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.     

Cytochrome P-450 and the activation of promutagens in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be related to the growth phase of aerobic cultures when glucose was the carbon source. When grown on glucose medium the log-phase cells of the diploid strain D5 contained about 9× more cytochrome P-450 than log-phase cells of the diploid strain D4. The D4 cells grown on medium containing glucose contained about 10× more cytochrome P-450 than D4 cell grown on medium containing galactose as carbon source. Cells of strain D4, harvested from log-phase cultures grown on glucose, were capable of metabolizing aflatoxin B₁, dimethylnitrosamine, β-naphthylamine, ethyl carbamate, cyclophosphamide and dimethylsulphoxide to products active genetically in the same cells. The metabolism of the compounds tested was attributed to cyctochrome P-450-dependent mixed-function oxidation since genetic activity was high in log cells grown on medium containing glucose but negligible in log cells grown on medium containing galactose. However, aflatoxin B₁ differed from the other promutagens tested since the genetic activity of this compound in cells grown on galactose medium was similar to the activity in cells grown on glucose medium. This result is discussed in relation to enzyme systems which could metabolize aflatoxin B₁. The results of treating log-phase cells of the strain D5, grown on medium containing glucose, with aflatoxin B₁ and dimethylnitrosamine are presented and compared with the results from the strain D4.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

Distribution and characteristics of aflatoxin-producingAspergillus flavus in the soil in Kanagawa Prefecture,Central Japan

In Kanagawa Prefecture, located in central Japan, aflatoxin-producingAspergillus flavus was isolated in 4 (2.5%) of 160 field soil samples. In the 4 fields, whose soil contained aflatoxin-producingA. flavus, the annual average temperature of the sampling sites of the soil ranged from 13.8 to 15.1°C. Of all the isolated strains of aflatoxin-producingA. flavus, 4 strains, isolated from a single soil sample, produced large amounts of aflatoxin B₁ and B₂ when incubated in coconut agar, peanut agar, peanuts or trilaurin-added rice, although they did not produce aflatoxin when incubated in rice, yeast extract-sucrose broth or sucrose-low salts broth.     

Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B₁ and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 μm in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B₁, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B₁ production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B₁ and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B₁ per ml. The percentages of isolates producing >10 μg of aflatoxin B₁ per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B₁ or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Rapid On-Site Sensing Aflatoxin B1 in Food and Feed via a Chromatographic Time-Resolved Fluoroimmunoassay

Aflatoxin B₁ poses grave threats to food and feed safety due to its strong carcinogenesis and toxicity, thus requiring ultrasensitive rapid on-site determination. Herein, a portable immunosensor based on chromatographic time-resolved fluoroimmunoassay was developed for sensitive and on-site determination of aflatoxin B₁ in food and feed samples. Chromatographic time-resolved fluoroimmunoassay offered a magnified positive signal and low signal-to-noise ratio in time-resolved mode due to the absence of noise interference caused by excitation light sources. Compared with the immunosensing performance in previous studies, this platform demonstrated a wider dynamic range of 0.2-60 μg/kg, lower limit of detection from 0.06 to 0.12 µg/kg, and considerable recovery from 80.5% to 116.7% for different food and feed sample matrices. It was found to be little cross-reactivity with other aflatoxins (B₂, G₁, G₂, and M₁). In the case of determination of aflatoxin B₁ in peanuts, corn, soy sauce, vegetable oil, and mouse feed, excellent agreement was found when compared with aflatoxin B₁ determination via the conversational high-performance liquid chromatography method. The chromatographic time-resolved fluoroimmunoassay affords a powerful alternative for rapid on-site determination of aflatoxin B₁ and holds a promise for food safety in consideration of practical food safety and environmental monitoring.     

On the binding of aflatoxin B 1 and its metabolites to hepatic microsomes

The metabolism of aflatoxin B₁ was studied using the cytochrome P450-dependent mixed function oxidase system of rat liver microsomes. An aflatoxin metabolite produced in the presence of microsomes and NADPH and not produced in the presence of SKF-525A seems to become covalently bound to microsomes. The bound metabolite is observed as a spectral peak at 412 nm by means of difference spectroscopy. This metabolite appears to be related to either aflatoxin B_2a or its precursor.     

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.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

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.     

Aflatoxin B<Subscript>1</Subscript> Contamination of Traditionally Processed Peanuts Butter for Human Consumption in Sudan

A survey was carried out to detect the presence of aflatoxin B₁ in 60 duplicated samples (120 samples) of peanuts butter purchased from the local markets and other traditionally prepared and distributed by the street sellers in Khartoum state, Sudan. AflaTest-P affinity column was used to extract the toxin from the samples, and the concentration was measured by calibrated Vicam fluorometer. Aflatoxin B₁ was detected at variable levels in 100% of the screened samples. Traditionally prepared samples showed the highest incidence of aflatoxin B₁ which is above the internationally regulated tolerance levels (5–20 ppb). The means and the ranges of the aflatoxin B₁ recovered were as follows: 63.9 ppb (29–128 ppb), 54.5 ppb (21–131 ppb) and 101 ppb (17–170 ppb) for samples collected from Khartoum, Khartoum North and Omdurman areas, respectively. Samples from retail stores presented relatively low aflatoxin B₁ incidences 14.5 ppb (1–57 ppb), but only 30% of the samples revealed aflatoxin level below 10 ppb. Laboratory segregated and carefully prepared butter from good grade nuts showed the lowest levels of this toxin (3.3 ppb; 2–6 ppb). The results showed that peanuts butter prepared by the street sellers and distributed by the retail stores are evidently hazardous to human health. There is therefore urgent need for strong form of quality control measures and public awareness. The use of excellent grade peanuts and care during processing and storage are priority.     

Effect of temperature on production of aflatoxin on rice by Aspergillus flavus

Summary The effect of temperature on formation of aflatoxin on solid substrate (rice) byAspergillus flavus NRRL 2999 has been studied in some detail. The optimum temperature for production of both aflatoxin B₁ and G₁ under the conditions employed is 28° C. Comparable yields of B₁ were obtained at 32° C, but considerably less G₁ was produced at this temperature. Both B₁ and G₁ were found in lesser amounts at temperatures above 32° C, and the aflatoxin content of rice incubated at 37° C was low (300–700 ppb) even though growth was good.Reducing the temperature from 28° to 15° C resulted in progressively less aflatoxin, but 100 ppb of B₁ was detected in cultures incubated 3 weeks at 11° C. No aflatoxin was produced at 8° C.The ratio of the four aflatoxins is affected by temperature. At the lower temperatures, essentially equal amounts of aflatoxin B₁ and G₁ were produced, whereas at 28° C, approximately four times as much B₁ was detected as G₁. At the higher temperatures, relatively less G was formed, until at 37° C, less than 10 ppb was detected.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase,isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus

The relationship between the activities of 3 cytosolic enzymes with aflatoxin biosynthesis in Aspergillus parasiticus cultured under different conditions has been investigated in order to find out the role of each enzyme in aflatoxin biosynthesis. Basically the activity of isocitrate dehydrogenase (IDH) was higher in non-toxigenic strains as compared to its counterpart toxigenic fungi (p < 0.05). In contrast, the activities of fatty acid synthase (FAS) as well as glutathione S-transferase (GST) were higher (P < 0.05) in toxigenic strains than that of the non-toxigenic fungi. Aflatoxin production was inhibited in fungi grown in presence of various concentrations of neem leaf extract. Aflatoxin was at its lowest level (>90% inhibition) when the concentration of neem extract was adjusted to 50% (v/v). No significant changes in FAS and IDH activities were observed when aflatoxin synthesis was under restraints by neem (Azadirachta indica) leaf extract. During a certain period of time of culture growth, when aflatoxin production reached to its maximum level, the activity of FAS was slightly induced in the toxigenic strains fed with a low concentration (1.56% v/v) of the neem leaf extract. At the time (96 h) when aflatoxin concentration reached to its maximum levels, the activity of GST in the toxigenic fungi was significantly higher (i.e., 7–11 folds) than that of non-toxigenic strains. The difference was highest in mycelial samples collected after 120 h. However unlike FAS and IDH, GST was readily inhibited (67%) in mycelia fed with 1.56% v/v of the neem extract. The inhibition reached to maximum of 80% in samples exposed to 6.25–12.5% of the extract. These results further substantiate previous finding that there is a positive correlation between GST activity and aflatoxin production in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Synergistic effect ofSacoglottis gabonensis bark extract,

Aflatoxin B₁ metabolism was studied using microsomal and cytosolic fractions isolated from weanling male Fischer F344 rats given in drinking water for 7 days an aqueous extract ofSacoglottis gabonensis bark, 0.1% ethanol solution, or a solution containing both extract and ethanolad libitum. Microsomal production of aflatoxin B₁-dihydrodiol, aflatoxin Q₁, aflatoxin M₁, aflatoxin Pi and proteinaflatoxin adduct formation, and cytosolic aflatoxin B₁-glutathione conjugation were assayed. Pretreatment with the extract alone or together with ethanol caused significant increases in aflatoxin M₁ production as compared to controls given only water, but aflatoxin Q₁ production was enhanced only by pretreatment with both extract and ethanol. All the three treatments caused significant reductions in liver glutathione content. The highest aflatoxin B₁ metabolising activity as determined by aflatoxin M₁ and aflatoxin Q₁ production was observed in rats pretreated with both ethanol and the extract, suggesting synergism. The findings suggest that at relatively mild doses,S. gabonensis extract alone or in concert with ethanol may influence response to aflatoxin.     

A novel glass fiber disc culture system for testing of small amounts of compounds on growth and aflatoxin production byAspergillus flavus

A new method for growingAspergllius flavus for experimental studies is presented. The system consists of a humidified vial with a thick septum pierced by a pin on which a glass fiber disc is affixed. The disc contains the test solution and inoculum plus medium. The method has been used to assess the effect of variations in culture conditions on production of aflatoxin B₁ (AFB₁). The AFB₁ level was affected by the amount of medium placed on the disc and type of disc material. The results for different types of glass fiber and quartz discs were compared with AFB₁ produced by fungus grown in liquid medium or on paper discs. When compared to a liquid medium culture there was a 15 to 20-fold increase in AFB₁ for one type of disc. Incubations with less than 14 µl of medium gave satisfactory results. A crude phosphatidylcholine preparation at a concentration of 0.7% of the medium resulted in a 4-fold increase in AFB₁.Abbreviations AFB₁ aflatoxin B₁ - CV coefficient of variation - PC phosphatidylcholine - SD suspended disc     

The role of zinc in the aflatoxigenic potential of Aspergillus flavus NRRL 3251 on foodstuffs

The production of total aflatoxin (B₁, B₂, M₁ and M₂) in ten tropical foodstuffs (with and without zinc enrichment) inoculated with Aspergillus flavus NRRL 3251 was examined to determine the effect of zinc on aflatoxigenic potential. Aflatoxin production was not linearly correlated with zinc levels of the food substrates. The data presented indicate that optimal zinc requirement for maximal aflatoxin production was substrate specific.     

Aflatoxins B1 and G1 solubility in standard solutions and stability during cold storage

In the present work we study the use of different solvents to store aflatoxins B₁ and G₁ standard solutions. We have obtained significant differences between aflatoxin B₁ and G₁ In ethyl acetate, methanol and water, with aflatoxin G₁ being less stable. We recommend chloroform as the election solvent to store the aflatoxin solutions. The fact that aflatoxins are highly stable in water may have a potential use in experiments of biological activity.     

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.     

Interrelationship of kernel water activity,soil temperature,maturity, and phytoalexin production in preharvest aflatoxin contamination of drought-stressed peanuts

Samples of Florunner peanuts were collected throughout a period of late-season drought stress with mean geocarposphere temperatures of 29 and 25 °C, and determinations of maturity, kernel water activity (a_w), percent moisture, capacity for phytoalexin production, and aflatoxin contamination were made. Results showed an association between the loss of the capacity of kernels to produce phytoalexins and the appearance of aflatoxin contamination. Kernel a_w appeared to be the most important factor controlling the capacity of kernels to produce phytoalexins. Mature peanuts possessed additional resistance to contamination that could not be attributed solely to phytoalexin production. Kernel moisture loss was accelerated in the 29 °C treatment compared to the 25 °C treatment, and data indicated that the higher soil temperature also favored growth and aflatoxin production by Aspergillus flavus in peanuts susceptible to contamination.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.