Flavus NRRL Research Articles

Effect of Carotenoids on Aflatoxin B1 Synthesis by Aspergillus flavus

ABSTRACT Carotenes and xanthophylls occurring in yellow corn and related terpenoids were tested for their effect on growth and aflatoxin B(1) production by Aspergillus flavus NRRL 3357, using the suspended disc culture method. Aflatoxin synthesis was inhibited at concentrations of beta-carotene, lutein, and zeaxanthin comparable to those found in the horny endosperm of mature corn. Usually growth was not significantly affected. Inhibition of aflatoxin biosynthesis was greater for compounds with an alpha-ionone-type ring (alpha-carotene, lutein, or alpha-ionone) compared with compounds with a beta-ionone ring. The presence of hydroxy groups on the rings tended to decrease inhibition, but did not override the effect of the ring type; lutein was similar to alpha-carotene and zeaxanthin was similar to beta-carotene in inhibition. A mutant accumulating norsolorinic acid (NA), A. parasiticus SRRC 162, incubated with alpha-carotene produced reduced levels of both NA and aflatoxin, indicating that inhibition occurred before NA. Additional A. flavus strains tested against 50 mug/ml of beta-carotene had 89 to 96% inhibition, which was significantly more sensitive than NRRL 3357. A. parasiticus strains were less sensitive and generally had similar or lower inhibition than NRRL 3357. The results indicate that the presence of carotenoids in endosperm may decrease the amount of aflatoxin produced by A. flavus.

Inhibition of Production of Cyclopiazonic Acid and Ochratoxin A by the Fungicide Iprodione

Aspergillus flavus NRRL 1290 and Aspergillus ochraceus NRRL 3174 were grown on a glucose-salts medium and yeast extract-sucrose broth containing the fungicide iprodione at concentrations of 0, 1,3,5, 10, 15, and 20 μg of active ingredient per ml of growth medium. Cultures were analyzed for cyclopiazonic acid, ochratoxin A, and mycelium production after 4,7, 10, 14, and 21 days of incubation at 25°C. Increasing concentrations of iprodione in the growth media resulted in greater reduction of cyclopiazonic acid, ochratoxin A, and mycelium production at the end of each incubation period. More than 50% reduction of cyclopiazonic acid, ochratoxin A, and mycelium production was observed when iprodione was added to growth media at a concentration of 5 μg/ml of medium. Higher concentrations of iprodione (10 to 20 μg/ml of growth medium) inhibited the production of cyclopiazonic acid and mycelium by A. flavus NRRL 1290 almost completely, but not the production of ochratoxin A and mycelium by A. ochraceus NRRL 3174.

Growth and aflatoxin B 1 production on olives and olive paste by moulds isolated from ‘Turkish-style’ natural black olives in brine

The growth and aflatoxin production of five Aspergillus flavus and two Aspergillus parasiticus strains isolated from natural black olives in brine for table consumption, fresh whole black olives, fresh damaged black olives and fresh black olive paste were examined. Aspergillus flavus NRRL 6555 and A. parasiticus NRRL 2999 strains were also studied for comparison. All aflatoxigenic strains inoculated onto fresh damaged black olives and fresh black olive paste grew extensively but on fresh whole black olives they grew weakly. Strong aflatoxigenic strains produced low levels of aflatoxins on fresh whole black olives, fresh damaged black olives and fresh black olive paste. Weak aflatoxigenic strains did not produce detectable amounts of aflatoxin.

Effect of Steryl Cinnamic Acid Derivatives from Corn Bran on Aspergillus flavus, Corn Earworm Larvae, and Driedfruit Beetle Larvae and Adults

Steryl esters of ferulic and p-coumaric acids were tested for their effect on corn fungal pathogens and insects. The steryl ester fraction from corn bran had no effect on corn earworm larvae or driedfruit beetle adults, but driedfruit beetle larvae showed a significant increase in weight and the triglyceride fraction was active. Bioassays of pure ferulate esters and the ester fraction from both corn and rice showed no appreciable effect at 100 ppm on spore germination or germ tube growth of Aspergillus flavus, Fusarium moniliforme, or Diplodia maydis. Sclerotinia sclerotiorum was inhibited 75−90% by the total steryl ester fraction from corn and approximately 50% by the ester fraction from rice bran oil. Stigmastanyl and campestanyl ferulate showed little or no inhibition. The steryl ester fraction increased aflatoxin B1 level in A. flavus NRRL 6536 significantly at 0.33 and 1.0 mg/mL. However, the esters had no effect on A. flavus NRRL 3357. The steryl ferulate and p-coumarate ester fraction from corn was...

Investigation of the Aflatoxin Production Capacity on Rice of Aflatoxigenic Molds Isolated From Natural Black Olive Samples

The aflatoxin production capacity on rice of 13 aflatoxigenic molds isolated from natural black olive samples was investigated. In addition, the aflatoxigenic Aspergillus parasiticus NRRL 2999 and Aspergillus flavus NRRL 6555 strains were used for comparison. In the study it was found that the total aflatoxine production of seven of the strains was 0,106 µg/g - 5,9 µg/g and that no aflatoxin production was detected in the other six strains.

Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus.

Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification. A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques. Chromosomal DNA of A. flavus var. flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector. Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A. flavus var. flavus digested with either EcoRI or PstI. One of these clones was chosen for further analysis and was subcloned into pUC19. The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K. E. Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups. The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI. The cloned probe may be species specific as it hybridized with the DNA of all isolates of A. flavus tested in addition to strains recognized as varieties of A. flavus (e.g., A. flavus var. oryzae, A. flavus var. parasiticus, and A. flavus var. sojae). pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.

Evaluation of Paecilomyces lilacinus, chitin, and cellulose amendments in the biological control of Aspergillus flavus fungi

This study was undertaken to investigate the feasibility of using Paecilomyces lilacinus NRRL 13 866, chitin, and cellulose amendments, in order to reduce survival and prevent germination of sclerotia from Aspergillus flavus NRRL 6556, NRRL 13 048, and A. parasiticus NRRL 13 005, NRRL 13 539 buried in sandy soil in Georgia and Illinois (April–October, 1990). The number of sclerotia that germinated sporogenically in moist chambers following burial in Illinois was twice that of sclerotia buried in Georgia and varied among the four sclerotium-producing Aspergillus spp. strains. Sclerotium viability, as measured by Aspergillus spp. colony formation on potato dextrose agar, was high (>84%) for all strains and treatments (e.g. amendment, location). Even so, A. parasiticus NRRL 13 539 sclerotia were apparently more susceptible to P. lilacinus colonization than sclerotia from other strains. Treatment with Paecilomyces sp. caused a small but significant reduction in germination among sclerotia buried in Georgia.

Polyacrylamide gel electrophoretic mobility of alkaline proteinase produced by Aspergillus flavus isolated from a penguin.

The electrophoretic mobility of alkaline proteinase produced by four isolates from a penguin (Spheniscus magellanicus) was same in all of them and was consistent with that of Aspergillus (A.) flavus NRRL 1957 used as a reference strain, but not consistent with that of A. parasiticus NRRL 2999. Therefore, four isolates were confirmed as A. flavus.

Inhibition of aflatoxin B 1 production by emetine in stationary cultures of Aspergillus flavus

Emetine (1 m m ), a known inhibitor of protein synthesis, was found to inhibit aflatoxin B 1 biosynthesis in Aspergillus flavus NRRL 6515 stationary broth cultures without appreciably affecting growth. Maximal levels of inhibition (60–80%) were observed when the inhibitor was added at inoculation or 3 d post-inoculation (p.i.), and a 40% reduction was observed when emetine was added as late as 6 d p.i. Inhibition was reversible, if exposure to emetine was limited to the first 3 d of growth, but irreversible if cultures were exposed to emetine subsequently. Total percent nitrogen was also inhibited by the addition of emetine, but the amount of soluble protein per gram of mycelium was unaffected. Thus, reduced levels of aflatoxin B 1 , due to the addition of emetine, possibly resulted from the inhibition of insoluble, perhaps membrane-bound, enzymes formed 3–6 d p.i. Since aflatoxin precursors did not accumulate in the presence of emetine, the inhibitor presumably affects enzymes involved in initial or acetate condensation stages of aflatoxin biosynthesis.

Inhibition by gamma‐irradiation and antimicrobial food additives of aflatoxin B1 production by Aspergillus flavus in poultry diet

AbstractThe effect of gamma‐irradiation and the antimicrobial food additives on growth and aflatoxin B1 production by A. flavus NRRL 5520 in poultry diet was investigated. By increasing the irradiation doses the viable population as well as aflatoxin B1 production decreased greatly. No growth and no detection of aflatoxin production occurred in poultry diet after treatment with 6 kGy. The addition of the antimicrobial food additives resulted in a significant decrease in aflatoxin B1. Growth and toxin production was completely inhibited by 0.64% sodium propionate, 0.134% potassium sorbate and 0.1% sodium bisulfite. The combination treatments between gamma‐irradiation (3 kGy) and sodium propionate (0.48%), potassium sorbate (0.022% and 0.044%) and sodium bisulfite (0.0334% and 0.0667%) inhibited completely the fungal growth and aflatoxin B1 production. At 5 kGy, no growth was recorded and aflatoxin B1 production was completely inhibited at all tested levels of priopionate, sorbate and bisulfite. The combined effects between gamma‐irradiation and the antimicrobial agents had a synergistic effect on the mould growth and aflatoxin production in poultry diet.

Paecilomyces Lilacinus, a Colonist of Aspergillus Flavus Sclerotia Buried in Soil in Illinois and Georgia

(1990). Paecilomyces Lilacinus, a Colonist of Aspergillus Flavus Sclerotia Buried in Soil in Illinois and Georgia. Mycologia: Vol. 82, No. 3, pp. 393-395.

Ammonium Bicarbonate Inhibition of Mycotoxigenic Fungi and Spoilage Yeasts

Sodium bicarbonate inhibits growth and aflatoxin production by Aspergillus parasiticus. This survey determined that other mycotoxigenic fungi were also sensitive to bicarbonates. Sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, ammonium sulfate, and sodium chloride were added to buffered or unbuffered potato-dextrose agar to determine the bicarbonate effect on growth and morphology of six mycotoxigenic fungi. Three nonmycotoxigenic fungi and four yeast species were also examined. Ammonium bicarbonate at 0.11M completely inhibited the growth of Fusarium tricinctum NRRL 13442, F. tricinctum NRRL 13426, F. graminearum NRRL 5883, F. sporotrichioides NRRL 3249, Penicillium griseofulvum NRRL 989, Aspergillus ochraceus NRRL 3174, A. flavus NRRL 1957, A. niger, and P. notatum. Sodium chloride and pH elevated through the use of ampso-NaOH, capso-NaOH, or glycine-NaOH buffer did not display an inhibitory effect on the filamentous fungi examined. Buffered ammonium sulfate treatments (pH approximately 9.0) completely inhibited all of the mycotoxigenic fungi, but at pH 5.6, ammonium sulfate treatments were not inhibitory. Sodium bicarbonate (0.11M) was effective only against P. griseofulvum, A. flavus NRRL 1957, A. niger, and P. notatum, causing viability reductions of 5.6, 3.7, 4.9, and 2.9 log cycles, respectively. Potassium bicarbonate was generally as inhibitory as the sodium salt. In contrast, elevated pH, alone, appeared to account for the >6 log reduction observed for the yeasts Lipomyces starkeyi, Geotrichum candidum, Kluyveromyces marxianus, and Debaryomyces hansenii.

Inhibitory strains of Bacillus subtilis for growth and aflatoxin-production of aflatoxigenic fungi.

We isolated a bacterial strain, NK-330, from Japanese soil which inhibited the growth of Aspergillus parasiticus NRRL 2999 on a PDA plate. Later, we isolated an inhibitory strain, NK-C-3, as a contaminant. Both strains inhibited not only the growth of A. parasiticus NRRL 2999 but also that of A. flavus NRRL 3357. These strains were gram-positive rods, and were identified as Bacillus subtilis. They inhibited the growth and aflatoxin-production of NRRL 2999 or NRRL 3357 in peanuts or corn. Moreover, cultures of these strains markedly inhibited the growth of NRRL 2999 or NRRL 3357, and also inhibited the aflatoxin-production completely.

Effects of Nutrients and Inhibitors in Olives on Aflatoxigenic Molds

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus NRRL 6555 were studied on fresh olives, fresh olives supplemented with nutrients, and fresh olives treated with heat, lye, and freezing temperatures. Studies were also done on yeast extract sucrose agar (YESA) either mixed with chopped fresh olives or made with aqueous extracts of fresh and treated olives. Samples were incubated at 25°C for 7 d. Olive paste supplemented with zinc and sucrose supported little growth and no aflatoxin B1 production. Amino acids, yeast extract, and a combination of zinc, carbohydrate, and amino acids exhibited extensive growth and moderate amounts of aflatoxin. Fresh and frozen olive pastes supported poor growth and no aflatoxin production. Heat- and lye-treated olives supported extensive growth and little aflatoxin production. Heavy growth and moderate amounts of aflatoxin B1 were supported by YES A mixed with olive pastes. YES A made with aqueous extracts of olives supported extensive growth and moderate toxin production, except on YES A made with extract from frozen olives which exhibited poor growth and low toxin amounts. A. flavus grew similarly to A. parasiticus but was unable to produce any aflatoxin except on heat- and lye-treated olives, where traces were detected. Olives are a poor substrate for mold development and may contain inhibiting substances against growth and aflatoxin production.

Influence of inoculum size of Aspergillus flavus link on the production of aflatoxin B 1 in maize medium before and after exposure to combination treatment of heat and gamma radiation

Vegetative growth of Aspergillus flavus Link NRRL 5906 was independent of inoculum size. Reduction in inoculum size by serial dilution (3–4 log cycles) resulted in 3–12 fold increase in aflatoxin production. Irradiation with 3.5 kGy reduced inoculum size by 3–5 log cycles and resulted in enhanced aflatoxin B 1 production but this effect was similar to the effect obtained by dilution of unirradiated spores. It is concluded that aflatoxin B 1 formation is inversely related to the inoculum size of A. flavus NRRL 5906. Maximum accumulation of aflatoxin B 1 was generally in 4 days in amended maize meal broth and 8 days in maize meal broth. A combination of moist heat (60°C for 30 min under > 85% R.H.) and 4.0 kGy of gamma irradiation inhibited aflatoxins production in flasks containing either broth.

Preliminary studies of the effects of heat and gamma irradiation on the production of aflatoxin B 1 in static liquid culture, by Aspergillus flavus link NRRL 5906

Aflatoxin B 1 production by a strain of Aspergillus flavus NRRL 5906 was examined in static liquid culture in maize meal broth (MMB) and maize meal broth supplemented with 2% glucose and 2% peptone (AMMB). Erlenmeyer flasks were inoculated with 1.0 ml aliquots of fungal spores which had been heat-treated (60°C for 30 min) under low humidity (< 45% R.H. dry heat) or high humidity conditions (>85% R.H., moist heat) followed by gamma irradiation with either 0.0, 3.5 or 4.0 kGy. AMMB supported 6–17 times more vegetative growth (depending on the heat and dose combination) than spores incubated in MMB alone. High inoculum size of control unheated spores (log CFU/g, 6.9) yielded the least aflatoxin B 1 in flasks containing AMMB (8.2–19.3 μg/ml). A dose of 3.5 kGy reduced by 3.2–3.8 log cycles the viable inoculum of control unheated spores, resulting in 2–5 fold increase in aflatoxin B 1 formed in flasks containing AMMB. Increasing the applied load to 4.0 kGy, however, reduced aflatoxin B 1 levels formed in AMMB to similar or lower levels than found in flasks inoculated with control unirradiated spores. Combination treatment of A. flavus with dry heat and 3.5 kGy reduced the spore inoculum size by about 4 log cycles and yielded the highest amount (41.1 μg/ml) of aflatoxin B 1 in AMMB. However, moist heat treatment of spores receiving the same dose (3.5 kGy) reduced toxin level formed by 25%. Aflatoxin B 1 formation by A. flavus spores incubated in AMMB was completely prevented by a combination treatment of moist heat and 4.0 kGy of gamma irradiation. This same treatment attenuated aflatoxins B 2, G 1 and G 2 production which are formed with B 1 by A. flavus NRRL 5906. Spores raised in all flasks containing MMB did not form aflatoxin except when the medium MMB was autoclaved twice at 121°C for 15 min.

Production and Isolation of Aflatoxin M1 for Toxicological Studies

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.

Toxic Effects of Aflatoxin B 1 in Chickens Given Feed Contaminated with Aspergillus flavus and Reduction of the Toxicity by Activated Charcoal and Some Chemical Agents

Aspergillus flavus NRRL 3357 was grown on enriched long-grain rice for 7-10 days to produce aflatoxin B1 (AFB1). The quantity of AFB1 in moldy rice was determined by thin-layer chromatography using ultraviolet light. When the dried moldy rice powder was fed to day-old Hubbard X Hubbard broiler chicks in unmedicated feed (AFB1 level 10 ppm) for 8 weeks, there was a profound reduction in weight gain and feed consumption. Chickens fed AFB1 developed severe liver damage, as determined by the concentration of hepatic microsomal cytochrome P-450 and by the activities of microsomal benzphetamine N-demethylase and serum glutamic oxalacetic transaminase. However, activated charcoal, reduced glutathione, cysteine, selenium (as sodium selenite), beta-carotene, and fisetin administered orally considerably reduced the toxicity of AFB1 in the experimental chickens.

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger.

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.

Susceptibility of Strawberries, Blackberries, and Cherries to Aspergillus Mold Growth and Aflatoxin Production

The susceptibility of blackberries, cherries, and strawberries to Aspergillus growth and aflatoxin production has been examined. Three aflatoxigenic isolates of Aspergillus, A. flavus ATCC 15548 and NRRL 3251 as well as A. parasiticus NRRL 2999, were cultured on homogenates of the fruits for 14 days at 28 +/- 2 degrees C. Percent mycelial growth and spore infestation were determined each day with a calibrated grid. At day 14 each culture was frozen at -5 degrees C until aflatoxins were extracted with methylene chloride and water. Aflatoxins were separated by thin layer chromatography (TLC) with benzene-methanol-acetic acid (90 + 5 + 5). This extraction and solvent system provided satisfactory separations of the aflatoxins and was free of background interference on the TLC plates. Although all fruits served as substrates for both Aspergillus growth and aflatoxin production, cherries appeared to be a more favorable substrate than did blackberries, and the latter was more favorable than strawberries. Whereas A. flavus produced both B1 and G1 on all substrates, it yielded B2 and G2 only on cherries. Although A. parasiticus NRRL 2999 synthesized B1, B2, G1, and G2 on both blackberries and cherries, no aflatoxins were detected on strawberries. In contrast, A. flavus NRRL 3251 failed to produce detectable levels of aflatoxin on any substrate. All substrates supported both mycelial growth and subsequent sporulation with cherries greater than blackberries greater than strawberries.