An LC-MS method was developed for the analysis of zearalenone-4-beta-D-glucopyranoside and zearalenone in wheat (Triticum aestivum). The limit of determination for zearalenone-4-beta-D-glucopyranoside and zearalenone was 10 microg/kg. The recovery rates were calculated to be 69% and 89% at a concentration of 100 microg/kg for zearalenone-4-beta-D-glucopyranoside and zearalenone, respectively. Twenty-four Bavarian wheat samples from a 1999 harvest were analyzed. Zearalenone was present in 22 of 24 field samples, the levels ranged from 11 to 860 microg/kg. Zearalenone-4-beta-D-glucopyranoside was found in 10 of the zearalenone positive samples (42%) at levels ranging from 17 to 104 microg/kg. The amounts of zearalenone-4-beta-D-glucopyranoside were correlated to those of zearalenone (r2 = 0.86, b = 0.10). After gastrointestinal hydrolyzation, zearalenone-4-beta-glucopyranoside might be implicated in the development of a zearalenone-syndrome. Therefore, more attention should be focused on conjugated mycotoxins in food and feed.

We examined 233 silage samples and found that molds were present in 206 samples with counts between 1 ؋ 10 3 and 8.9 ؋ 10 7 (mean, 4.7 ؋ 10 6 ) CFU/g. Mycophenolic acid, a metabolite of Penicillium roqueforti, was detected by liquid chromatography-mass spectrometry in 74 (32%) of these samples at levels ranging from 20 to 35,000 (mean, 1,400) g/kg. This compound has well-known immunosuppressive properties, so feeding with contaminated silage may promote the development of infectious diseases in livestock.Silage is frequently contaminated with fungi of the genera Monascus, Aspergillus, and Penicillium (14). One of the most common molds is Penicillium roqueforti, which can produce secondary metabolites such as roquefortine C, isofumiclavines A and B, PR toxin, macrofortines, and mycophenolic acid (5, 6, 10, 13). Roquefortine C has been detected frequently in silage (3,11,16), but little is known about the natural occurrence of the other mycotoxins, especially mycophenolic acid.Mycophenolic acid [6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid] is a weak organic acid with antifungal, antibacterial, and antiviral activities (1, 2, 5). Its acute toxicity to mammals seems to be low: the calculated oral 50% lethal doses for rats and mice are 700 and 2,500 mg/kg, respectively (6). Mycophenolic acid is also a noncompetitive inhibitor of eukaryotic inosine monophosphate dehydrogenase (12) and blocks the conversion of inosine-5-phosphate and xanthine-5-phosphate to guanosine-5-phosphate. As T and B lymphocytes rely primarily on the de novo biosynthesis of purine rather than on the purine salvage pathway, mycophenolic acid blocks their proliferative response and inhibits both antibody formation and the production of cytotoxic T cells (9).Consumption of immunosuppressive compounds increases the risk of infectious diseases in livestock, but this risk cannot be accurately estimated without knowledge of naturally occurring immunosuppressants such as mycophenolic acid in silage. Therefore, we analyzed samples of grass and maize silage for the presence of P. roqueforti and mycophenolic acid.Samples. Samples of grass (n ϭ 98) and maize (n ϭ 135) silage partly visibly contaminated with molds were collected in Bavaria during 1997 and 1998. The mycobiota of the samples was determined quantitatively and qualitatively, and an aliquot of each silage type (ϳ500 g) was stored at Ϫ18°C until the analysis of mycophenolic acid.Mycological examination. An aliquot of 10 g of mechanically minced silage was suspended in 90 ml of sterile peptone water (10 g of casein peptone [Merck, Darmstadt, Germany], 8.5 g of sodium chloride [Merck], 1,000 ml of distilled water) and shaken at 20°C for 30 min. From this initial dilution (10 Ϫ1 ), subsequent dilutions (1:10) were made in sterile peptone water. For mold count determinations, 0.1-ml aliquots from the dilutions (10 Ϫ2 to 10 Ϫ4) were plated on Sabouraud 2% dextrose agar (Merck) supplemented with 400,000 IU of penicillin G (Sigma, Deisenhofen, Germany) and 40 mg of strept...

From 1999-2001 three different varieties of wheat [Contur (susceptible to Fusarium), Batis and Petrus (less susceptible to Fusarium)] were cultivated under organic and conventional conditions in order to determine mycotoxin burden. Soil quality, preceding crop and weather conditions were comparable in the different production systems. The wheat batches were analysed for moulds, and the contents of zearalenone (ZEN) and deoxynivalenol (DON). Feeding trials were carried out with growing pigs (n = 96; average initial live weight 22.2 +/- 1.5 kg [mean +/- SD]) to examine a possible influence on the animal performance and on mycotoxin residues. The data recorded were clinical conditions, performance, biochemical and hematological data. Residues of ZEN, alpha- and beta-zearalenol (ZEL) and of DON were determined in bile, liver and muscle after slaughtering. Conventionally cultivated wheat was more frequently contaminated with Fusarium and contained more frequently ZEN and DON in higher concentrations than the organically produced wheat. Hematological and biochemical parameters of pigs fed with organically cultivated diets were not different from those of conventionally fed pigs. Pigs fed with organically produced wheat showed a slightly higher daily weight gain, but a lower carcass yield than the conventionally fed animals. The highest residues of DON and total-ZEN (ZEN + alpha-ZEL + beta-ZEL) were found in bile. Bile samples of organically fed pigs contained lower concentrations of total-ZEN than those of conventionally fed pigs. Altogether, these data suggest that wheat from an organic farming does not have higher mycotoxin-contamination than wheat from the conventional farming system.

A total of 233 silages were examined and found that Monascus ruber was present in 43 samples with counts between 1 x 10(3) and 9 x 10(6) colony-forming units (CFU)/g (mean: 2 x 10(5) CFU/g). Monacolin K(L) and the hydroxy acid monacolin K(A) were detected by liquid chromatography-mass spectrometry in 45 and 50 of 233 samples at levels ranging from 25-15 600 and 28-65 400 microg/kg, respectively. Citrinin was found with high-performance liquid chromatography-fluorescence detection (FLD) in 14 (6%) samples, the concentrations varied between 2.4 and 64.2 microg/kg. The concentrations of citrinin were low and toxic effects are not anticipated. Monacolin K(A) and monacolin K(L) occur frequently and in considerable amounts in silages. These metabolites are believed to influence the metabolic activity of rumen anaerobic fungi resulting in a poorer digestion of crude fibre.

Maize (Zea mays) cell cultures were used for the production of zearalenone-4-β-D-glucopyranoside as standard compound. Wheat samples were extracted with acetonitrile: water, applied to a florisil column and eluted with methanol:ethyl acetate. For determination and quantification of zearalenone-4-β-D-glucopyranoside and zearalenone a LC-MS method was developed. A concentration of 10 μg/kg zearalenone-4-β-D-glucopyranoside and zearalenone was detectable. The recovery rates were calculated to be 69% and 89% at a concentration level of 100 μg/kg for zearalenone-4-β-D-glucopyranoside and zearalenone, respectively.24 Bavarian wheat samples from harvest 1999 were analyzed. Zearalenone was present in 22 out of 24 field samples, the levels ranged from 11-860 μg/kg. Zearalenone-4-β-D-glucopyranoside was found in 10 out of the zearalenone positive samples (42%) at levels ranging from 17 to 104 μg/kg. The amounts of zearalenone-4-β-D-glucopyranoside were correlated to those of zearalenone (r2=0,86; b=0,10).