultivar CM-82036. Biotransformation reactions were observed already at the earliest tested time point (6 h after treatment), and formed metabolites showed different kinetic profiles. After ripening, less than 15% of the toxins added to the plants were determined to be unmetabolized.

Determination of Fusarium‐Mycotoxins Beauvericin and Enniatins with Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

Abstract A liquid chromatography–mass spectrometric method was developed and validated to determine Fusarium mycotoxins beauvericin and enniatins (A, A1, B, B1) in grain samples. For the first time, a triple quadrupole mass spectrometer with multiple reaction monitoring was used for the analysis of these mycotoxins, which allowed the simultaneous structural identification of the analytes. The sample preparation involved direct purification of sample extracts with reversed‐phase–solid phase extraction, enabling the avoidance of time consuming and loss‐causing liquid–liquid extractions. The method validation included the determination of selectivity, repeatability, limit of detection, limit of quantification, recovery, and linearity. The developed method proved to be sensitive and repeatable for the analysis of the mycotoxins in grain matrix. Since the method was originally developed solely for beauvericin, the recoveries of some enniatins remained rather low, except at the lowest spiking level. This was due to the natural contamination of grain matrix with enniatins B and B1, which resulted in higher recoveries for these mycotoxins. The mean recoveries for beauvericin and enniatins A, A1, B, and B1 were 76–82%, 55–66%, 71–80%, 57–103%, and 68–116%, respectively. The calculated limits of quantification for beauvericin and enniatins A, A1, B, and B1 were 0.2, 0.2, 0.7, 0.9, and 1.5 µg/kg, respectively.

Determination of ergot alkaloids from grains with UPLC-MS/MS.

A simple and rapid method for determining six ergot alkaloids and four of their respective epimers was developed for rye and wheat. The analytes were extracted from the sample matrix with ACN/ammonium carbonate solution. The extract was purified with a commercial push-through SPE column (Mycosep 150 Ergot). After concentration and filtration steps, the final separation of the analytes was achieved with ultra-performance LC-MS/MS. The chromatographic separation of the ergot alkaloids was achieved in 4.5 min. The method performance proved satisfactory in the preliminary validation. The calculated LOQs were low ranging from 0.01 to 1.0 microg/kg for wheat and from 0.01 to 10.0 microg/kg for rye. At the concentration levels of 10, 50 and 200 microg/kg, the recoveries were between 80 and 120% in most cases and the within-day repeatability (expressed as RSD) ranged between 1.3 and 13.9%. Despite the cleanup of the samples, some matrix effect was observed in the MS, highlighting the necessity of using matrix-assisted standards. This is the first article to describe the application of the push-through columns and ultra-performance LC in the analysis of ergot alkaloids.

Updated survey of Fusarium species and toxins in Finnish cereal grains

ABSTRACT The aim of the project was to produce updated information during 2005–14 on the Fusarium species found in Finnish cereal grains, and the toxins produced by them, as the last comprehensive survey study of Fusarium species and their toxins in Finland was carried out at the turn of the 1960s and the 1970s. Another aim was to use the latest molecular and chemical methods to investigate the occurrence and correlation of Fusarium species and their mycotoxins in Finland. The most common Fusarium species found in Finland in the FinMyco project 2005 and 2006 were F. avenaceum, F. culmorum, F. graminearum, F. poae, F. sporotrichioides and F. langsethiae. F. avenaceum was the most dominant species in barley, spring wheat and oat samples. The occurrence of F. culmorum and F. graminearum was high in oats and barley. Infection by Fusarium fungi was the lowest in winter cereal grains. The incidence of Fusarium species in 2005 was much higher than in 2006 due to weather conditions. F. langsethiae has become much more common in Finland since 2001. F. graminearum has also risen in the order of importance. A highly significant correlation was found between Fusarium graminearum DNA and deoxynivalenol (DON) levels in Finnish oats, barley and wheat. When comparing the FinMyco data in 2005–06 with the results of the Finnish safety monitoring programme for 2005–14, spring cereals were noted as being more susceptible to infection by Fusarium fungi and the formation of toxins. The contents of T-2 and HT-2 toxins and the frequency of exceptionally high DON concentrations all increased in Finland during 2005–14. Beauvericin (BEA), enniatins (ENNs) and moniliformin (MON) were also very common contaminants of Finnish grains in 2005–06. Climate change is leading to warmer weather, and this may indicate more changes in Finnish Fusarium mycobiota and toxin contents and profiles in the near future.