Inhibitory effect of molybdenum and vanadium salts on aflatoxin B1 synthesis by Aspergillus flavus

The effects of the elements zinc, manganese, iron, copper, molybdenum, and vanadium, added in various salt forms, on mycelial weights and aflatoxin B1 accumulation in the mycelium of Aspergillus flavus were investigated in liquid shake cultures. Ammonium heptamolybdate, when added to a complete medium at concentrations of 50-100 mg/L, appreciably reduced aflatoxin B1 accumulation without affecting growth of the fungus. Sodium molybdate and sodium monovanadate also reduced aflatoxin B1 yields without affecting mycelial growth but to a lesser extent. The addition of zinc sulphate stimulated aflatoxin B1 production in all media used. The influence of the other trace elements on aflatoxin production depended on the level of trace elements present in the basal medium. In general, manganese chloride had a stimulatory effect, whereas copper sulphate depressed yields. Mycelial levels of aflatoxin had peaked and then declined before mycelial dry weights had reached maximum. High yields of aflatoxin B1 were obtained in media having a final pH as low as pH 2.8.     

Factors affecting aflatoxin production by Aspergillus parasiticus in a chemically defined medium

The optimum levels of sucrose, (NH4)2SO4, MgSO4, KH2PO4 and ZnSO4 for aflatoxin production in a chemically defined medium have been established. The last two were found to be essential for fungal growth and aflatoxin production. The effect of various carbon sources on aflatoxin production was tested using the defined medium. Asparagine was found to be essential for aflatoxin production. Very little aflatoxin was produced in the absence of asparagine with any of the other inorganic nitrogen sources tested. Supplementation with yeast extract, Casamino acids, Casitone and peptone increased the aflatoxin yield, but omission of asparagine led to decreased aflatoxin yields even when complex nitrogen sources were present. Asparagine could be replaced by aspartic acid or alanine.     

Aflatoxin formation,lipid synthesis,and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation

Glucose utilization, growth of mold, and synthesis of aflatoxin and total lipid by Aspergillus parasiticus were studied with cultures that were incubated statically and with agitation. With both cultural conditions, maximal toxin formation occurred at 5 days which coincided with the end of rapid mold growth and rapid uptake of glucose. The toxin concentration decreased as incubation continued. The pattern for formation and depletion of total lipid was similar to that for aflatoxin. Maximal yields of toxin and of total lipid did not coincide with maximal production of mold mycelium. Incubation with agitation enhanced mold growth, consumption of glucose, and production of aflatoxin and total lipid during the first 3 days. Generally, more growht occured in agitated cultures, but maximal yields of aflatoxin and total lipid were lower than in quiescent cultures. The need for limited, but not excessive, O₂ for synthesis of aflatoxin and lipid also was demonstrated by varying the volume of medium in flasks that were incubated quiescently. Incorporation of [1-¹⁴C] glucose into aflatoxin indicated that limiting the O₂ supply and thereby favoring glucose catabolism via the Embden-Meyerhof pathway enhanced toxin formation. Aflatoxin formation also was greater when oxidative respiration of the mold was restricted by a metabolic inhibitor. Results suggest that the degree of aeration of the culture is important in controlling biosynthesis of aflatoxin.     

Production of aflatoxins B1 and G1 in chemically defined medium

Summary Aflatoxins B₁ and G₁ were produced in a chemically defined liquid medium in stationary culture. Glucose, sucrose, and fructose were satisfactory carbon sources. Organic nitrogen compounds were essential for production of high levels of aflatoxins. Complex nitrogen sources, such as yeast extract and peptone, gave higher yields than single amino acids. Aspartate, glycine, glutamine, and glutamate were good sources of nitrogen for toxin production. Little or no aflatoxin was produced when zinc, iron, or magnesium were omitted from the medium. Manganese appeared to reduce yields of aflatoxin.     

Production of Aflatoxin on Soybeans

Probable factors influencing resistance to aflatoxin synthesis in soybeans have been investigated by using cultures of Aspergillus parasiticus NRRL 3240. Soybeans contain a small amount of zinc (0.01 mug/g) bound to phytic acid. Autoclaving soybeans at 15 pounds (6803.88 g) for 15 min increases the aflatoxin production, probably by making zinc available. Addition of zinc to both autoclaved and nonautoclaved soybeans promotes aflatoxin production. However, addition of varying levels of phytic acid at a constant concentration of zinc depresses aflatoxin synthesis with an increase in the added phytic acid. In a synthetic medium known to give good yields of aflatoxin, the addition of phytic acid (10 mM) decreases aflatoxin synthesis.     

Influence of trace elements and nitrogen sources on versicolorin production by a mutant strain of Aspergillus parasiticus

A mutant strain of Aspergillus parasiticus blocked in aflatoxin biosynthesis accumulates versicolorin A and versicolorin C. The effect of trace elements on the growth and versicolorin production by this strain was studied in a defined medium. The omission of manganese was slightly stimulatory to versicolorin production; when zinc was omitted from the medium, no detectable versicolorins were produced. Experiments on nitrogen sources in a highsucrose medium indicated that fourfold to fivefold increases in versicolorin yields could be obtained by substituting 3 ml/l corn steep liquor or 0.1 M NH₄NO₃ for the 0.023 M (NH₄)₂SO₃ used previously as the nitrogen source in studies on versicolorin production by this strain. These improved yields will facilitate attempts to accumulate enough versicolorin A and versicolorin C for toxicity and carcinogenicity testing. Chromatographic profiles of mycelial extracts of cultures grown in a defined medium with 0.1 M NH₄NO₃ as the nitrogen source revealed 2 previously unrecognized compounds. The accumulation of these new metabolites in a mutant blocked in aflatoxin production may indicate that they are biosynthetically related to aflatoxin.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Aflatoxin Production and Degradation by Aspergillus flavus in 20-Liter Fermentors

Yields of from 200 to 300 mg per liter of aflatoxins B(1) and G(1) were produced by two strains of Aspergillus flavus in 20-liter fermentors under proper conditions of inoculum (well-dispersed growth) and aeration (0.5 volume per volume per min of air, 300 rev/min, 30 psi back pressure, baffles). Peak yields were usually attained in 72 hr, after which the aflatoxin concentration declined rapidly. Degradation of aflatoxin depended primarily on mycelial lysis and high-aeration conditions. Cultures previously reported not to degrade aflatoxin could be induced to do so under these conditions. The percentage and rate of toxin degradation were independent of toxin concentration, and appeared to be nonenzymatic and nonspecific. Degradation simulating that occurring in the fermentor was achieved by reacting aflatoxin with peroxidized methyl esters of vegetable oil; initial degradation was rapid and appeared to involve a complex series of reactions.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values

Aspergillus parasiticus was grown in a modified Lab-Lemco tryptone broth both as a single culture and in association with Lactococcus lactis. Total aflatoxin (B1 + G1) production was higher in the mixed cultures. This stimulation persisted when different batches of media, inoculation procedures and makes of ingredients were used. Aflatoxin yields increased in media with an initial pH of 4.2 compared with a pH close to neutrality. Hydrochloric and/or lactic acid had little effect. The substitution of half the carbon content of the medium by lactate resulted in stimulation or reduction on aflatoxin production when the initial pH was 4.2 or 6.8, respectively.     

High Aflatoxin Production on a Chemically Defined Medium

Aspergillus parasiticus ATCC 15517 produced 28 to 30 mg of aflatoxin per 100 ml of a medium containing sucrose, asparagine, and salts in stationary and shaken cultures. In the absence of asparagine in the medium, the toxin yields fell drastically, and the thin-layer chromatograms of the chloroform extracts of the cultures indicated the total absence of aflatoxin G₁ and the presence of new intense blue and green fluorescent bands having R_F values lower than aflatoxins. Initial pH was critical and had to be around 4.5 for good growth and high toxin production on this medium. Optimum concentrations of KH₂PO₄ and MgSO₄·7H₂O in the medium were much lower than those normally used in fungal growth media.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucroselow salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucrose-low salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values

L uchese , R.H. & H arrigan , W.F. 1990. Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values. Journal of Applied Bacteriology 69 , 512–519.
Aspergillus parasiticus was grown in a modified Lab-Lemco tryptone broth both as a single culture and in association with Lactococcus lactis . Total aflatoxin (B1 + G1) production was higher in the mixed cultures. This stimulation persisted when different batches of media, inoculation procedures and makes of ingredients were used. Aflatoxin yields increased in media with an initial pH of 4.2 compared with a pH close to neutrality. Hydrochloric and/or lactic acid had little effect. The substitution of half the carbon content of the medium by lactate resulted in stimulation or reduction on aflatoxin production when the initial pH was 4.2 or 6.8, respectively.     

Effect of aluminium and nickel on aflatoxin production byAspergillus flavus

Effect of nickel and aluminium was studied on aflatoxin and lipid production by two strains ofAspergillus flavus in a sucrose—asparagine—salts medium. Inclusion of aluminium in the medium established an inverse relationship between aflatoxin and lipid production. At lower concentrations aluminium stimulated aflatoxin production, whereas at higher concentrations it stimulated total lipid production. Nickel at higher concentrations resulted in an increase in total aflatoxin production. However, no definite correlation was observed between total aflatoxin and total lipid production when nickel was included in the medium. *** DIRECT SUPPORT *** A00FP116 00002     

Cowpeas as growth substrate do not support the production of aflatoxin byAspergillus sp

A number of 21Aspergillus sp. strains isolated from cowpeas from Benin (Africa) were characterized by RAPD methodology. Seven of these strains grouped withA. flavus in the dendrogram generated with the RAPD data. Only three were able to produce aflatoxin in significant amounts. Twelve other isolates grouped withA. parasiticus. All of these strains except 3 produced aflatoxin. Two additional strains neither fit with theA. flavus group, nor theA. parasiticus group according to their RAPD pattern. Both did not produce aflatoxin in measurable amounts. Generally the aflatoxin positive strains produced high amounts of aflatoxin after growth on YES medium. However after growth on cowpea based medium aflatoxin biosynthesis was strongly ceased, albeit the growth of the colony was only partly reduced. This was true for media made either with the whole cowpea seed or with cowpea seed without seed coat. Interestingly when the cowpea medium was heat sterilized the fungus was able to produce high amounts of aflatoxin. This, however, was not the case after the use of gamma irradiation as sterilization method for the medium. The expression of thenor- 1 gene, which is one of the early genes involved in aflatoxin biosynthesis, was significantly repressed after growth on gamma irradiated cowpea medium in contrast to YES medium. This study was part of the project “Capability Building for Research and Quality Assurance in Traditional Food Processing in West Africa”     

Production of Aflatoxins in Submerged Culture

Aflatoxins can be produced on a synthetic medium in submerged culture. Glucose, sucrose, or fructose are the preferred carbon sources, and Casamino Acids are the preferred nitrogen source. Ammonia is almost as good a nitrogen source. Zinc is required at levels of at least 0.4 mg per liter. Concentrations of aflatoxin of 60 to 80 mg per liter (as determined by optical-density measurements of a chloroform extract of the unfiltered broth) can readily be obtained in indented shake flasks; somewhat lower yields were obtained in 5-liter fermentors.     

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins.

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity.     

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity.