Microbial Hydroxylation of 5-Anilino-1,2,3,4-Thiatriazole

Two hundred eighty-five fungi, including 100 basidiomycetes and 35 yeasts, 75 actinomycetes, and 40 bacteria were screened for their ability to convert 5-anilino-1,2,3,4-thiatriazole (AT) to 5-(p-hydroxyanilino)-1,2,3,4-thiatriazole (p-HT). Eleven cultures were found that formed p-HT, which was isolated and whose structure was determined. Aspergillus tamarii NRRL 3280 formed 8.6 g of p-HT/liter from 10 g of AT/liter (78.9% conversion) in shaken flasks and 4.57 g of p-HT/liter from 6 g of AT/liter (69.8% conversion) in 30-liter fermentors. Washed cells of A. tamarii NRRL 3280 also carried out this conversion. 5-(o-hydroxyanilino)-1,2,3,4-thiatriazole (o-HT) was identified as a second product formed by Aspergillus terreus NRRL 1960.     

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.     

Production of Aflatoxin on Wheat and Oats: Measurement with a Recording Densitometer

A method has been developed for the production of aflatoxin by growing Aspergillus flavus NRRL 3145 on solid substrate wheat. Optimal yields of 900 mug of aflatoxin G(1) and 900 mug of aflatoxin B(1) per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavus strains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 mug of B(1) and 450 mug of G(1) per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Inhibition of aflatoxin production by selected insecticides

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

Inhibition of aflatoxin production by selected insecticides.

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

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).     

Mycotoxicological control on raw material and tablets of cascara sagrada (Rhamnus purshiana)

Among phytotherapic medicines, tablets of cascara sagrada (Rhamnus purshiana) dried bark, usually used as laxative, are commercially widespread in our market. Taking into account natural origin and/or inappropriate procedures that may allow the occurrence of toxinogenic Aspergillus flavus group, a study on susceptibility to aflatoxin contamination and natural aflatoxin incidence was performed by TLC and HPLC methods. This survey allows one to conclude that bark of Cascara Sagrada is a good substrate for the growth of A. parasiticus NRRL 2999 and for aflatoxins production. Natural anatoxins presence was detected on 2 from 9 raw material samples. One of them (irradiated sample) had only aflatoxin B1 (10 μg/kg) and the other (pasteurized) was positive for aflatoxin B1 (19 μg/kg); G1 (6 μg/kg) and B2 (1.46 μg/kg). Only one from 10 lots of tablets analyzed was positive for aflatoxin B1 (5.42 μg/kg) and B2 (0.32 μg/kg).Therefore, adequate quality control including an aflatoxins assay must be performed to guarantee the harmlessness of natural drugs.     

Aflatoxin is degraded by mycelia from toxigenic and nontoxigenic strains of aspergilli grown on different substrates

The ability of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 to degrade aflatoxin varied depending on the substrate used to grow the mold. Substrates which allowed substantial mycelial growth yielded mycelia which actively degraded aflatoxin. Substrates which allowed minimal growth of mycelia yielded mycelia with little ability to degrade aflatoxin. Biodegradation of aflatoxin was also strain-dependent. A. parasiticus NRRL 2999 and NRRL 3000 actively degraded aflatoxin, A. flavus NRRL 3353 was less active, and A. flavus NRRL 482 and A. parasiticus NRRL 3315 degraded minimal amounts of aflatoxins. Those aspergilli producing greatest amounts of aflatoxin also degraded aflatoxins most rapidly, whereas those strains which produced minimal amounts of aflatoxin generally degraded aflatoxins less effectively. Substrates which allowed maximum aflatoxin production also yielded mycelia which actively degraded aflatoxins, whereas media which allowed limited production of aflatoxin generally yielded mycelia with minimal ability to degrade the toxin. Although exceptions exist, generally as aflatoxin production increased so did the ability of mycelia to degrade the toxin.     

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

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.     

Biosynthetic relationship among aflatoxins B1, B2, G1, and G2.

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B1 (AFB1) and G1 (AFG1) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB1-AFG1 was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB1 and AFG1) were cultured with DHST, the amounts of AFB1 and AFG1 produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFB1-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB1-AFG1 and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium.     

Biosynthetic relationship among aflatoxins B1, B2, G1, and G2

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B1 (AFB1) and G1 (AFG1) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB1-AFG1 was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB1 and AFG1) were cultured with DHST, the amounts of AFB1 and AFG1 produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFB1-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB1-AFG1 and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium.     

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii (“Clostridium butylicum”)

Clostridium beijerinckii (“Clostridium butylicum”) NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.     

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum")

Clostridium beijerinckii ("Clostridium butylicum") NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.