Mycotoxins of fungal strains from stored herbal plants and mycotoxin contents of Nigerian crude herbal drugs

The ability of fungi isolated from stored herbal drug plants to produce mycotoxins in semisynthetic media was studied. The results obtained show that aflatoxins and ochratoxin A, were produced by Aspergillus flavus, A. parasiticus and A. ochraceus isolates. The time-production courses of aflatoxins B₁, B₂, ₁ and ochratoxin A in crude herbal drug preparations show that more of these toxins were produced with increase in time of storage of the drugs. The results indicate that the potential exists for the toxigenic strains to elaborate mycotoxins in a large quantity in herbal drug substrates than in semisynthetic media.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. III. Cellulose-decomposing and mycotoxin-producing fungi

From 40 peanut seed samples collected in Egypt, forty-three species and one variety of fungi, belonging to 16 genera, were collected. The most dominant genera were Aspergillus (11 species + one variety), Penicillium (11 species) and Fusarium (4 species). From the preceding genera A. fumigatus, A. flavus, A. niger, P. chrysogenum and F. oxysporum were the most frequent species.Forty-nine isolates belonging to 12 species and one variety were tested for production of mycotoxins, after growth on liquid medium containing two carbon sources (sucrose or cellulose). Thin layer chromatographic analysis revealed that the quality and quantity of mycotoxins was higher on sucrose than cellulose. Mycotoxins identified were aflatoxins B₁, B₂, G₁ & G₂, citrinin; fumagillin; diacetoxyscirpenol T-2 toxin; satratoxin H; and zearalenone.     

Fungal flora and mycotoxins of six kinds of nut seeds for human consumption in Saudi Arabia

A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960–7704 and 1948–7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28°C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media wereAspergillus flavus, A. niger andPenicillium chrysogenum. On glucose-Czapek's agar,Rhizopus stolonifer andAspergillus flavus var.columnaris were isolated from all 6 kinds of nut,A. parasiticus from 5 kinds andA. fumigatus from 4 kinds with high frequencies.Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B₁, B₂, G₁ & G₂, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B₁ & G₁ were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 µg/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.     

Survey of aflatoxins and ochratoxin A in stored tubers ofCyperus esculentus L.

The mold incidence, moisture contents, pH and levels of mycotoxins (aflatoxins B₁, G₁ and ochratoxin A) on/of/in rootstock snack (tubers ofCyperus esculentus L.) samples were monitored during a 150-day storage period. Whereas the mold incidence, moisture and mycotoxin levels increased with storage time, the pH declined during the same period. Altogether, 12 fungal species, mostly toxigenic, includingAspergillus flavus, A. parasiticus andA. ochraceus were isolated. At collection period only 3 of the 9 snack samples analysed contained trace amounts of aflatoxins. By 120th day, all the 9 samples were contaminated and the average levels were 454 and 80 ppb for aflatoxin B₁ and aflatoxin G₁ respectively on the 150th day. Ochratoxin A was not detected before 120th day and then only at low levels, occuring in a maximum of four samples and ranging between 10 and 80 ppb.     

Comparison of the ability of three Aspergillus strains to form aflatoxins on bakery products and on nutrient agar

The growth of Aspergillus parasiticus NRRL 2999, A. parasiticus NRRL 3000 and A. flavus NRRL 3251 on whole wheat bread and on cake (Rührkuchen) was compared and the formation of the aflatoxins B₁, B₂, G₁, G₂ and M₁ on these substrates and, for purpose of comparison, on malt extract agar was determined. On cake the moulds grew better than on bread and formed the highest yields of aflatoxins. Malt extract agar was the most unfavourable substrate for toxin production. The ratio M₁/B₁ on bread and cake was in the order of 0.1–0.4 and was higher than the data reported for grains. The highest yields of aflatoxin B₁ (1.0 g/g) were produced by A. flavus NRRL 3251 on cake.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

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.     

Book reviews

Sesame seeds (Sesamum indicum L) from four different geographic locations in Sierra Leone were sampled for their mycoflora. Three toxigenic Aspergillus species: A. flavus Link ex Fries, A. ochraceus Wilhelm, and A. tamarii Kita were common to all samples. Penicillium citrinum Thom and two Fusarium sp. were found in samples from two localities. The mycotoxins aflatoxin B₁ and G₁, ochratoxin A and B, and citrinin were positively identified.     

Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

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. purasiticus is/are enzyme(s) or at least influenced by enzyme(s).     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

New examinations of mycotoxin carryover to cocoa beans

Mycotoxins are secondary metabolites which can form on various foodstuffs through the growth of certain fungi. Ochratoxin A (OTA) and the aflatoxins B₁ B₂, G₁ and G₂ have been detected in low concentrations in cocoa and cocoa products. As regards the question of in what stages of the cocoa production process a contamination with the mycotoxin-producing moulds and the formation of mycotoxins takes place, it is assumed that in the case of cocoa the contamination is not concerning the individual beans but the fermentation units. A model test was carried out to provide information on the process by which a possible carryover of the above-mentioned mycotoxins to cocoa beans occurs during the fermentation process. For this purpose fresh cocoa beans were left to soak in an artificial mycotoxin-containing fermentation solution. The mycotoxin levels in the cocoa beans were regularly determined over a period of 12 days. New findings were made as regards the migration of mycotoxins during the fermentation process. We interpret the divergent uptake behaviour of the mycotoxins to indicate that the transport of OTA and that of aflatoxins does not take place in the same manner. This is possibly caused by chemico-physical effects, such as the different polarities of the mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

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.     

Inhibitory effect of hena (Lawsonia inermis leaves) and carrot root on aflatoxin production byAspergillus parasiticus

Experiments were undertaken to evaluate the effect of some natural products (hena, and carrot root) on growth and aflatoxins production byAspergillus parasiticus FRR 2752. Powdered hena (0.5 and 5%) inhibited mycelial growth and delayed 1 sporulation ofA parasiticus during 7 days. The inhibition of growth was increased with increasing the added amount. Aflatoxins production byA parasiticus was reduced with 40–100% in the presence of hena (Lawsonia inermis leaves). Carrot root extract stimulated the fungal growth and aflatoxin production, whereas carrot root fibers slightly enriched fungal growth, inhibited aflatoxins production (B₁, G₁, and G₂), but there was no inhibition of aflatoxin B₂ production byA parasiticus.     

Mycotoxins producing fungi and mycoflora of air-dust from Taif,Saudi Arabia

Using the dilution plate method, 70 species and 31 genera were collected from 20 dust samples on glucose (28 genera and 64 species) and cellulose Czapek's agar (22 genera and 46 species) at 28° C. The most common fungi were Aspergillus niger, A. flavus, A. flavus var. columnaris, Phoma glomerata, Fusarium oxysporum, Penicillium chrysogenum and Mucor racemosus; and A. nidulans, Phoma humicola, Drechslera spicifera and Stachybotrys chartarum on the two media, respectively.Toxicity test showed that about 85% of the isolates tested were toxic to brine shrimp (Artemia salina). Thin layer chromatographic analysis revealed that 13 out of 23 toxic isolates produced known mycotoxins. Toxins identified were: aflatoxins B₁ and B₂, Kojic acid and trichodermine.     

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.