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Aflatoxin is degraded by heated and unheated mycelia, filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus.

Steaming one-half of a lot of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 for 6 min resulted in little or no subsequent degradation of aflatoxin B1 or G1 by these mycelia. The other half of these mycelia was not heat-treated and degraded aflatoxins B1 and G1. Filtrates of the growth substrate which remained after the mycelium was removed from 8- to 15-day old cultures of A. parasiticus NRRL 2999 did not degrade substantial amounts of aflatoxin B1 or G1, whereas mycelia originally produced on these filtrates degraded substantial amounts of both aflatoxins. The supernatant fluid from homogenates of 9-day-old mycelia of A. parasiticus NRRL 2999 degraded aflatoxins B1 and G1 when 0.1 M or 1.0 M phosphate buffer, pH 6.5, was used to suspend the homogenate. These data support the hypothesis that the aflatoxin degrading factor(s) present in the mycelium of A. parasiticus is/are enzyme(s) or at least influenced by enzyme(s).

Aflatoxins↗

Search for B-1 aflatoxin and similar fluorescent compounds in 'misso'.

Following previous studies on the investigation of B-1 Aflatoxin in food samples (4,5) we report in this paper the results obtained using thin layer chromatography (TLC) with three different solvent systems and spectrophotometric analysis in order to differentiate actual B-1 Aflatoxin and Aflatoxin-like substances. Twenty 'Misso' samples were studied and we could detect Aflatoxin in 3 of them. An Aflatoxin-suspected substance was also isolated. No Aflatoxin-producing Aspergillus was isolated from the samples analysed.

Aflatoxins↗

Experimental aflatoxin production in commercial yoghurt.

The production of aflatoxins in commercial yoghurt inoculated with Aspergillus parasiticus NRRL 2999 was studied, using different incubation conditions. In all of the experiments, the level of aflatoxins was higher at 28 degrees C than at 15 degrees C and higher in a "damaged" container than in an "intact" container (related to microaerophilic conditions). No fungal growth or aflatoxin production was seen at 10 degrees C. Both fungal growth and aflatoxin concentration vary throughout the incubation period instead of progressively increasing. The ratio of aflatoxin B and G (B:G) at 28 degrees C was almost 1:1, but generally more aflatoxin G was detected at 15 degrees C. The distribution in mycelium/substrate was approximately 1:1 at both 28 degrees C and 15 degrees C.

Aflatoxins↗

Analytical methods for aflatoxins in corn and peanuts.

Aflatoxin determinations can be approached many ways. Peanuts and corn are more often contaminated with aflatoxins B1 and B2 than with aflatoxins B1, B2, G1, and G2. Some countries are only interested in B1 content and others are interested in the total aflatoxin content. It is essential to safely handle all experimental materials associated with aflatoxin analyses or the aflatoxigenic fungi. Visual screening of suspect peanut lots, based on the presence of conidial heads of the Aspergillus flavus group, and screening corn for the presence of bright greenish yellow fluorescence (BGYF) are not chemical tests and such screening techniques may allow aflatoxin contaminated lots into commerce. Minicolumn screening procedures should always be used in conjunction with a quantitative method. Several thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods are suitable for quantitation and are in general use. Immunochemical methods such as the ELISA or affinity column methods are being rapidly developed. The chemical and immunochemical methods can be reliable if care is taken, using suitable controls and personnel that are well-trained. All analytical laboratories should stress safety and include suitable analytical validation procedures.

Aflatoxins↗

Evaluation of silica cartridge purification and hemiacetal formation for liquid chromatographic determination of aflatoxins in corn.

Rapid silica cartridge cleanup, acid-catalyzed conversion of aflatoxins B1 and G1 to hemiacetals, and reverse-phase liquid chromatography with fluorescence detection were evaluated for effectiveness in determining aflatoxins B1, B2, G1, and G2 in corn at concentrations ranging from 2 ng/g to 100 micrograms/g. In testing the method, aflatoxins applied to silica cartridges were recovered at greater than 97.1% overall. Conversion of aflatoxins B1 and G1 to their hemiacetals was shown to be complete (150 micrograms of toxin per extract). Application of these techniques to spiked corn extracts yielded data indicating excellent repeatability of derivatization relative to paired standards; coefficients of variation ranged from 1.08% to 5.81%. The repeatability of the method with naturally contaminated corn was also excellent; coefficients of variation ranged from 1.15% to 3.97%. Liquid chromatographic determination of aflatoxins in corn using fluorescence detection was sensitive, accurate, and precise resulting in applicability from less than 1 ng/g of aflatoxin B1 to greater than 100,000 ng/g.

Aflatoxins↗

The effect of zinc and phytic acid on the incorporation of 1-14C-acetate into aflatoxin by resting mycelia of Aspergillus parasiticus.

The effect of zinc and phytic acid on [1-14C]-acetate incorporation into aflatoxins by resting mycelium was studied. When different levels of ZnSO4 were used to study its effect on the incorporation of [1-14C]-acetate into aflatoxins, it was found that the specific radioactivity incorporation into aflatoxins was maximum at the level of 10 mM-ZnSO4. At this concentration the change in the specific radioactivities of aflatoxins B1 + B2 and aflatoxins G1 + G2 were +74.61% and +29.66%, respectively. On the other hand, phytic acid had an inhibitory effect on the incorporation of [1-14C]-acetate. These observations have been correlated in order to find out why soyabean is unable to produce aflatoxins by Aspergillus parasiticus.

Acetates↗

[Influence of raw milk processing on the aflatoxin M content of milk products (author's transl)].

Several contracdictory results in the literature lead us to the conclusion, that the influence of raw milk processing on aflatoxin content should be tested under as close as possible practical conditions. Even though storage at 5 degrees C for 1-3 days does not greatly influence aflatoxin content (11-25% reduction), this might still be important for food inspection. Although experiments on exposure to light resp. oxygen were repeated ten times, the results could not be definitely be shown as due to oxidation. Part of the contradictory literature references also in the case of freeze drying in our opinion caused by the way in which aflatoxins was added. The loss was less pronounced when milk was contaminated naturally than when it was added artificially. Heating of milk, depending on the conditions, caused a decrease of the aflatoxin-content of between 12 and 35% when making butter from naturally contaminated cream 23% (18-28%) of the aflatoxin M1 appeared in the butter, whereas the buttermilk contained the major amount of aflatoxin.

Aflatoxins↗

[Sampling of cheese for aflatoxins (author's transl)].

Due to the greatly differing incidence of molds on cheese and thus the extremely differing amounts of aflatoxin reliable sampling in cheeses is difficult, particularly if only one semi-hard of hard cheese is available. Experiments were undertaken now to sample--hopefully without losses and suitable for commercial application--in order to be able to ascertain the possible aflatoxin content of a cheese. Based on storage results with artificially contaminated Provolone and Tilsit cheeses as a pretest determination of aflatoxins in scrapings from a 100 cm2 surface are is recommended. At high levels (20-30 microgram/kg) the sampling of several borings at different locations on the cheese is necessary. The aflatoxin content on the surface is not constant, a fact which can be explained by biochemical reactions and by migration of aflatoxins into deeper layers. Washing of cheeses seems not to be of any significant influence on the aflatoxin-content.

Aflatoxins↗

The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.

Aflatoxin B1↗

Effect of fatty acids on aflatoxin production by Aspergillus parasiticus.

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.

Aflatoxins↗

Synthesis of allylthiopyridazine derivatives and inhibition of aflatoxin B1-induced hepatotoxicity in rats.

Five kinds of allylthiopyridazine derivatives were synthesized and their chemoprotective activities examined in rats exposed to aflatoxin B1-toxicant. Rats were pretreated with five allylthiopyridazine derivatives at daily oral doses of 50 mg/kg for 10 consecutive days, and during this period with one or three repeated doses of the potent hepatotoxin, aflatoxin B1. The hepatoprotective effects of the allylthiopyridazine derivatives against aflatoxin B1 (1 mg/kg, three times at intervals of 3 days, i.p., or at 3 mg/kg, once at final days, i.p.) administration were showed the significantly normal as compared with control in body and liver weights. Aspartate aminotransferase and alanine aminotransferase levels were markedly elevated after aflatoxin B1 administration, and pretreatment with allylthiopyridazine derivatives, before aflatoxin B1 administration, resulted in decreased levels of these enzymes. In addition, the allylthiopyridazine derivatives, K6 (3-methoxy-), K8 (3-chloro-), K16 (3-ethoxy-) and K17 (3-n-propoxy), induced elevated hepatic GSH levels. Four kinds of allylthiopyridazine derivatives investigated were effective against aflatoxin B1-induced hepatotoxicity.

Aflatoxin B1↗

Molecular genetic analysis and regulation of aflatoxin biosynthesis.

Aflatoxins, produced by some Aspergillus species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular mechanism of AFB biosynthesis showed that the genes required for biosynthesis are in a 70 kb gene cluster. They encode a DNA-binding protein functioning in aflatoxin pathway gene regulation, and other enzymes such as cytochrome p450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid synthases. Information gained from these studies has led to a better understanding of aflatoxin biosynthesis by these fungi. The characterization of genes involved in aflatoxin formation affords the opportunity to examine the mechanism of molecular regulation of the aflatoxin biosynthetic pathway, particularly during the interaction between aflatoxin-producing fungi and plants.

Aflatoxins↗

An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene.

Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.

Aflatoxins↗

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in Aspergillus flavus NRRL 3357 and Aspergillus parasiticus SRRC 143.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B(1) and B(2) biosynthesis, while A. parasiticus cultures had significantly increased B(1) and G(1) biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.

Aflatoxins↗

Aflatoxin genes and the aflatoxigenic potential of Koji moulds.

Sixty-four Aspergillus isolates, 54 of which originated from food fermentations, and 18 Aspergillus reference strains were identified and screened for the presence of aflatoxin genes aflR and omt-1. Among the Koji moulds, not only A. oryzae but also A. flavus strains were found. Furthermore, 27% of A. oryzae and 93% of A. flavus strains lacked either aflR or both aflR- and omt-1. A selection of 29 strains was also checked for the presence of pksA and nor-1. This revealed large deletions in the aflatoxin gene cluster of some strains. The hybridisation patterns also suggested a polarity in the deletion events, originating in the vicinity of pksA and extending towards omt-1. Other strains exhibited BamHI restriction fragment length polymorphisms (RFLPs) for either aflR or for aflR and omt-1. All aflR and/or omt-1 deletion strains turned out to be unable to produce aflatoxin. The RFLP-carrying strains either produced only traces of aflatoxin or none at all. In 73% of the A. oryzae strains, no apparent deletions were detected with the aflR and omt-1 probes. Nevertheless, after incubation in aflatoxin-inducing media, no aflatoxin B1 production could be detected in those A. oryzae strains.

Aflatoxins↗

Aflatoxin deposition and clearance in the eggs of laying hens.

Hens fed a diet containing 3310 micrograms of AFB1 and 1680 micrograms of AFB2 per kg feed for 28 days showed a significant decrease in egg production and egg weights by wk 3 and 4 of feeding, respectively. Transfer of aflatoxins to the eggs occurred rapidly, reaching maximum levels after 4-5 days, and remained relatively constant throughout aflatoxin feeding. The mean values for combined residue levels in eggs were less than 0.5 microgram/kg. Levels of AFB2, AFM1 and AFM2 were similar in yolk and albumen while levels of B1 and B2a were higher in the yolk. Upon removal of the aflatoxin-containing diet, residues in eggs decreased rapidly. Clearance of aflatoxin residues from the albumen occurred faster than from the yolk. Thus, no residues were detected in the albumen and in the yolk after 5 and 7 days of withdrawal, respectively. No aflatoxin residues could be recovered from whole eggs after feeding the aflatoxin-free diet for 4 days.

Aflatoxins↗

Levels of fumonisin B1 in corn naturally contaminated with aflatoxins.

Aflatoxins and fumonisin B1 are hepatotoxic and carcinogenic metabolites produced by Aspergillus flavus and Fusarium moniliforme, respectively. These fungi are common natural contaminants of corn, and both aflatoxins and fumonisin B1 have been implicated as aetiological agents in animal and human diseases. To determine whether these mycotoxins co-exist on corn under natural conditions, 28 samples from the 1991 Georgia (USA) corn crop were assayed for (total) aflatoxin and fumonisin B1. 27 samples were positive for aflatoxin, 24 samples were positive for fumonisin B1, and 23 samples had detectable levels of both. In the positive samples, the mean aflatoxin concentration was 73 ppb (SD = 86), and the average fumonisin B1 concentration was 0.87 ppm (SD = 0.65). A correlation between aflatoxin and fumonisin B1 concentrations was not evident. None the less, these results demonstrate that exposure to both mycotoxins can occur simultaneously by consumption of co-contaminated corn.

Aflatoxins↗

Effects of phyllosilicate clay on the metabolic profile of aflatoxin B1 in Fischer-344 rats.

The phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS), has been shown to prevent aflatoxicosis in farm animals by reducing the bioavailability of aflatoxin. The present study was designed to determine the effects of HSCAS on the metabolism of aflatoxin B1 (AFB1) in an aflatoxin-sensitive species. Male Fischer-344 rats were orally dosed with 1.0, 0.5, 0.25 and 0.125 mg AFB1/kg body weight alone and in combination with 0.5% HSCAS. Urine samples were collected after 6, 24, 36, and 48 h. Aflatoxin M1 (AFM1) and aflatoxin P1 (AFP1) were detected in most urine samples, with or without HSCAS. AFM1 was found to be the major metabolite. Metabolite concentrations were significantly decreased in the presence of HSCAS, and more importantly, no additional metabolites were detected. Our results suggest that the AFB1-HSCAS complex was not significantly dissociated in vivo, and support earlier findings that HSCAS tightly binds aflatoxin.

Aflatoxin B1↗