Mold occurrence and aflatoxin B(1) and fumonisin B(1) determination in corn samples in Venezuela

Fumonisins are mycotoxins produced mainly by Fusarium moniliforme and Fusarium proliferatum, which have been associated with several animal and human diseases. Aflatoxins are hepatotoxic, mutagenic, and teratogenic metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Both have been reported at high levels in corn. This study was pursued to determine mold, aflatoxin B(1) (AFTB(1)), and fumonisin B(1) (FB(1)) levels in white and yellow corn. Mold levels were determined using potato dextrose agar and identification of the main genus of molds present in corn, AFTB(1) levels by immunoaffinity chromatography, and FB(1) levels by a Bond-Elut SAX cartridge and HPLC. AFTB(1) an     

Evaluation of conventional and organic italian foodstuffs for deoxynivalenol and fumonisins B(1) and B(2)

Two lots of human foodstuffs from conventional and organic brand foods were purchased from supermarkets and analyzed for three Fusarium toxins, deoxynivalenol, by GC-ECD, and fumonisins B(1) and B(2) (FB(1)-FB(2)), by LC-MS. The occurrence of deoxynivalenol contamination was higher than 80% in both organic and conventional foods; fumonisin B(1) was found in 20% of organic foods and in 31% of conventional ones and fumonisin B(2) in more than the 32% of the food samples from both the agricultural practices. The highest median concentration of deoxynivalenol occurred in conventional rice-based foodstuffs (207 microg/kg): that of fumonisin B(1) in conventional maize-based foods (345 microg/kg) and that of fumonisin B(2) in organic wheat-based foods (210 microg/kg).     

Surveillance of fumonisins in maize-based feeds and cereals from spain.

A survey has been carried out to determine the levels of fumonisins in 171 samples of maize-based feeds and cereals available in Spain. Also, the samples were examined for mold count and fungal species. Aspergillus, Penicillium, and Fusarium were the most frequent genera, and Fusarium and Aphanocladium had the highest individual percentage counts. Regarding Fusarium species, F. moniliforme (47. 4%) was the predominant species; F. proliferatum (5.3%) and F. subglutinans (7.0%) were isolated at low frequency. The high-performance liquid chromatography-o-phthaldialdehyde fluorescence method was used for the analysis of fumonisins. The highest levels of fumonisins were detected in maize. Overall, fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) were detected in 79.5 and 14.6%, of samples respectively, with average FB(1) levels of 3.3 microg/g and average FB(2) levels of 1.7 microg/g. Low levels of fumonisins in wheat, barley, and soybean were detected. This would appear to be the first report of concentrations of fumonisins in these commodities.     

Reduction of fumonisin B1 in corn grits by single-screw extrusion

This study was designed to determine the efficacy of extrusion in reducing fumonisin B1 in corn flaking grits in the presence and absence of glucose. In addition, degradation products of fumonisin B1 during extrusion were identified and quantitated with a mass balance approach. Uncontaminated clean corn grits, grits spiked with 30 microg/g fumonisin B1, and grits fermented with Fusarium verticillioides M-2552 (40-50 microg/g fumonisin B1) were extruded in the presence and absence of glucose (10%, w/w) using a single-screw extruder. Extrusion decreased fumonisin B1 by 21-37%, whereas the same process with added glucose further decreased fumonisin B1 by 77-87%. LC-fluorescence and LC-MS showed that most fumonisin in the extruded samples without added glucose was the fumonisin B1 form, whereas the main degradation product in grits extruded with glucose was N-(deoxy- d-fructos-1-yl)fumonisin B1. The formation of hydrolyzed fumonisin B1 was not significant during extrusion. Results suggest that extrusion in the presence of glucose may reduce fumonisin B1 in corn grits significantly.     

Incidence of Fusarium verticillioides and levels of fumonisins in corn from main production areas in Iran

A total of 52 corn samples collected in 2000 from four main corn production provinces of Iran (Fars, Kermanshah, Khuzestan, and Mazandaran) were analyzed for contamination with Fusarium verticillioides and fumonisins (FB(1), FB(2), FB(3), and 3-epi-FB(3)). The mean incidence of F. verticillioides (percent of kernels infected) for these four areas was 26.7, 21.4, 24.9, and 59.0%, respectively. The incidence in Mazandaran was significantly (p < 0.05) above that of the other areas. All samples from Mazandaran were contaminated with fumonisins with a mean level of total fumonisins of 10674 microg/kg. In contrast, the incidence of fumonisin contamination above 10 microg/kg was 53 (8/15), 42 (5/12), and 57% (8/14) in the samples from Fars, Kermanshah, and Khuzestan, respectively, and the corresponding mean total fumonisin levels were 215, 71, and 174 microg/kg, respectively. No statistical differences (p > 0.05) were observed in the fumonisin levels of the corn samples from these three provinces, which were significantly (p < 0.05) lower than the fumonisin contamination in samples from Mazandaran.     

Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn silage

Corn silage was dried, ground, and then extracted with 0.1 M ethylenediaminetetraacetic acid. The filtrate was applied to a FumoniTest immunoaffinity column. Fumonisins were derivatized with naphthalene-2,3-dicarboxaldehyde, separated on a C(18) liquid chromatographic column, and detected by fluorescence. The detection limits for fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 50, 25, and 25 ng/g of dried silage, respectively. Recoveries of fumonisin B(1), fumonisin B(2), and fumonisin B(3) from wet and dried corn silage spiked over the range of 100-5000 ng/g averaged 91-106%. The method was applied to corn silage samples collected from the midwestern area of the United States during 2001-2002. Of 89 corn silage samples, fumonisin B(1), fumonisin B(2), and fumonisin B(3) were found in 86 (97%), 64 (72%), and 51 (57%) of the samples. The mean positive levels of fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 615, 93, and 51 ng/g, respectively, in dried silage. This suggests that fumonisins may be frequent low level contaminants in corn silage.     

Natural occurrence of fumonisins B(1) and B(2) in domestic maize of Taiwan.

Samples of maize grown in various districts of Taiwan were collected and analyzed for the presence of fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) using high-performance liquid chromatography. Forty-nine (44.5%) and 2 (1.8%) of 110 samples were found to contain FB(1) (109-1148 ng/g) and FB(2) (222-255 ng/g), respectively. The frequency of detection and also the maximum FB(1) concentration were found in samples from Penton (2/2, 262 ng/g), followed by Chiayi (18/26, 264 ng/g), Tainan (8/16, 160 ng/g), Hualinen (5/14, 1148 ng/g), Taitung (7/20, 109 ng/g), and Yunlin (9/26, 361 ng/g). Of the 110 samples examined, only 2 samples from Hualinen had been detected containing FB(2). During an analysis of the distribution pattern of FB(1), it became apparent that >79% of tested samples had FB(1) concentrations <100 ng/g, whereas 2.7% (or 3 samples) contained FB(1) >300 ng/g. These results clearly illustrated that domestically produced maize for human consumption is frequently contaminated with FB(1).     

Development of a sensitive ELISA for the determination of fumonisin B(1) in cereals

Monoclonal fumonisin B(1) antibodies with high titer were raised by using FB(1)-glutaraldehyde-keyhole limpet hemocyanin immunogen prepared by a short cross-linker reagent (glutaraldehyde). Mean cross-reactivities of the selected monoclonal antibody for FB(1), FB(2), and FB(3) were 100, 91.8, and 209%, respectively; no reactivity was found with hydrolyzed fumonisin. A direct, competitive enzyme-linked immunosorbent assay for the quantitative determination of FB(1) in cereals has been developed with this antibody. Fifty percent acetonitrile-based solvent with some additives was used for extraction of cereals, and the diluted extracts were used without cleanup in the test. The mean within-assay and interassay coefficients of variation for the standard curve were <10%. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB(1). The toxin recovery from cereals infected with 50-200 ng/g of FB(1) varied between 61 and 84%. According to the comparable results of naturally infected maize samples, this test proved to be suitable for the rapid screening of food and feed samples for the presence of FBs.     

Occurrence of fumonisins B(2) and B(4) in retail raisins

Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.     

Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi

Resistance to mycotoxin contamination was compared in field samples harvested from 45 commercial corn (maize) hybrids and 5 single-cross aflatoxin-resistant germplasm lines in years with high and moderate heat stress. In high heat stress, mycotoxin levels were (4.34 +/- 0.32) x 10(3) microg/kg [(0.95-10.5 x 10(3) microg/kg] aflatoxins and 11.2 +/- 1.2 mg/kg (0-35 mg/kg) fumonisins in commercial hybrids and 370 +/- 88 microg/kg (140-609 microg/kg) aflatoxins and 4.0 +/- 1.3 mg/kg (1.7-7.8 mg/kg) fumonisins in aflatoxin-resistant germplasm lines. Deoxynivalenol was detected (one-fourth of the samples, 0-1.5 mg/kg), but not zearalenone. In moderate heat stress, mycotoxin levels were 6.2 +/- 1.6 microg/kg (0-30.4 microg/kg) aflatoxins and 2.5 +/- 0.2 mg/kg (0.5-4.8 mg/kg) fumonisins in commercial hybrids and 1.6 +/- 0.7 microg/kg (0-7 microg/kg) aflatoxins and 1.2 +/- 0.2 mg/kg (0.5-3.0 mg/kg) fumonisins in aflatoxin-resistant germplasm lines. The results are consistent with heat stress playing an important role in the susceptibility of corn to both aflatoxin and fumonisin contamination, with significant reductions of both aflatoxins and fumonisins in aflatoxin-resistant germplasm lines.     

Substrate suitability of different genotypes of sorghum in relation to Aspergillus infection and aflatoxin production

Grain sorghum is often damaged by rain in the field and severely infected by grain mold, which includes Aspergillus infection and aflatoxin production. The objective of the study is to investigate the extent of aflatoxin production with Aspergillus infection in vitro in different sorghum genotypes with different pericarps, red, yellow, and white, the physical and chemical characteristics of grain during infection, and the changes in grain polyphenols and phytic acid in comparison to maize and groundnut. The physical characters and biochemical composition of sorghum grain contribute to make it less susceptible to Aspergillus infection and aflatoxin contamination compared to maize and groundnut. The lowest amounts of aflatoxin and ergosterol were observed in genotypes with red pericarp, whereas higher amounts of aflatoxin and ergosterol were found in white genotypes followed by maize and groundnut. All of the red genotypes differ in polyphenol composition and aflatoxin produced, showing resistance to mold damage. Another indication of resistance in red genotypes was the delayed peaking of aflatoxin production (9 days after infection). In red sorghum genotypes there was a significant, positive correlation existing between polyphenol content and aflatoxin produced at 3 and 6 days after infection, the r values being 0.589 and 0.513, respectively. The starch content decreased whereas the protein content in all sorghum genotypes increased during infection. Maximum phytic acid was observed in white sorghum genotypes. Phytic acid in yellow genotypes was found to have a significant negative correlation (r = -0.569) with aflatoxin produced.     

Excretion of (14)C-fumonisin B(1), (14)C-hydrolyzed fumonisin B(1), and (14)C-fumonisin B(1)-fructose in rats.

14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form.     

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.     

Maize (Zea mays L.) genetic factors for preventing fumonisin contamination

Fusarium moniliforme and Fusarium proliferatum are the most frequently isolated fungi from maize (Zea mays L.) in Spain. Both Fusarium species produce toxins potentially dangerous for animals and humans, the fumonisins being the most significant of those toxins. White maize is preferred for human consumption, and extra care should be taken to avoid kernel mycotoxin contamination. The objectives of this study were to identify and quantify kernel infection by Fusarium spp. and contamination by fumonisin on white maize hybrids, to search for white maize sources of resistance to infection by Fusarium spp. and mycotoxin contamination, and to preliminarily study the genetics involved in such resistances. Ten F(1) single crosses derived from a diallel mating design among five white maize inbreds were evaluated in a randomized complete block design with three replications in 2002 at two locations. Fusarium verticilloides and F. proliferatum were detected on kernels of white maize hybrids cultivated in northwestern Spain. No differences in fungal infection were found among maize genotypes, but differences in fumonisin contamination were significant and could be related, in part, to differences in husk tightness. Among the genotypes studied, general combining ability (GCA) effects were the most important for resistance to fumonisin contamination. Inbreds EP10 and EC22 showed the most favorable GCA effects for husk tightness and fumonisin content, and the cross between them, EP10 x EC22, had the most favorable specific combining ability (SCA) effect for husk tightness. Inbreds EP10 and EC22 showed favorable GCA effects for fumonisin contamination and husk tightness, and the cross EP10 x EC22 was the only one with an average fumonisin level below 1 mug/g. Although this should be confirmed with more extensive studies, white maize inbreds developed from white maize landraces could be sources of resistance to fumonisin contamination.     

Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B(1), 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B(1), 67%; G(1), 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.     

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).     

Analysis of fumonisin B(1) in Fusarium proliferatum-infected asparagus spears and garlic bulbs from Germany by liquid chromatography-electrospray ionization mass spectrometry

Fusarium proliferatum is one of a group of fungal species that produce fumonisins and is considered to be a pathogen of many economically important plants. The occurrence of fumonisin B(1) (FB(1)) in F. proliferatum-infected asparagus spears from Germany was investigated using a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB(1)-d(6) as internal standard. FB(1) was detected in 9 of the 10 samples in amounts ranging from 36.4 to 4513.7 ng/g (based on dry weight). Furthermore, the capability of producing FB(1) by the fungus in garlic bulbs was investigated. Therefore, garlic was cultured in F. proliferatum-contaminated soil, and the bulbs were screened for infection with F. proliferatum and for the occurrence of fumonisins by LC-MS. F. proliferatum was detectable in the garlic tissue, and all samples contained FB(1) (26.0-94.6 ng/g). This is the first report of the natural occurrence of FB(1) in German asparagus spears, and these findings suggest a potential for natural contamination of garlic bulbs with fumonisins.     

Aflatoxins and fumonisins in corn from the high-incidence area for human hepatocellular carcinoma in Guangxi, China

A comparative study on the natural occurrence of aflatoxins and Fusarium toxins was conducted with corn samples from high- and low-incidence areas for human primary hepatocellular carcinoma (PHC) in Guangxi, China. In samples from the high-risk area, aflatoxin B(1) was the predominant toxin detected in terms of quantity and frequency, with its concentration ranging between 9 and 2496 microg/kg and an 85% incidence of contamination. Among the samples, 13 (76%) exceeded the Chinese regulation of 20 microg/kg for aflatoxin B(1) in corn and corn-based products intended for human consumption. Significant differences in aflatoxin B(1), B(2), and G(1) and total aflatoxin concentrations in corn between the areas were found (P < 0.05). The average daily intake of aflatoxin B(1) from corn in the high-risk area was 184.1 microg, and the probable daily intake is estimated to be 3.68 microg/kg of body weight/day, 3.20 times the TD(50) in rats. Corn samples from both areas were simultaneously contaminated with fumonisins B(1), B(2), and B(3). Aflatoxin B(1) may play an important role in the development of PHC in Guangxi.     

Inhibition of aflatoxin B(1) biosynthesis in Aspergillus flavus by anthocyanidins and related flavonoids.

Anthocyanidins and precursors or related flavonoids were tested at concentrations from 0.3 to 9.7 mM ( approximately 0.1-3.0 mg/mL) for activity against growth and aflatoxin B(1) biosynthesis by Aspergillus flavus Link:Fr. NRRL 3357. Aflatoxin B(1) production was inhibited by all anthocyanidins tested, and 3-hydroxy compounds were more active than 3-deoxy forms. Monoglycosides of cyanidin were 40% less inhibitory than the aglycon, whereas a monoglucoside and a diglucoside of pelargonidin were 80 and 5%, respectively, as active as the aglycon. Of eight flavonoids tested, only kaempferol was moderately active, whereas luteolin and catechin were weakly inhibitory. Binary combinations of delphinidin and three other aflatoxin inhibitors acted independently of each other. Results with an aflatoxin pathway mutant indicated that anthocyanidin inhibition occurred before norsolorinic acid synthesis.     

Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin

Aflatoxicol (AFL) and aflatoxins B1 and M1 were found in tissues (kidney, liver, and muscle) of feeder pigs given an estimated LD50 oral dose of B1 (1.0 mg/kg body weight) provided as a rice culture of Aspergillus flavus and of market-weight pigs fed a naturally contaminated feed, containing aflatoxin B1 at a level of 400 ng/g from corn, for 14 days. The residues in all tissues decreased with time after treatment in both groups, with no detectable residues (approximate detection limits, ng/g, B1 0.03, M1 0.05, AFL 0.01) in pig tissues from the feeding experiment 24 h after withdrawal of aflatoxin-contaminated feed. B1 and M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the dominant aflatoxin. The level of AFL, when detected, was about 10% of the B1 level.