Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

Production of fumonisin B1 and B2 byFusarium moniliforme isolated from Korean corn kerneis for feed

The natural occurrence of fumonisin B₁ (FB₁) and B₂ (FB₂), a promoter for hepatocarcinogenesis, was investigated in Korean corn kernels for feed by HPLC with fluorescence detection. From the 12 corn kernel samples, FB₁ was detected in 5 samples at levels ranging from 53 to 1327ng/g, while FB₂ was found in 4 samples from 69 to 680ng/g. In the positive samples, the average concentrations of FB₁ and FB₂ were 506 and 288ng/g, respectively. One sample (No. K3) showed the highest FB₁ and FB₂ contents as 1327 and 680ng/g, respectively. In the micrological survey on 5 positive samples for FB₁ and FB₂, 6 strains ofFusarium monlliforme were isolated, and all these isolates had a producibility of FB₁ and FB₁, with maximum levels of 80.7 to 180.9 g/g.Thisis the first report on the natural co-contamination of FB₁ and FB₂ in Korean corn kernels for feed, and on the ability ofF. moniliforme isolated from corn kernels for feed in Korea to produce FB₁ and FB₂.     

Determination of fumonisins in corn: Evaluation of two purification procedures

The performance of two solid phase extraction (SPE) purification procedures, used in the determination of fumonlsin B₁ (FB₁), B₂ (FB₂) and B₃ (FB₂) In corn, was evaluated using both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Fewer interferences were observed In extracts prepared using the strong anion exchange (SAX) media, in contrast to those purified on C₁₈ media, where on occasions, visual discernment of the TLC bands was hampered by the presence of interfering compounds. Precipitate formation, resulting In the blocking of SPE cartridges was also encountered when using the C₁₈ procedure. HPLC analyses of extracts prepared by both media indicated that they gave comparable fumonlsin recoveries from naturally contaminated corn samples. The results suggest that the C₁₈ procedure, originally developed for the TLC analyses of FB₁ in mixed feeds, may also be applied to the determination of FB₂ and FB₂. However, where TLC is used quantitatively for fumonlsin levels <1 μg/g, purification of sample extracts on SAX media is recommended.     

A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates

During the 1989 corn harvest season, numerous reports of equine leukoencephalomalacia (ELEM) outbreaks and a pulmonary edema (PPE) syndrome in swine from several regions of the United States were received by the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Previous and concurrent research linked Fusarium moniliforme and fumonisin-contaminated feeds to both diseases. Chemical and mycological investigations revealed fumonisin B₁ (FB₁) concentrations of 20 to 360 ppm in suspect swine feeds and 8 to 117 ppm in suspect equine feeds. Nonproblem feeds contained concentrations below 8 ppm. Fusarium moniliforme and Fusarium proliferatum were isolated from both problem and nonproblem equine and swine feeds. When cultured on autoclaved corn, the F. moniliforme and F. proliferatum isolates produced respective FB₁ and fumonisin B₂ (FB₂) that range from less than 5 to more than 2450 ppm and less than 5 to more than 1000 ppm, respectively. Isolates from both problem and nonproblem feeds produced high levels (greater than 500 ppm) in culture. Reported here is a review of chemical and mycological data resulting from the study of several cases of PPE and ELEM.     

Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina

Fusarium species and fumonisin production by toxigenic strains were investigated. During 1996–1998, 158 samples of poultry feeds were collected from a factory located in the department of Río Cuarto Córdoba province, Argentina. The most common species of Fusarium were F. moniliforme (60.7%) and F. nygamai (35.4%) followed by F. semitectum, F. subglutinans, F. proliferatum, F. dlamini, F. solani, F. oxysporum and F. napiforme. Fungal counts ranged from 1 × 10³ to 8 × 10⁵ CFU/g with mean values from 1.5 × 10³ to 2.3 × 10⁵ CFU/g. The highest counts were for F. dlamini, F. subglutinans, F. moniliforme and F. nygamai. Strains of F. moniliforme, F. nygamai, and F. proliferatum were screened for their potential to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) in corn grain. The samples were analysed using a modified high performance liquid chromatography method. The strains assayed, 43 strains, produced three fumonisins. There was a high degree of variability in the quantities of FB₁, FB₂, and FB₃ produced. The toxin produced in highest levels by the majority of the strains was FB₁. The range of concentration varied from 5.4 to 3,991, 1.01 to 189 and 0.4 to 765 ppm per gram of corn for FB₁, FB₂ and FB₃ respectively. The toxigenic pattern of strains was normal, although two strains of F. moniliforme produced exceptionally high concentrations of FB₃ and minor concentrations of FB₂ and FB₁. This is the first report from Argentina on Fusarium species in poultry feeds and fumonisin production by these strains.This revised version was published online in October 2005 with corrections to the Cover Date.     

Thermostability of Fumonisin B(1), a Mycotoxin from Fusarium moniliforme, in Corn

Fumonisin B(1) (FB(1)) is a mycotoxin from Fusarium moniliforme that is frequently associated with corn. Thermal treatments are used in many processes concerning this cereal and its derivatives. The thermostability of this toxin in dry contaminated corn, resulting from F. moniliforme culture, was studied in different time-temperature combinations. FB(1) was quantified by instrumentalized thin-layer chromatography after a two-step sequential development and postchromatographic derivatization by p-anisaldehyde. The identity of FB(1) in extracts, before and after heat treatments, was confirmed by high-pressure liquid chromatography. For each temperature, the natural logarithm of the ratio of resulting FB(1) on initial content (In C/C(0)) is linearly correlated to exposure time. The calculated half-lives (L(50)), corresponding to the 50% value, were 10 min, 38 min, 175 min, and 8 h at 150, 125, 100, and 75 degrees C, respectively. There is a linear relationship between calculated L(50)s on a logarithmic scale and temperature. Therefore FB(1) is not significantly destroyed by the main drying processes of corn or thermal treatments used for its derivatives. Other associated means are required for detoxification.     

Fumonisins: Isolation,chemical characterization and biological effects

The fumonisin B mycotoxins (FB₁ and FB₂) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B₁ (FB₁, the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB₁ have led to the identification of two new fumonisins, FB₃ and FB₄. Fumonisins A₁ and A₂, the N-acetyl derivatives of FB₁ and FB₂ respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05–0.1%, FB₂ and FB₃ closely mimic the toxicological and cancer initiating activity of FB₁ and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA₁ under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.     

Production of fumonisins B1, B2 and B3 byFusarium proliferatum Isolated from rye grains

Using the seed- plate technique, we have isolated a strain ofF. proliferatum from rye grains that produces 3 fumonisins, fumonisin B₁ (FB₁), FB₂ and FB₃ on inoculated rice and corn. Inoculated corn and rice were extracted with an aqueous methanol solution and fumonisin concentrations estimated using high performance liquid chromatography. Production of all 3 fumonisins (FB₁, FB₂ and FB₃) was much higher on rice than corn; ranging from 3816, 1068 and 985 ppm to 1643, 350 and 162 ppm respectively. We conclude that all natural substrates whereF. proliferatum is present as a component of the mycoflora should be monitored for the presence of fumonisins.     

Evalution of liquid media for fumonisin production byFusarium moniliforme MCR 826

Production of fumonlsins B₁ (FB₁) and B₂ (FB₂) by 5 lyophillzation batches ofFusarium moniliforme strain MRC 826 was studied in several liquid media and vermlculite supplemented with liquid media. In addition the effect of different parameters including pH, Inert material, shake versus stationary cultures as well as different carbon sources on the production of the fumonlsins were investigated. Fumonlsin production in liquid cultures was significantly (P<0.01) correlated (r=0.92–0.98) with fungal growth, which in turn is affected by the pH of the medium as well as the carbon source utilized. The highest FB₁ yields (approximately 40 mg/l) over the incubation period of 14 days were produced in a chemically defined medium with glucose as carbon source set at an initial pH value of 4. FB₁ production in “corn patty” cultures (approximately 1 to 3 g/kg), however, by far exceeded that obtained in the liquid media, while poor fungal growth and fumonlsin production was obtained in vermlculite supplemented cultures. From these studies it became clear that the ability of a culture to produce fumonlsins is determined by the interaction of a variety of physiological and nutritional factors regarding the inoculum and the culture medium.     

Bioaccessibility of total bound fumonisin from corn flakes

Fumonisin B₁ (FB₁) is often found as a natural contaminant of corn and corn-based food. Several publications have demonstrated the presence of fumonisin bound to proteins and to other compounds of the matrix. In spite of the low oral bioavailability of FB₁ in rats, pigs, chickens, cows, and monkeys, FB₁ can cause agriculturally significant disease and possibly human cancer. The aim of this work was to determine the bioaccessibility of total bound FB₁ (TB FB₁) (percentage of TB FB₁, released from corn flakes to the chyme) after in vitro digestion. Two samples of corn flakes washed with solvents were incubated with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices. After hydrolysis of the chyme with KOH, TB FB₁ was determined as hydrolyzed FB₁ (HFB₁). The bioaccessibility of TB FB₁ in chyme from corn flakes was 37–64%, indicating that these derivatives should be considered in evaluation of exposure to fumonisin.     

Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market

Corn-based human foodstuffs purchased in Taiwan were analyzed for fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) using high-performance liquid chromatography. Fifty-two (33.9%) and 32 (20.9%) of 153 samples were found to contain FB₁ (73–2395 ng/g) and FB2 (120–715 ng/g), respectively. The highest frequency of detection and also the highest FB1 concentrations were found in sweetcorn (50%, 1089 ng/g) and cornflour (50%, 608 ng/g), followed by corn snacks (33.3%, 2395 ng/g), miscellaneous corn products (33.3%, 73 ng/g), popcorn (31.8%, 1003 ng/g) and cornflakes (23.5%, 1281 ng/g). 16 corn snacks (= approximately 20.5% of the samples) had an average FB₁ and FB₂ content of 456 and 145 ng/g, respectively, while six sweetcorn (= 25%) samples were contaminated with an average of 400 ng/g of FB₁ and 65 ng/g of FB₂. Of the 22 pop-corn samples examined, 7 had an average of 347 ng/g and 116 ng/g of FB₁ and FB₂, respectively. During an analysis of the distribution pattern for the combined fumonisin levels of FB₁ and FB₂, it became apparent that more than 69% of tested samples had fumonisin concentrations below 100 ng/g, while 11.1% (or 17 samples) contained in excess of 600 ng toxins per g. These results clearly illustrated that commercially available corn-based foodstuffs for human consumption in Taiwan are frequently contaminated with FB₁ and FB₂.This revised version was published online in October 2005 with corrections to the Cover Date.     

Survival of Aspergillus flavus and Fusarium moniliforme in High-Moisture Corn Stored Under Modified Atmospheres

Freshly harvested high-moisture corn with 29.4% moisture and corn remoistened to 19.6% moisture were inoculated with Aspergillus flavus Link ex Fr. and stored for 4 weeks at about 27 C in air (0.03% CO₂, 21% O₂, and 78% N₂) and three modified atmospheres: (i) 99.7% N₂ and 0.3% O₂; (ii) 61.7% CO₂, 8.7% O₂, and 29.6% N₂; and (iii) 13.5% CO₂, 0.5% O₂, and 84.8% N₂. Kernel infections by A. flavus, Fusarium moniliforme (Sheld.) Snyd. et Hans., and other fungi were monitored weekly. The modified-atmosphere treatments delayed deterioration by A. flavus and F. moniliforme, but their growth was not completely stopped. A. flavus survived better in the remoistened than in the freshly harvested corn. F. moniliforme survived in both. A. flavus and F. moniliforme were the dominant fungi in corn removed from the modified atmospheres and exposed to normal air for 1 week.     

Mycoflora and fumonisin-producing strains ofFusarium moniliforme in mixed poultry feeds and component raw material

In a survey of the mycoflora and mycotoxins in foods and feeds, 66 samples of mixed poultry feeds and some component raw materials were investigated. Fungal counts ranged from < 10² to 1.3 × 10⁶ CFU/g.Fusarium spp. counts ranged from 10² to 1.0 × 10⁶ CFU/g. TheFusarium spp. strains isolated were screened for their potential to produce fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in maize cultures. Samples and maize cultures were analysed for FB₁ and FB₂ using TLC and fluorescamine-derivative HPLC. No fumonisins were detected in the samples (<6 ppm).Fusarium moniliforme was isolated in 59.1% of samples, and 97.4% of the strains produced FB₁ and 79.4% of strains produced FB₂ in maize cultures. Some isolates produced higher FB₁ and FB₂ levels than the reference strainF. moniliforme MRC 826.     

Effect of<Emphasis Type="Italic">in vitro</Emphasis> digestion on fumonisin B<Subscript>1</Subscript> in corn flakes

Low levels of fumonisins have been found frequently in corn based breakfast cereals and can occur bound to protein and other matrix components.In vitro digestion of two samples of corn flakes was carried out under "fed conditions." Fumonisins were measured as o-phthaldialdehyde/mercaptoethanol derivatives by LC-fluorescence. One sample of corn flakes (FN12) had high concentrations of fumonisin B₁ (FB) (average 125 ng/g) and total bound FB₁, (TB FB₁) (average 92 ng/g) and the other (FN11) had a low level of free FB₁ (average 29 ng/g) and no detectable TB FB₁. After incubation of the samples with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices, chyme was analysed for FB₁, hydrolyzed FB₁ (HFB₁) and partially hydrolyzed fumonisin B₁ (PHFB₁). The bioaccessibility (percentage of FB₁ released from corn flakes into chyme) was 38-78% for incurred FB₁ in FN12, 8-54% for incurred plus spiked FB₁ in FN12, and 19-66% for incurred plus spiked FB₁ in FN11. HFB₁ and PHFB₁ were not detected. If free FB₁ was first extracted from sample FN12, no FB1 was detected in the chyme, indicating no contribution from TB FB₁. Concentrations were corrected for method recovery of FB₁ or, for bound FB₁, partial method recovery of HFB₁ Presented at the XIIth IUPAC International Symposium on Mycotoxins and Phycotoxins, Istanbul, Turkey, 21–25 May, 2007     

Preliminary determination of mycotoxin binding to soil when leaching through soil with water

Mycotoxin-contaminated crops that are left in the field are potential contaminants of groundwater. Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) distribution in soil-water systems and the comparative response of aflatoxin-contaminated corn and pure aflatoxin when leached through soil were investigated using columns. Each experiment was repeated once. Eluates and soil extracts were analyzed for AFB₁ and its metabolites and FB₁ along with different amounts of pure FB₁ and water mixtures. AFB₁ was detected in water samples from columns containing 10% and 20% silty clay loam soil and aflatoxin-contaminated corn mixtures and in the upper (top) 2.5 cm of soil from the 10 cm soil column. Aflatoxin B₂ (AFB₂) was detected in eluates from the column containing 10% soil and aflatoxin-contaminated corn mixture and from the column containing aflatoxin-contaminated corn alone. No AFB₂ was detected in eluates from the column containing 20% soil and aflatoxin-contaminated corn mixture. No detectable amount of aflatoxin was observed in eluates from the containing 50% silty clay loam soil and aflatoxin contaminated corn. No detectable amount of FB₁ was observed in eluates or soil extracts, but FB₁ was detected in the mixtures of pure FB₁ and water.     

Screening toxicity study in young carp (Cyprinus carpio L.) on feed amended with fumonisin B1

Fumonisin B₁ (FB₁) is one of several mycotoxins produced by Fusarium moniliforme, a major fungal pathogen of corn and widely spread throughout the world. FB₁ produces a wide range of biological effects, some of which are specific for particular organs or species and some are common to all investigated animals. In this study we have evaluated subchronic toxicosis features in young carp (Cyprinus carpio L.) exposed to 0.5 and 5.0 mg FB₁ kg^–1 body weight for 42 days through nutritionally balanced diet. During the trial we observed loss of body weight in both treated groups, together with higher incidence of infective bacterial dermatological lesions erythrodermatitis cyprini (Aeromonas salmonicida subsp. nova) in the group treated with the higher FB₁ dose. Several hematological parameters (erythrocyte count, platelet count) and serum chemical concentrations (creatinin, total bilirubin) and activities (aspartate aminotransferase, AST and alanine aminotransferase, ALT) were greater in the fumonisin treated groups than in the control group. Our results indicate that long-term dietary exposure to 0.5 and 5.0 mg FB₁ kg^–1 body weight is not lethal to young carp, but can produce adverse physiological effects. These findings also suggest that primary target organs of FB₁ in the carp are kidney and liver, as it has already been observed in other animal species tested. Specifically changed red blood cell-parameters reveal that FB₁ probably causes erythrocyte membrane defect or interferes with carp's respiratory process.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.     

Factors affecting the growth of Fusarium proliferatum and the production of fumonisin B1: oxygen and pH

Fumonisins are mycotoxins produced primarily by Fusarium moniliforme and Fusarium proliferatum in corn. In liquid culture, production of fumonisin B₁ (FB₁), the most common moiety of the family of fumonisins, can be obtained using a defined medium that is nitrogen-limited. Under nitrogen-limited conditions both growth and the production of FB₁ were greatly influenced by pH and aeration. At pH above 5.0, F. proliferatum grew normally but produced little FB₁ (<100 μg m⁻¹). At pH below 5.0, there was less growth but substantially more FB₁. Below a pH of 2.5, both growth and metabolism were slower with very little FB₁ produced. When the optimal pH range of between 3.0 and 4.0 under well-aerated conditions was used, both growth and FB₁ production were high. However, under oxygen-limited conditions, less growth occurred, glucose consumption was increased, and no FB₁ was produced. Received 16 May 1997/ Accepted in revised form 03 September 1997