Production and Analytical Properties of Antibodies with High Specificity to Zearalenone

The time course of production, specificity, and analytical potential of antizearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; -zearalenole, 0.15%; and -zearalenole, <0.02%. With synthetic analogues: zearalanone, 31.7% and -zearalanone, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in enzyme immunoassay

Zearalenone-6′-carboxymethyloxime was synthesized, and its conjugates to albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (a-zearalenol, 28%; ß-zearalenol, 6%; zearalanone, 12%; and α-zearalanol, 5%). The sensitivity of enzyme immunoassay with immobilized gelatin-based conjugates for determination of zearalenone in solution was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 μg/kg.     

Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in immunoenzyme analysis

Zearalenone-6'-carboxymethyloxime was synthesized, and its conjugates with albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (alpha-zearalenol--28%, beta-zearalenol--6%, zearalanone--12%, and alpha-zearalanol--5%). The sensitivity of enzyme immunoassay using gelatin-based immobilized conjugates for determination of zearalenone in solutions was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 micrograms/kg.     

Nachweis von Zearalenon-4-β-D-Glucopyranosid in Weizen

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).     

Fermentation of wort containing deoxynivalenol and zearalenone

Wort containing deoxynivalenol and zearalenone, each added at a level of 1.9 μg/mL, was fermented by 3 strains ofSaccharomyces cerevisiae for 7 or 9 days to make beer. Analysis showed that deoxynivalenol was stable during this process. The major metabolite of zearalenone was β - zearalenol, which formed in up to 69% of the initial zearalenone concentration, while up to 8.1% of the initial zearalenone was converted to α - zearalenol. The major part of the metabolism of zearalenone occurred by 1 – 2 days. Control experiments, where the yeasts were omitted and deoxynivalenol, zearalenone and α - and β - zearalenol were added, showed good recovery and stability of the mycotoxins over the 7–9 day time period. No deoxynivalenol, zearalenone, α-zearalenol or β-zearalenol was detected in control yeast fermentations where they were not added to the wort.     

Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection

A fast, cost-efficient, sensitive and accurate assay method for zearalenone in edible oils is described, as an alternative to gel permeation chromatography (GPC). Oil samples were extracted with an alkaline mixture of methanol and water (methanol +10 g/l aqueous ammonium carbaminate solution, pH 9; 9 + 1, v+v). The pH of the extract was neutralized with hydrochloric acid and then concentrated to dryness. The residue was dissolved with HPLC solvent, and zearalenone was determined by high-performance liquid chromatography with fluorometric detection (HPLC-FLD). The method was successfully validated for two matrices, maize oil and rapeseed oil. The recovery rate was 87%, and the coefficient of variation was 2.8% in a rapeseed oil sample contaminated with 27 μg zearalenone/kg. At a signal-to-noise ratio of 3:1, the method detection limit was 10 μg/kg, which was considered to be adequate in view of the present European Union maximum level of 400 μg/kg.     

Mycotoxins and Fusarium spp. associated with infected ears of corn in Minnesota

Five Fusarium species were isolated from the grain of dent corn (Zea mays) selected from 20 of 32 damaged fields in 10 counties in Minnesota on the basis of hyphal growth visible on kernels in the field. Three mycotoxins were identified in the infected ears: zearalenone, deoxynivalenol, and 15-acetyl-deoxynivalenol. This is the first report of the presence of 15-acetyl-deoxynivalenol on corn ears in the field prior to harvest and in combination with deoxynivalenol and zearalenone. Ninety-nine cultures were selected from colonies growing from kernels on an agar medium; 30% of the cultures were F. graminearum, 23% were F. subglutinans, 20% were F. moniliforme, 14% were F. oxysporum, and 12% were F. proliferatum.     

Mycotoxins and Fusarium spp. associated with infected ears of corn in Minnesota.

Five Fusarium species were isolated from the grain of dent corn (Zea mays) selected from 20 of 32 damaged fields in 10 counties in Minnesota on the basis of hyphal growth visible on kernels in the field. Three mycotoxins were identified in the infected ears: zearalenone, deoxynivalenol, and 15-acetyl-deoxynivalenol. This is the first report of the presence of 15-acetyl-deoxynivalenol on corn ears in the field prior to harvest and in combination with deoxynivalenol and zearalenone. Ninety-nine cultures were selected from colonies growing from kernels on an agar medium; 30% of the cultures were F. graminearum, 23% were F. subglutinans, 20% were F. moniliforme, 14% were F. oxysporum, and 12% were F. proliferatum.     

Adsorption and degradation of zearalenone by bacillus strains

Two Bacillus strains; Bacillus subtilis 168 and Bacillus natto CICC 24640 separately adsorbed and degraded zearalenone in liquid media, in vitro. Viable, autoclaved (121°C, 20 min) and acid-treated cells of both strains separately bound more than 55% of zearalenone (ZEN, 20 μg/L) after 30 min and 1-h incubation at 37°C under aerobic conditions, and the amount of ZEN adsorbed was dependent on initial cell volume. In addition, ZEN was degraded by the culture extract of both strains. Degradation by B. subtilis 168 and B. natto CICC 24640 culture extract after 24-h aerobic incubation at 30°C was 81% and 100%, respectively. B. natto CICC 24640 culture extract comprehensively degraded ZEN and, for both strains, no oestrogenic ZEN analogues were present. ZEN degradation was accompanied by carbondioxide emission indicating a decarboxylation reaction. ZEN degradation by the salient B. natto CICC 24640 culture extract varied with initial ZEN concentration, incubation time, temperature and pH. Degradation was enhanced by Mn²⁺, Zn²⁺, Ca²⁺ and Mg²⁺ but impeded by Hg²⁺, Cu²⁺, Pb²⁺, ethylenediaminetetraacetic acid and 1,10-phenanthroline. The degradation reaction is associated with a metalloproteinase of molar mass in the range 31–43 kDa. Overall, the two generally recognised as safe Bacillus strains can, potentially, be utilised for detoxification of zearalenone in food.     

Effect of whole and fractionated dietary alfalfa meal on zearalenone toxicosis and metabolism in rats and swine

Experiments were conducted to determine the mechanism by which dietary alfalfa can protect against zearalenone toxicosis. Female weanling rats were fed semipurified diets containing whole alfalfa meal, fractionated alfalfa meal (fiber, solvent extract, and water extract), and purified components of alfalfa (coumestrol, saponin, lignin, coumestrol + lignin, and saponin + lignin) with and without 250 mg zearalenone/kg of diet. All ingredients were provided for 2 weeks at levels corresponding to those found in diets containing 15 and 25% alfalfa. Yorkshire gilts were fed 15 and 25% alfalfa meal with and without 10 mg zearalenone/kg of diet for 4 weeks. The feeding of zearalenone to rats reduced growth and food consumption but this was overcome by 25% alfalfa. Zearalenone also increased the activity of hepatic 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), the enzyme believed to metabolize zearalenone to alpha- and beta-zearalenols. Dietary alfalfa did not overcome this effect. Alfalfa fiber was the only fraction to partially overcome the growth-depressing effects of zearalenone while the other fractions had no beneficial effects and 3 alpha-HSD was not affected by diet. None of the purified components affected growth parameters or 3 alpha-HSD. The enzyme was also not affected by zearalenone or alfalfa in swine diets. Coumestrol, alpha-zearalenone, and beta-zearalenone were shown to be competitive inhibitors of 3 alpha-HSD in rat liver. It was concluded that the fiber fraction of alfalfa protects against zearalenone toxicity, and that this effect is not dependent on coumestrol or saponin and is not likely mediated through 3 alpha-HSD.     

Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

Natural occurrence of zearalenone in rice and soybean produced in Korea

88 rice and 75 soybean samples were collected from 8 provinces of Korea from March through September in 1988. The Fusarium mycotoxins, zearalenone was analyzed by direct competitive enzyme linked immunosorbent assay. 10.2% of rice and 9.3 % of soybean samples contained detectable zearalenone. The average levels of zearalenone of rice and soybean samples were 11.78μg/kg and 7.70μ/kg, respectively.     

The effects of zearalenone on reproduction in swine. I. The relationship between ingested zearalenone dose and anestrus in non-pregnant, sexually mature gilts

Ninety-nine sexually mature, non-pregnant gilts were checked for estrus daily with a mature boar and then allocated at estrus (D O) to receive 2 kg/d of a diet containing 0, 1, 5 or 10 ppm purified zearalenone between D 5 and 20 of the estrous cycle during two seasons of the year (winter and summer). None of the gilts exhibited any visual signs of "hyperestrogenism" and there was no effect of season on interestrous interval (P > 0.05). A significant effect of zearalenone dose on inter-estrous interval was detected (P < 0.001). Gilts receiving 0 or 1 ppm had similar inter-estrous intervals (21.0 +/- 0.3 and 21.5 +/- 0.8 d, respectively) whereas gilts receiving 5 and 10 ppm had extended cycles (29.2 +/- 2.9 and 32.7 +/- 3.3 d, respectively). Plasma progesterone concentrations at D 19 to 21 were higher in gilts with extended cycles (P < 0.001) and corpora lutea (CL) were present at laparotomy. Some 86% of these retained CL underwent spontaneous regression resulting in the onset of estrus within the next 30 d. Fecal zearalenone concentrations rose during ingestion of contaminated diets and declined to pretreatment values within 2 d (1 ppm) to 8 d (10 ppm) of the cessation of treatment. These data show that feeding zearalenone at concentrations of 5 to 10 ppm from D 5 to 20 of the estrous cycle causes luteal maintenance and extended inter-estrous intervals. Spontaneous regression of these CL usually occurs within 30 d after zearalenone is removed from the diet. Fecal zearalenone analysis does not appear to be an effective method for determining prior exposure to zearalenone when carried out more than a few days following the last ingestion of zearalenone.     

Deoxynivalenol, acetyl deoxynivalenol, and zearalenone formation by Canadian isolates of Fusarium graminearum on solid substrates.

Three isolates of Fusarium graminearum (DAOM 180377, 180378, and 180379) were screened for their ability to produce mycotoxins on the solid substrates corn and rice. They all produced deoxynivalenol and zearalenone on corn. On rice, only DAOM 180378 and 180379 produced significant amounts of these mycotoxins, with levels of deoxynivalenol being much higher than those of zearalenone. The effects of the initial moisture content before autoclaving, incubation temperature, and time were studied with isolate DAOM 180378. At 19.5 degrees C the main product was zearalenone, whereas at 25 degrees C both deoxynivalenol and zearalenone were formed. Higher incubation temperatures (28 degrees C) favored deoxynivalenol formation, the maximum amount being 515 ppm (515 micrograms/g) formed after 24 days at an initial moisture content of 40%. The maximum level of zearalenone produced at the same temperature was 399 ppm, but at an initial moisture content of 35%. Other factors, such as pH, oxygen and carbon dioxide concentrations, and size of the culture flask also appeared to affect the production of mycotoxins.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Zearalenone-producing species of Fusarium on barley seed

Isolates of Fusarium were obtained from samples of barley grain at harvest in 1971, 1973 and 1974. The ability of these isolates to produce the myco-toxin zearalenone was examined, the grain also being tested for its presence in 1974. The most common isolate was F. culmorum, 60% of which in 1974 produced zearalenone. Other species isolated which produced this myco-toxin were F. monilijorme, F. graminearum, F. avenaceum, F. nivale and F. sambucinum var coeruleum, the last three species have not previously been recorded as producing zearalenone.     

Role of Zearalenone Lactonase in Protection of Gliocladium roseum from Fungitoxic Effects of the Mycotoxin Zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of ≤10 μg/ml. The fungitoxic effect declined in the order zearalenone > α-zearalenol > β-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

A simple method of determining zearalenone in cereals by liquid chromatography

Summary The paper described a simplified method for the separation, identification and determination of zearalenone by liquid chromatography in cereals. The cereal medium is dried, homogenised and extracted with ether. From the ether extract zearalenone is transferred into ethyl acetate, whereafter the sample is analysed using an external standard. The detection limit of the method is 500 g/kg. Recoveries of zearalenone varied from 94.5% to 96.2%.The conditions of the liquid chromatography are described.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.