Occurrence of type A, B and D trichothecenes, zearalenone and stachybotrylactam in straw.

Straw is the main by-product of grain production, used as bedding material and animal feed. If produced or stored under adverse hygienic conditions, straw is prone to the growth of filamentous fungi. Some of them, e.g. Aspergillus, Fusarium and Stachybotrys spp. are well-known mycotoxin producers. Since studies on mycotoxins in straw are scarce, 192 straw samples (wheat n = 80; barley n = 79; triticale n = 12; oat n = 11; rye n = 12) were collected across Germany within the German official feed surveillance and screened for the presence of 21 mycotoxins. The following mycotoxins (positive samples for at least one mycotoxin n = 184) were detected: zearalenone (n = 86, 6.0-785 µg/kg), nivalenol (n = 51, 30-2,600 µg/kg), deoxynivalenol (n = 156, 20-24,000 µg/kg), 15-acetyl-deoxynivalenol (n = 34, 20-2,400 µg/kg), 3-acetyl-deoxynivalenol (n = 16, 40-340 µg/kg), scirpentriol (n = 14, 40-680 µg/kg), T-2 toxin (n = 67, 10-250 µg/kg), HT-2 toxin (n = 92, 20-800 µg/kg), T-2 tetraol (n = 13, 70-480 µg/kg). 15-monoacetoxyscirpenol (30 µg/kg) and T-2 triol (60 µg/kg) were only detected in one barley sample. Macrocyclic trichothecenes (satratoxin G, F, roridin E, and verrucarin J) were also found in only one barley sample (quantified as roridin A equivalent: total 183 µg/kg). The occurrence of stachybotrylactam was monitored for the first time in four samples (n = 4, 0.96-7.4 µg/kg). Fusarenon-X, 4,15-diacetoxyscirpenol, neosolaniol, satratoxin H and roridin-L2 were not detectable in the samples. The results indicate a non-negligible contribution of straw to oral and possibly inhalation exposure to mycotoxins of animals or humans handling contaminated straw.

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.

Ochratoxin A in brewer's yeast used as food supplement.

Brewer's yeasts are rich in vitamins of the B-group and contain other nutritive factors; therefore, they are recommended as valuable food supplements for people with special dietary requirements like pregnant women, children, and adolescents, or for people with high physical activity. Additionally, certain strains of brewer's yeast are known to be capable of adsorbing xenobiotics such as mycotoxins. Because of that, these yeasts are regarded as having positive effects in food, beverage, and feed technology. Their potential to bind mycotoxins such as ochratoxin A (OTA), however, can subsequently lead to a contamination of such brewer's yeasts used as food supplements. In the present study, we analyzed 46 samples of brewer's yeasts for the occurrence of OTA by HPLC with fluorescence detector (HPLC-FLD) and for confirmatory measurements by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nearly 90% of the samples were contaminated with OTA, the levels ranging from the limit of detection (LOD, 0.01 µg/kg) to 4.2 µg/kg. The mean and median levels of contamination were 0.49 and 0.27 µg/kg, respectively. Based on these results, the additional weekly OTA exposure by regularly consuming such supplements was assessed. Depending on different subpopulations (adults, children) and levels of contamination used for calculation, the additional OTA intake via brewer's yeast products ranged from 9.3% (mean case) to 114% (worst case) of the published mean weekly OTA intake in Germany (adults 279.3 ng, children 195.3 ng). At present, maximum levels for OTA in nutritional supplements like brewer's yeast do not exist. Based on our results, however, it is recommended that producers of these dietary supplements should include mycotoxin analyses in ongoing and future self-monitoring programs and in product quality checks.

Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs.

Stachybotrys (S.) chartarum is an omnipresent cellulolytic mould which produces secondary metabolites, such as the highly toxic macrocyclic trichothecenes. While it is known to occur in animal feed like hay and straw as well as in water-damaged indoor environments, there is little knowledge about the occurrence of S. chartarum and its secondary metabolites in food. The objective of the present study was to examine selected dried culinary herbs for the presence of S. chartarum chemotype S, to assess the potential risk of a contamination of foods with macrocyclic trichothecenes. In total, 50 Stachybotrys isolates from different types of culinary herbs (n=100) such as marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) were examined by MTT-cell culture test (effect-based bioassay), ELISA, and by liquid chromatography tandem mass spectrometry (LC-MS/MS). Selected toxic and non-toxic isolates (n=15) were genetically characterized by PCR and sequencing. Five isolates (10%) were highly toxic in the MTT-cell culture test, and the production of macrocyclic trichothecenes was proven by ELISA and LC-MS/MS. These five isolates were genetically confirmed as S. chartarum chemotype S. To the best of our knowledge, this is the first report about a contamination of dried culinary herbs with toxigenic S. chartarum.