Natural occurrence of mycotoxins in cereals

Besides peanuts and cottonseed, cereal grains are the most important feed and food source that occasionally are naturally contaminated with mycotoxins. The problem of mycotoxins occurring naturally in cereals, especially in corn, has become trouble-some because of changing agricultural technology. The mycotoxin problem in cereals is not restricted to any geographic or climatic region. Toxins are produced on cereals, both in the field and in storage; they involve both the grain and the whole plant. The genera of fungi most involved areAspergillus, Fusarium, Penicillium andClaviceps. Mycotoxins known to occur naturally in cereals include aflatoxins B₁, B₂, G₁ and G₂-as well as aflatoxins M₁ and M₂-ochratoxins A and B, penicillic acid, patulin, ergot, zearalenone, citrinin, T-2, tenuazonic acid, kojic acid and sterigmatocystin. Of these mycotoxins, aflatoxins, patulin, penicillic acid and sterigmatocystin are carcinogens.     

Screening methods to detect toxigenic fungi in liquid medium

Rapid and sensitive methods for identifying toxin production by toxigenic fungi cultivated in liquid medium were developed. The production of aflatoxins B₁, B₂, G₁, G₂, sterigmatocystin, ochratoxin A, patulin, penicillic acid, citrinin and zearalenone was detected employing thin layer chromatography and high performance liquid chromatography detecion with or without extraction and purification procedures. No significant variation was found and toxins could be detected after 2–4 days incubation. The sensitivity of the methods and recovery after extraction have been estimated.     

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B₁, B₂, G₁, G₂, and M₁. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B₁, B₂, and G₁ in the 96 h and aflatoxins M₁ and G₂ in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B₁, B₂, and M₁ in the 96 h and aflatoxin G₁ in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.     

Liquid Chromatography of Aflatoxins

There are numerous reports on studies of aflatoxins, but there are only a few reports on the isolation and mutual separation of aflatoxins by liquid chromatography. Following the previous reports on the liquid chromatography of four kinds (B₁, B₂, G₁ and G₂) of aflatoxins, the authors carried out the chromatography of six kinds of aflatoxins including aflatoxins B_2a and G_2a. Various kinds of adsorbents and eluting solvents were examined, and then the good mutual separation of aflatoxins was obtained by using the Sephadex G-10 (CM), a newly prepared adsorbent containing carboxymethyl group, and pure water for eluting solvent. Aflatoxins G_2a, B_2a, G₂, B₂, G₁ and B₁ were eluted in this order.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

Aflatoxin biosynthetic pathway: Elucidation by using blocked mutants of Aspergillus parasiticus

Two mutant strains of Aspergillus parasiticus, both deficient in aflatoxin production, were used to elucidate the biosynthetic pathway of this mycotoxin. One of the mutants, A. parasiticus ATCC 24551, was capable of accumulating large amounts of averufin, and the other, A. parasiticus 1-11-105 wh-1, accumulated versicolorin A. The averufin producing mutant efficiently converted ¹⁴C-labeled versiconal acetate, versicolorin A, and sterigmatocystin into aflatoxin B₁ and G₁, indicating that averufin preceded these compounds in the aflatoxin biosynthetic pathway. In the presence of dichlorvos (dimethyl 2,2-dichlorovinyl phosphate), a known inhibitor of aflatoxin biosynthesis, the conversion of versicolorin A and sterigmatocystin was unaffected, but the conversion of versiconal acetate was markedly inhibited. The mutant accumulating versicolorin A incorporated ¹⁴C-labeled acetate, averufin, and versiconal acetate into versicolorin A. In the presence of dichlorvos, however, the major conversion product was versiconal acetate. This strongly suggested that dichlorvos inhibited the conversion step of versiconal acetate into versicolorin A. This mutant resumed production of aflatoxin B₁ if sterigmatocystin was added to the resting cell cultures, indicating that the mutant was blocked at the enzymatic step catalyzing the conversion of versicolorin A into sterigmatocystin, and as a result was incapable of aflatoxin production. The experimental evidence is thus provided for the involvement and interrelationship of three anthraquinones (averufin, versiconal acetate, and versicolorin A) and a xanthone (sterigmatocystin) in aflatoxin biosynthesis. A pathway for the biosynthesis of aflatoxin B₁ is proposed to be: acetate →→→ averufin → versiconal acetate → versicolorin A → sterigmatocystin → aflatoxin B₁.     

Products of Spontaneous Conjugation of Aflatoxins with Bovine Serum Albumin: Immunochemical Properties

Products of spontaneous conjugation of aflatoxins B₁, G₁, and G₂ with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by antiaflatoxin antibodies. Antisera against BSA–B₁, BSA–G₁, and BSA–G₂ were obtained and their specificity determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Detection and estimation of aflatoxins using both chemical and biological techniques

Production of aflatoxins on both natural (rice and corn) and semisynthetic (YES) media was conducted using an identified toxin-producing strain ofAspergillus flavus. TheA flavus strain was able to produce 4 types of aflatoxins, namely B₁, B₂, G₁, and G₂ on rice, corn, and YES media. Quantitative data showed that the concentrations of aflatoxins B₁ and G₁ produced were 52, 40.3, and 39.6; and 64.7, 45.0, and 58.0jug for 50g of rice, corn, and YES media, respectively. In comparison, the yielded amounts of aflatoxins B₂ and G₂ were much lower: 11.5, 17.9, and 17.5; and 28.S, 40.3, and 39.5 μg for 50 g of rice, corn, and YES media, respectively. A bioassay was conducted using the following 5 standard bacterial strains:Bacillus megaterium. Bacillus subtilis, Streptococcus faecal is, Staphylococcus epidermidis, andParacoccus denitrificans as well as a field strain of Candida albicans. All strains exceptP denitrificans showed varied degrees of inhibition when applied with crude aflatoxins at 5 to 40μg/mL. The minimum concentration of crude aflatoxins needed to inhibitP denitrificans was 10μg/mL. Moreover,Candida albicans was not inhibited at any concentration of aflatoxins applied in this work. Both undiluted and diluted (1/10, 1/100, and 1/1000) bacterial broth cultures showed a direct relationship between the diameter of inhibition zones and the concentrations of crude aflatoxins. Mean diameters of (7.0–20.5), (5–14), (4.5–13.0), (3.0–12.0), and (1.5–11.0) mm were observed when various concentrations of aflatoxins were applied usingB megaterium, S epidermidis, S faecal is, B subtilis, andP denitrificans, respectively. Field trials were applied to testify the validity of our data. A 1/100 dilution was prepared from each strain of 4 different species to estimate aflatoxins in samples of contaminated corn. Both chemical and biological assays were carried out at the same time. Data revealed that the most sensitive organism inhibited by as low as 7.5μg aflatoxins/mL wasB megaterium giving an inhibition zone of 10.5 mm, followed byS epidermidis with an inhibition zone of 7.5mm. In relation, the other 2 organisms were less sensitive to crude aflatoxins. Similarly, the biological assay was applied to detect aflatoxins in some samples of wheat, corn, peanut, rice, and poultry rations. Of the 14 wheat and 10 corn samples, only 4 wheat and 2 corn samples were found to be positive. The same results were obtained using TLC analysis.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

We detected biosynthetic activity for aflatoxins G₁ and G₂ in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B₁, aflatoxin B₂, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G₂ from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G₁ was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G₁ and G₂. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.     

Role of caffeine and tannin in anti-toxigenic properties of coffee and tea

Aflatoxins B₁, B₂, G₁ and G₂ produced by Aspergillus parasiticus var. globosus IMI 120920 in detannin-caffeinated coffee and black tea were five times more concentrated than in normal tea and coffee. Extracts of normal coffee and tea powders significantly reduced aflatoxins production in liquid broth at 1 and 3 % concentrations, with tea extract. having a more pronounced effect than coffee extract Relevant anti-aflatoxigenic properties appear to be due to tannin and caffeine. These induced 95 % inhibition in aflatoxins at 0.3 % and 0.6 %, respectively. Roasting of contaminated coffee beans at 200 °C for 20 min is effective in the reduction of aflatoxins.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles     

Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B₁, G₁, B₂, and G₂ requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B₁. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B₁, G₁, B₂, and G₂. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B₁ in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G₁, and G₂ in A. parasiticus suggests that enzymes required for the formation of aflatoxins G₁ and G₂ are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.     

An enzyme immunoassay system for aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B₁ carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B₁ with a low relative cross-reactivity against aflatoxin B₂, G₁, G₂, M₁ B_2a, and G_2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.     

Studies on the ability of mycotoxins to diffuse in bread

Summary The moulds Aspergillus parasiticus (aflatoxins B₁, B₂, G₁, G₂, and M₁), A. ochraceus (ochratoxin A) and Penicillium chrysogenum (citrinin) were grown on whole wheat bread either in the presence or absence of oxygen. In the presence of oxygen, both A. parasiticus and A. ochraceus developed dense colonies and formed considerable amounts of mycotoxins whereas Penicillium chrysogenum only grew and produced citrinin on the surface of the bread. In the absence of oxygen fungal growth did not occur and most of the toxins were undetectable even in regions of bread immediately adjacent to the moulds although a very slight diffusion of the aflatoxins B₁ and G₁ through 1 cm was observed. It is concluded that diffusion of the tested mycotoxins from hyphae into bread is not a problem for food safety.     

Fungal flora and aflatoxin contamination of feedstuff samples in Beida Governorate,Libya

Fungi of 19 genera, 30 species, and one variety were isolated from 25 samples of sheep-, cattle- and camel feedstuffs collected from different farms in the Beida Governorate, Libya.Aspergillus, Penicillium andFusarium were the most common genera in the three substrates tested. TLC was used to establish the identity of aflatoxins in the chloroform extract of all samples and the ability to produce aflatoxins byAspergillus flavus in a synthetic liquid medium. Twenty samples out of 25 tested were naturally contaminated and 21 isolates ofA. flavus out of 30 produced at least one of the following aflatoxins: B₁; B₁, G₁; and B₁, B₂, G₁, G₂. This is the first report about the natural occurrence of aflatoxins and aflatoxin-producers of the genusAspergillus in Libya.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Aflatoxin-producing Potential of Isolates of the Aspergillus flavus-oryzae Group from Peanuts (Arachis hypogaea)

Seventy-eight samples of farmer stock peanuts, representing peanuts grown in nine different geographical areas during 1964, were assayed for aflatoxin and examined for associated microflora. Only two samples contained more than 50 ppb of aflatoxin. Infestation by members of the Aspergillus flavus-oryzae group varied from 35 to 100% of the kernels per area and from 1 to 100% of the kernels per sample. Aflatoxin production by individual isolates ranged from 0 to 349,143 ppb under the test conditions employed. In general, the isolates produced 8 to 10 times more B₁ than B₂, and no isolate producing aflatoxins G₁ or G₂ was found. The importance of proper postharvest handling of peanuts is emphasized by the prevalence of isolates of A. flavus-oryzae capable of producing aflatoxins on farmers stock peanuts.