Cytotoxicity of Fusarium mycotoxins to mammalian cell cultures as determined by the MTT bioassay.

Fusarium mycotoxins occur worldwide in cereal grains and animal feeds and cause outbreaks of Fusarium mycotoxicoses in humans and animals. In this study mammalian cell cultures were used to screen the cytotoxicity of the most common Fusarium mycotoxins; deoxynivalenol (DON), zearalenone (ZEN), fumonisin B(1) (FB(1)) and moniliformin (MON). The most sensitive cell line for each Fusarium mycotoxin was determined for further toxicological investigations as an alternative to whole animal testing. Chinese hamster ovary cells (CHO-K1) were found to be the most sensitive for DON and FB(1) with IC(50) values of 0.27 and 85.5 microg/ml, respectively, after 48-h exposure. The hepatocellular carcinoma cells (HepG2) showed the highest sensitivity to MON with IC(50) values of 39.5 for 48 h and 26.8 microg/ml for 72-h exposure. Balb/c mice keratinocyte cell line (C5-O) was found to be the most sensitive to ZEN with IC(50) of 24.1 microg/ml after 72-h exposure. DON was found the most cytotoxic to the cell cultures of all the mycotoxins tested, followed by MON, ZEN, and FB(1). The results indicated that CHO-K1, C5-O, and HepG2 cells were found to be the sensitive cell lines for preliminary screening of DON, ZEN and MON contaminated feed and food extracts, respectively.

Antifungal activity of sodium acetate and Lactobacillus rhamnosus.

The inhibition of molds by sodium acetate in deMan Rogosa Sharpe (MRS) medium, along with the antifungal activity of Lactobacillus rhamnosus VT1, was studied by the slope agar plate method. MRS agar prepared with and without sodium acetate was used as the agar substrate. A total of 42 strains of Aspergillus, Penicillium, Fusarium, Alternaria, Cladosporium, and Rhizopus were used to compare sensitivities to the inhibitory activity of sodium acetate and L. rhamnosus VT1. It was found that sodium acetate in MRS medium affected the growth of 33 of the 42 mold strains tested to various degrees. The highest sensitivity to sodium acetate was shown by strains of Fusarium, followed by strains of Penicillium, Aspergillus, and Rhizopus. L. rhamnosus VT1 also inhibited mold growth. A significant finding was that sodium acetate and L. rhamnosus VT1 in combination exhibited a possible synergistic action. Thirty-nine of the 42 mold strains tested were completely inhibited by the presence of both antifungal agents. This finding confirms that sodium acetate, a basic component of commercial MRS medium, has strong antifungal properties, and this must be taken into consideration when evaluating the antifungal activity of Lactobacillus cultures grown in MRS broth.

Relationship of mould count, ergosterol and ochratoxin A production.

The relationship between viable mould count, ergosterol content and ochratoxin A (OA) formation was studied at different inoculum concentrations of Aspergillus ochraceus NRRL 3174 and Penicillium verrucosum NRRL 3260 grown on sterile long-grain enriched white rice as the substrate. Ergosterol was determined by extraction, saponification and quantification using high performance thin layer chromatography (HPTLC) with UV detection. Ergosterol and ochratoxin A were detected after 3 days of incubation and reached their maximum at 7-10 days of incubation. After that, a decline in the concentrations in both ergosterol and ochratoxin was observed. Ergosterol measurement by HPTLC appeared to be a useful method to detect fungal activity, which corresponded to ochratoxin production. Thus, the ergosterol assay may have a use as an early indicator of potential mycotoxin production.

Thermal stability of moniliformin at varying temperature, pH, and time in an aqueous environment.

Moniliformin (MON) is a widely occurring mycotoxin, produced mainly by Fusarium proliferatum and Fusarium subglutinans in corn, that has been shown to be acutely toxic for various animal species and is a suspected cause of Keshan disease in China. The effects of temperature (100, 125, and 150 degrees C) and pH (4, 7, and 9) on the stability of MON were determined in aqueous buffer solutions at processing times ranging from 10 to 60 min. The percentage of MON reduction was positively related to increasing temperature and pH. MON was most stable at pH 4. After 60 min at pH 4 and 150 degrees C, MON was reduced by only 5%. Heating at pH 10 caused major reduction of MON. After 60 min at pH 10 and 100, 125, and 150 degrees C, MON was reduced by 56, 72, and 83%, respectively. One trial done at 175 degrees C and pH 10 showed that less than 1% MON remained after 60 min of processing.

Occurrence of fumonisins and moniliformin in corn and corn-based food products of U.S. origin.

Food-grade corn and corn-based food products intended for human consumption were analyzed for the incidence and levels of fumonisin B1 (FB1), fumonisin B2 (FB2), moniliformin, and Fusarium molds. A total of 100 food-grade commercial corn samples were obtained from two corn processing companies at five different locations in the United States. Seventy-one percent of the samples contained FB1 with concentrations ranging from 43 to 1,642 microg/kg. None of the samples contained FB2. Fifty percent of the samples contained moniliformin with concentrations ranging from 26 to 774 microg/kg. All samples were infected by Fusarium molds, and the infection rates ranged from 8 to 88%. Thirty-four samples of corn-based food products were purchased from supermarkets in Arizona, California, Nebraska, and Ohio. Sixty-five percent of the samples contained FB1, ranging in concentrations from 28 to 2,679 microg/kg. FB2 was detected in 29% of the samples with concentrations ranging from 30 to 797 microg/kg. Sixty-eight percent of the samples contained moniliformin with concentrations ranging from 31 to 858 microg/kg. Sixty-two percent of the samples contained viable Fusarium mold propagules ranging from 9.5 x 10(1) to 5.5 x 10(5)/g. The simultaneous occurrence of FB1 and moniliformin was observed in 34% of corn samples and 53% of corn-based food products. This study has shown co-occurrence of fumonisins and moniliformin in food-grade corn and corn-based foods that indicates a risk of simultaneous exposure of consumers to both toxins.

Fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum as affected by cycling temperatures.

The effects of temperatures cycling between 5 and 20 degrees C, 10 and 25 degrees C, and 15 and 30 degrees C on the production of fumonisin B1 (FB1) and ergosterol by Fusarium moniliforme and Fusarium proliferatum on rice was studied. Temperatures were cycled at 12-h intervals by manually moving cultures from one temperature to another. Constant temperature incubation at 25 degrees C and a low temperature stress were compared with the cycling temperature incubations. Low temperature stress was achieved by incubating rice cultures at 25 degrees C for 2 weeks followed by 15 degrees C for 4 weeks. The maximum yields of FB1 were found to be 247 microg/g by F. moniliforme at temperatures that cycled between 10 and 25 degrees C after 2 weeks and 284 microg/g by F. proliferatum when the temperatures cycled between 5 and 20 degrees C after 6 weeks. Ergosterol content of the rice cultures was also monitored. Overall, the two Fusarium species showed differences in production of FB1 and ergosterol under the various temperature treatments. The most notable differences were that the temperature treatments that stimulated greatest FB1 production were different for each species: cycling temperatures between 10 and 25 degrees C for F. moniliforme and cycling temperatures between 5 and 25 degrees C for F. proliferatum. At most temperatures, F. proliferatum produced more ergosterol than F. moniliforme. Maximum production of ergosterol by F. proliferatum occurred at 6 weeks, with temperatures that cycled between 10 and 25 degrees C, whereas F. moniliforme produced maximum amounts of ergosterol at 6 weeks, with temperatures that cycled between 15 and 30 degrees C.

Effect of cycling temperatures on the production of deoxynivalenol and zearalenone by Fusarium graminearum NRRL 5883.

The effects of three regimens of cycling incubation temperatures and incubation at constant 25 degrees C on the growth of Fusarium graminearum NRRL 5883 and production of deoxynivalenol (DON) and zearalenone (ZEN) on rice were compared. The effects of low-temperature stress were also studied by incubating rice cultures at a constant 15 degrees C for 4 weeks following incubation at constant 25 degrees C for 2 weeks. Both incubation temperature and time significantly (P < or = 0.05) affected growth of F. graminearum NRRL 5883 and production of DON and ZEN. The highest amount of free ergosterol (640 microg/g culture material) that was used as a measure of fungal growth was found in cultures incubated at temperatures cycling between 15 and 30 degrees C during a 6-week period. The highest amounts of DON (1,679 microg/g culture material) and ZEN (603 microg/g culture material) were produced in cultures incubated at a constant 25 degrees C for 2 weeks prior to incubation at a constant 15 degrees C for an additional 4 weeks. Under cycling incubation temperatures, maximum amounts of DON (850 microg/g culture material) and ZEN (98 microg/g culture material) were produced in cultures incubated at temperatures cycling between 15 and 30 degrees C for 6 weeks. Overall, there was no correlation between mold growth and production of either DON or ZEN. However, DON production and ZEN production were correlated.

Stability of zearalenone during extrusion of corn grits.

The effects of extrusion cooking on the stability of zearalenone (ZEN) in spiked (4.4 microg/g) food-grade corn grits were investigated using a twin screw extruder. A ground rice culture material containing a high level of ZEN was used to spike the corn grits. The extrusion variables were screw type (mixing and nonmixing), temperature (120, 140, and 160 degrees C), and moisture content (18, 22, and 26%). Both unextruded and extruded samples were analyzed for ZEN by high-performance liquid chromatography. Extrusion cooking of the corn grits resulted in significant reductions of ZEN in grits extruded with either mixing screws or nonmixing screws, but use of mixing screws was somewhat more effective (66 to 83%) overall than nonmixing screws (65 to 77%). Greater reduction of ZEN was observed at either 120 or 140 degrees C than at 160 degrees C. The moisture content of corn grits was not a significant factor affecting reduction of ZEN during extrusion with either mixing or nonmixing screws.

Stability of deoxynivalenol in heat-treated foods.

The effects of high-temperature and -pressure processing of foods spiked with deoxynivalenol (DON) were examined. In extruded corn grits, extruded dry dog food, and autoclaved moist dog food, there were no significant reductions (P < 0.05) in DON after processing. Autoclaved cream-style corn showed a reduction in DON of only 12%. Overall, DON was stabile to the high temperature and pressure processes tested. The use of an alpha-amylase in the extraction method for analysis by an enzyme-linked immunosorbant assay (ELISA) improved the recovery of DON from the spiked extruded and autoclaved products by as much as 26% over the standard ELISA method.

Effect of processing on Fusarium mycotoxins.

Mycotoxins are secondary metabolites produced by a wide variety of fungal species that contaminate food or feed. Fumonisins (FUM), deoxynivalenol (DON) and zearalenone (ZEN) are examples of common mycotoxins in grains that have been shown to affect human and/or animal health. Physical, chemical and biological methods have been used for decontaminating grains containing these toxins. Some treatments reduce the concentration of mycotoxins while others are ineffective. For example, removal of damaged grain by density segregation can reduce DON and ZEN concentrations in corn and wheat. In contrast, thermal processing is usually ineffective for reducing the FUM and ZEN content of foods. More work is needed to identify effective methods for detoxifying mycotoxin contaminated food.

Occurrence of fumonisin B1 and B2 in beer.

A total of 29 nationally distributed brands of beer, representing 25 domestic US and four imported brands, were purchased in retail outlets in Lincoln, Nebraska and analysed for concentrations of fumonisin B1(FB1) and B2(FB2). Immunoaffinity column extraction and cleanup of fumonisins from the beer samples, coupled with detection and analysis by gradient high performance liquid chromatography (HPLC), provided a limit of quantitation for each toxin of 0.3 ng/ml. Of the brands of beer sampled, 86% were positive for FB1 and 41% were positive for FB2. No beer contained a detectable quantity of FB2 without a detectable quantity of FB1. The total fumonisin (FB1 + FB2) content of positive samples ranged from 0.3 to 12.7 ng/ml, with a mean concentration for all positive samples of 4.0 +/- 3.4 ng/ml (n = 25). Considering that the level of fumonisin contamination of corn in recent harvest years has been minimal, the results of this limited survey could represent levels associated with current agricultural and brewing practices.

High-performance liquid chromatographic method for the determination of moniliformin in corn.

A high-performance liquid chromatographic method using UV absorption was developed for determining moniliformin in corn. The toxin was extracted with water containing 1% tetrabutylammonium hydrogen sulfate (w/v). Paired moniliformin was partitioned into dichloromethane, which was evaporated to dryness at 50 degrees C. The residue was dissolved in water and applied to a disposable strong-anion exchange solid-phase extraction tube. Adsorbed moniliformin was eluted from the tube with 0.05M sodium dihydrogen phosphate monohydrate (pH 5). It was determined by ion-pair reversed-phase chromatography and UV measurement at 229 nm. The minimum detectable amount of pure moniliformin was 0.25 ng/injection (signal-to-noise ratio = 3:1). The detector response was linear from 0.25 to at least 20 ng. The limit of determination was 0.025 microgram/g corn. Recoveries of moniliformin from corn spiked at 0.025, 0.05, 0.25, and 1.0 microgram/g averaged 96.5, 96.2, 97.2, and 97.8% respectively.

Heat and pH alter the concentration of deoxynivalenol in an aqueous environment.

The effects of heat (100, 120, and 170 degrees C and pH (4.0, 7.0, and 10.0) on the stability of deoxynivalenol (DON) were measured in an aqueous buffer solution for different periods of time (15, 30, and 60 min). At pH 4.0 DON appeared to be very stable showing no destruction at 100 or 120 degrees C and only partial destruction at 170 degrees C after 60 min. At pH 7.0 DON was still stable but showed more destruction at 170 degrees C after 15 min. At pH 10.0 DON was partially destroyed at 100 degrees C after 60 min and was totally destroyed at 120 degrees C after 30 min and at 170 degrees C after 15 min.

Occurrence of fumonisins in corn-based food products.

Corn-based food products obtained from commercial outlets in three different parts of the U.S., Maryland, Nebraska, and Arizona were analyzed for total fumonisins by a commercial competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and for fumonisin B1 (FB1) by high-performance liquid chromatography (HPLC). The highest fumonisin concentrations were found in samples collected in Maryland, where all 18 samples were found positive for fumonisins (200 to 7,450 ng/g of food) by CD-ELISA and 15 of the 18 samples (83%) were found positive for FB1 (< 75 to 5,916 ng/g) by HPLC. Fumonisins were also detected by CD-ELISA in 14 of 15 samples collected in Arizona with concentrations ranging from 200 to 1,450 ng/g, but analyses by HPLC showed that only 8 of 15 samples (53%) were positive for FB1 (< 75 to 1,565 ng/g of food). Of the 23 samples collected in Nebraska, 20 (87%) were positive for fumonisins (200 to 2,500 ng/g) by CD-ELISA, but only 10 (44%) were positive for FB1 (< 75 to 927 ng/g) by HPLC. The highest fumonisin and FB1 concentrations were found in cornmeal samples, ranging up to 7,450 ng/g of cornmeal by CD-ELISA and 5,916 ng/g by HPLC. These findings indicate that there may be a risk of human exposure to fumonisins through the consumption of some corn-based foods.

Isolation and partial characterization of antifungal metabolites of Bacillus pumilus.

Antifungal metabolites produced by Bacillus pumilus in Potato Dextrose Broth (PDB) were isolated from culture supernatant fluid by precipitation with ammonium sulphate. The antifungal metabolites inhibited mycelial growth of many species of Aspergillus, Penicillium and Fusarium. They also inhibited production of aflatoxins, cyclopiazonic acid, ochratoxin A and patulin. The metabolites were heat-stable and remained active after sterilization at 121 degrees C for 15 min. Their activity was stable over a wide range of pH (2-10). The metabolites were resistant to hydrolysis by various proteases, peptidases and other enzymes. They were also resistant to denaturation by many protein-denaturing detergents except Nonidet P-40. The metabolites were soluble in water and relatively polar organic solvents. Chromatographic bioassay revealed that a crude precipitate of the metabolites contained only one compound with antifungal activity. The active compound did not form a fluorescent derivative with fluorescamine suggesting that the compound is either a cyclic polypeptide or a non-peptidic compound.

Stability of fumonisins in thermally processed corn products.

Little is known about the stability of fumonisins in corn-based foods during heating. This study investigated the effects of canning, baking, and roasting (dry heating) processes on the stability of fumonisins in artificially contaminated and naturally contaminated corn-based foods. All samples were analyzed for fumonisin levels by both a commercial enzyme-linked immunosorbent assay (ELISA) and a high-performance liquid chromatographic (HPLC) method. Canned whole-kernel corn showed a significant (P < or = 0.05) decrease in fumonisins by both ELISA (15%) and HPLC (11%) analyses. Canned cream-style corn and baked corn bread showed significant (P < or = 0.05) decreases in fumonisin levels at an average rate of 9% and 48%, respectively, as analyzed by ELISA. Corn-muffin mix artificially contaminated with 5 micrograms of fumonisin B1 (FB1) per g and naturally contaminated corn-muffin mix showed no significant (P < or = 0.05) losses of fumonisins upon baking. Roasting cornmeal samples artificially contaminated with 5 micrograms of FB1 per g and naturally contaminated cornmeal samples at 218 degrees C for 15 min resulted in almost complete loss of fumonisins.

Anti-aflatoxigenic activity of Lactobacillus casei pseudoplantarum.

Lactobacillus casei pseudoplantarum 371 isolated from a silage inoculant was found to inhibit aflatoxins B1 and G1 biosynthesis by Aspergillus flavus subsp. parasiticus NRRI. 2999, in liquid medium. The inhibitory activity in the Lactobacillus cell-free supernatant was found to be sensitive to proteolytic enzymes such as trypsin and alpha-chymotrypsin, but resistant to pepsin. Lab-Lemco tryptone broth (LTB), supplemented with 20% of dialyzed protein concentrate of the supernatant, totally inhibited the production of aflatoxins B1 and G1. When the protein concentrate was digested with trypsin, the production of aflatoxins B1 and G1 was restored. The inhibitory activity of the supernatant was inactivated within 10 min at 100 degrees C. A. flavus grown in the Lactobacillus cell-free supernatant did not produce a mutagenic response in the Salmonella mutagenicity test. However, Lactobacillus casei pseudo plantarum 371 did not have an effect on aflatoxin production and mold growth as measured by ergosterol and plate count, when the organisms were inoculated together on sterile steamed rice.

Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus.

Six isolates of Bacillus pumilus were tested for their ability to inhibit aflatoxin production of Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) broth. Aflatoxin production was inhibited in both simultaneous and deferred antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolites(s) produced in cell-free supernatant fluids of cultured broth. The inhibition was not due to organic acids or hydrogen peroxide produced by B. pumilus since the inhibitory activity was not lost after pH adjustment or treatment of supernatant fluids with catalase. A range of media tested for the production of inhibitory metabolite(s) in supernatant fluids showed that all media supported bacterial growth and production of the metabolite(s). The metabolite(s) were produced over a wide range of temperature (25 to 37 degrees C) and pH (4 to 9) of growth of B. pumilus. They were stable over a wide range of pH (4 to 10) and were not inactivated after autoclaving at 121 degrees C for 30 minutes.

The use of the chicken embryo screening test and brine shrimp (Artemia salina) bioassays to assess the toxicity of fumonisin B1 mycotoxin.

Chicken embryos and brine shrimp naulpii were utilized in short-term toxicity bioassays to assess their sensitivity to the mycotoxin fumonisin B1 (FB1). Fertile chicken eggs (Cobb x) were dosed with FB1 on day 2 of incubation by the injection of 100 microliters of aqueous solution into the air space of each egg. Eggs were incubated with mechanical rotation until hatch, at which time mortality was assessed. Probit transformation of the mortality data produced a linear line of best fit (P < 0.05), from which an LD50 of 52 micrograms FB1/egg, equivalent to a concentration of 1.3 microns hatched in artificial seawater and exposed to FB1 in an optimized 96-well plate assay with a 48 hr mortality endpoint. Probit transformation of the mortality data resulted in an LC50 of 1.7 microns FB1, or 1.2 micrograms FB1/ml. Thus, at the cellular level, both bioassays appeared sensitive to FB1; however, from the standpoint of use as a screening assay, the chicken embryo bioassay is limited by the relatively high dose of FB1 required per egg. It is anticipated that the design and simplicity of the brine shrimp bioassay will accommodate screening for FB1 toxicity in contaminated samples.