Effect of ammoniation on the toxicity of corn artificially contaminated with aflatoxin B1

The effect of an aqueous ammonia treatment of aflatoxin B₁-containing corn was determined on toxicity of the corn administered orally to male Fischer rats. Corn was contaminated with aflatoxin B₁ (5 mg/g corn) and administered per se (2.0 or 4.0 g corn/kg) or after treatment with ammonium hydroxide. Body weight changes, liver weight, hexobarbital sleeping times, hepatic microsomal concentrations of protein and cytochromes P-450 and b₅, and mortality were determined 72 hr after dosing with corn, and serum alkaline phosphatase activity was determined 96 hr after dosing. The ammonia treatment of contaminated corn prevented the changes in these parameters caused by aflatoxin B₁. Ammoniation of corn free of aflatoxin had no adverse effect on the parameters, although ammoniation per se did raise the concentrations of hepatic microsomal protein. The results of the study indicate that ammoniation may prevent acute aflatoxicosis produced by aflatoxin-contaminated corn.     

Assessment of Maize Hybrids Resistance to Aspergillus Ear Rot and Aflatoxin Production in Environmental Conditions in Serbia

Aflatoxin, a naturally occurring toxin produced by the fungus Aspergillus flavus, is the most economically important mycotoxin in the world, with harmful effects on human and animal health. Preventive measures such as irrigation and planting dates can minimize aflatoxin contamination most years. However, no control strategy is completely effective when environmental conditions are extremely favorable for growth of the fungus. The most effective control method is growing maize hybrids with genetic resistance to aflatoxin contamination. The aim of this research was to evaluate the sensitivity of different maize hybrids to A. flavus infection and aflatoxin accumulation. Twenty commercial maize hybrids were evaluated in field trials with artificial inoculations using the colonized toothpicks method. The mycotoxin production potential of A. flavus isolates was confirmed by cluster amplification patterns (CAPs) analysis. The results of this research indicated the existence of significant differences in maize hybrids susceptibility to Aspergillus ear rot and aflatoxin B₁ accumulation. No hybrid included in this research showed complete resistance in all conditions, but some hybrids showed partial resistance. Different hybrids also responded differently depending on the sowing date. This research showed that infection intensity is not always consistent with aflatoxin levels, and therefore visual evaluation is not enough to assess maize safety.     

Relationship between the Fungal Incidence,Water Activity,Humidity, and Aflatoxin Content in Maize Samples from the Highlands and Coast of Ecuador

This study evaluated the fungal incidence through direct plating in Agar Dichloran Glycerol, and the presence of aflatoxins in maize samples from the Highlands and Coast of Ecuador by HPLC, investigating the influence of the temperature, altitude, water activity, and humidity of the collection regions on the maize samples’ contamination using Principal Components Analysis (PCA). The overall kernel infection by fungi was usually lower in samples from the Highlands, and no aflatoxins or Aspergillus series Flavi were detected in the samples from this region. In the coastal samples, Aspergillus sp. were isolated from all samples, while the potentially aflatoxigenic A. Flavi contaminated about 80% of them. Aflatoxins were present in 50% of these samples, in ranges from 0.42 to 107.69 µg/kg. PCA was able to segregate the samples according to their collection region, and showed that the maximum and minimum temperatures are closely and positively related to the presence of A. Flavi. A highly positive relationship was also observed between the water activity of the sample and aflatoxin contamination. On the other hand, the altitude had a very strong—but negative—relationship with the variables studied. This study is relevant because data regarding fungi and aflatoxin occurrence, as well the main factor influencing the contamination of Ecuadoran maize, are scarce; it clearly shows that aflatoxins are a hazard present in maize from the Ecuadorian Coast but not the Highlands.     

Aflatoxin Control in Maize by Trametes versicolor

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.     

Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM

Toxin-contaminated foods and beverages are a major source of illness, may cause death, and have a significant negative economic impact worldwide. Aflatoxin B1 (AFB1) is a potent toxin that may induce cancer after chronic low-level exposure. This study developed a quantitative recombinant AflR gene antiserum ELISA technique for aflatoxin B1 detection in contaminated food products. Aflatoxin B1 residuals from 36 food samples were analyzed with HPLC and VICAM. DNA was extracted from aflatoxin-contaminated samples and the AflR gene amplified using PCR. PCR products were purified and ligated into the pGEM-T vector. Recombinant plasmids were sequenced and transformed into competent E. coli (BL21). Molecular size and B-cell epitope prediction for the recombinant protein were assessed. The purified protein was used to induce the production of IgG antibodies in rabbits. Serum IgG was purified and labeled with alkaline phosphatase. Finally, indirect-ELISA was used to test the effectiveness of polyclonal antibodies for detection of aflatoxin B1 in food samples.     

Bio-detoxification of aflatoxin B1 in artificially contaminated maize grains using lactic acid bacteria

Aflatoxins are a group of carcinogenic mycotoxins produced by Aspergillus flavus, A. parasiticus, and A. nomius. Due to the ubiquitous occurrence of aflatoxins, preventive and remediative measures are necessary including detoxification techniques. Physical and chemical decontamination strategies are inconvenient. In this study a biological detoxification strategy was tested using bacteria of the Lactobacillus species collected from the biotechnology laboratory at University of Ibadan, Nigeria. Maize grains with moisture content of 17% were artificially inoculated with toxigenic A. flavus (LA 32G_28) and atoxigenic A. flavus (LA32_79) at ambient temperature and four samples of bulk maize grains were prepared at aflatoxin B1 concentrations of 50, 100, 200, and 500?ng/g. To evaluate the detoxifying potential of lactic acid bacteria five different cultures consisting of Lactobacillus acidophilus, L. brevis, L. casei, L. delbruekii, and L. plantarum were used to inoculate the aflatoxin B1–contaminated maize samples at 37°C. After 5 days, the residual aflatoxin B1 on maize was determined. All treatments showed significant reductions (P<?0.05) in aflatoxin B1. Lactic acid bacteria decreased the pH of the medium from 5.0 to 4.0. Pronounced aflatoxin B1 reduction was observed in maize contaminated at 50?ng/g (44.5%), while maize contaminated at 500ng/g was the least reduced (29.9%). L. plantarum was the most efficient organism in degrading aflatoxin B1. Use of lactic acid bacteria, which already has Generally Regarded As Safe (GRAS) status, should be encouraged for use as a bio-detoxification agent for aflatoxins.     

Assessment of Aflatoxin Contamination of Maize,Peanut Meal and Poultry Feed Mixtures from Different Agroecological Zones in Cameroon

Mycotoxins affect poultry production by being present in the feed and directly causing a negative impact on bird performance. Carry-over rates of mycotoxins in animal products are, in general, small (except for aflatoxins in milk and eggs) therefore representing a small source of mycotoxins for humans. Mycotoxins present directly in human food represent a much higher risk. The contamination of poultry feed by aflatoxins was determined as a first assessment of this risk in Cameroon. A total of 201 samples of maize, peanut meal, broiler and layer feeds were collected directly at poultry farms, poultry production sites and poultry feed dealers in three agroecological zones (AEZs) of Cameroon and analyzed for moisture content and aflatoxin levels. The results indicate that the mean of the moisture content of maize (14.1%) was significantly (P < 0.05) higher than all other commodities (10.0%–12.7%). Approximately 9% of maize samples were positive for aflatoxin, with concentrations overall ranging from <2 to 42 µg/kg. Most of the samples of peanut meal (100%), broiler (93.3%) and layer feeds (83.0%) were positive with concentrations of positive samples ranging from 39 to 950 µg/kg for peanut meal, 2 to 52 µg/kg for broiler feed and 2 to 23 µg/kg for layer feed. The aflatoxin content of layer feed did not vary by AEZ, while the highest (16.8 µg/kg) and the lowest (8.2 µg/kg) aflatoxin content of broiler feed were respectively recorded in Western High Plateau and in Rainforest agroecological zones. These results suggest that peanut meal is likely to be a high risk feed, and further investigation is needed to guide promotion of safe feeds for poultry in Cameroon.     

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard^® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.     

Safety and efficacy evaluation of aqueous citric acid to degrade B-aflatoxins in maize.

Chemical inactivation of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in maize grain by means of 1N aqueous citric acid was confirmed by the AFLATEST immunoaffinity column method, high performance liquid chromatography (HPLC), and the Ames test (Salmonella-microsomal screening system). The AFLATEST assay showed that aflatoxins in the maize grain with an initial concentration of 29 ng/g were completely degraded and 96.7% degradation occurred in maize contaminated with 93 ng/g when treated with the aqueous citric acid. Aflatoxin fluorescence strength of acidified samples was much weaker than untreated samples as observed in HPLC chromatograms. On the other hand, the Ames test results indicated that the mutagenic activity of acidified samples was greatly reduced compared with that of untreated samples based on his- --> his+ reversions in the Salmonella TA100 strain. Chemical inactivation appears to be a promising method of removing aflatoxin from food commodities.     

Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: Synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production

Occurrence of aflatoxin in Madhuca indica Gmel. seeds was determined by competitive ELISA. Eighty percent of mahua seed samples were found to be contaminated with aflatoxin. Total aflatoxin content ranged from 115.35 to 400.54 ppb whereas the concentration of AFB₁ was in the range of 86.43 to 382.45 ppb. Mahua oil was extracted by cold press expeller and analysed for contamination of aflatoxin in both the oil and cake samples. Total aflatoxin and aflatoxin B₁ were 220.66 and 201.57 ppb in oil as compared to that in cake samples where it was 87.55 and 74.35 ppb, respectively. Various individual and combined plant extracts were evaluated for their efficacy against growth of Aspergillus flavus and aflatoxin production in vitro. Combination of botanicals were found to be more effective in controlling fungal growth and aflatoxin production than individual extracts. Results of the present study suggests that synergistic effect of plant extracts can be used for control of fungal growth and aflatoxin production. These natural plant products may successfully replace synthetic chemicals and provide an alternative method to protect mahua as well as other agricultural commodities of nutritional significance from toxigenic fungi such as A. flavus and aflatoxin production.     

Characterization of the Aspergillus flavus Population from Highly Aflatoxin-Contaminated Corn in the United States

Aflatoxin contamination of corn is a major threat to the safe food and feed. The United States Federal Grain Inspection Service (FGIS) monitors commercial grain shipments for the presence of aflatoxin. A total of 146 Aspergillus flavus were isolated from 29 highly contaminated grain samples to characterize the visual phenotypes, aflatoxin-producing potential, and genotypes to explore the etiological cause of high aflatoxin contamination of US corn. Five of the isolates had reduced sensitivity (43–49% resistant) to the fungicide azoxystrobin, with the remainder all being over 50% resistant to azoxystrobin at the discriminating dose of 2.5 µg/mL. Only six isolates of the highly aflatoxigenic S morphotype were found, and 48 isolates were non-aflatoxigenic. Analysis of the mating type locus revealed 45% MAT 1-1 and 55% MAT 1-2. The A. flavus population originating from the highly aflatoxin contaminated grain samples was compared to a randomly selected subset of isolates originating from commercial corn samples with typical levels of aflatoxin contamination (average < 50 ppb). Use of simple sequence repeat (SSR) genotyping followed by principal component analysis (PCoA) revealed a similar pattern of genotypic distribution in the two populations, but greater diversity in the FGIS-derived population. The noticeable difference between the two populations was that genotypes identical to strain NRRL 21882, the active component of the aflatoxin biocontrol product Afla-Guard™, were ten times more common in the commercial corn population of A. flavus compared to the population from the high-aflatoxin corn samples. The other similarities between the two populations suggest that high aflatoxin concentrations in corn grain are generally the result of infection with common A. flavus genotypes.     

Development and Evaluation of Monoclonal Antibodies for Paxilline

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC₅₀s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC₅₀. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.     

Effect of soil on aflatoxin tissue retention in chicks when added to aflatoxin-contaminated poultry rations.

The effects of silty clay loam soil on aflatoxin B1 (AFB1) absorption were investigated when added to the diets of chicks fed aflatoxin-contaminated rations. Sixty 1-d-old White Leghorn chicks were fed a control ration (< 5 ng AFB1/g), a low aflatoxin-contaminated ration (55 ng AFB1/g), a high aflatoxin-contaminated ration (4,488 ng AFB1/g), or high aflatoxin-contaminated rations (4,488 ng AFB1/g) + 25% or 50% soil. Livers in each group were pooled and analyzed for AFB1 and metabolites. The addition of soil significantly reduced the levels of AFB1 in the livers, although the reduction was less when 25% soil was fed compared with the 50% soil feeding.     

Mould incidence and mycotoxin contamination in maize kernels from Swat Valley,North West Frontier Province of Pakistan

Mould incidence and aflatoxin B1 (AFB1) and ochratoxin A (OTA) contamination as well as proximate composition and minerals content of maize kernels from Swat Valley, North West Frontier Province of Pakistan was studied during the year, 2007. Results indicated that the mean moisture content of the kernels was within the recommended safe storage levels of ?15%. Across the whole valley, Aspergillus, Fusarium, Penicillium and Rhizopus were the most predominant fungal genera identified and amongst the mycotoxigenic species, Aspergillus flavus had the highest incidence. AFB1 content ranged from none to 30.92 μg/kg with the average values of 14.94 and 16.22 μg/kg for Upper and Lower Swat regions, respectively. Similar trend was observed for OTA with the contamination level ranged from <0.001 to 7.32 μg/kg. A significant numbers of samples contained AFB1 and OTA levels above the safe limits as recommended by the USFDA and EU but on the average the results were within the safe limit. These results indicate that maize consumers in Swat Valley may be exposed to the danger of aflatoxins and ochratoxins poisoning. Thus, there is a need for policy makers to establish and enforce maize quality standards and regulations related to moulds and mycotoxins across the area.     

Chemical investigation of different crude extracts from Teucrium ramosissimum leaves. Correlation with their antigenotoxic and antioxidant properties

The effect of extracts obtained from Teucrium ramosissimum leaves on genotoxicity and SOS response induced by aflatoxin B₁ (0.5 μg/assay) as well as nitrofurantoin (5 μg/assay) was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixture. However, all the extracts, particularly the total oligomers flavonoids (TOF) extract significantly decreased the genotoxicity induced by aflatoxin B₁ and nitrofurantoin. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non-enzymatic (NBT/Riboflavine assay) systems. TOF extract was the most effective one in inhibiting both xanthine oxidase activity and NBT reduction. Our findings emphasize the potential of T. ramosissimum to prevent mutations and also its antioxidant effect.     

Characterization of Ugandan Endemic Aspergillus Species and Identification of Non-Aflatoxigenic Isolates for Potential Biocontrol of Aflatoxins

Acute stunting in children, liver cancer, and death often occur due to human exposure to aflatoxins in food. The severity of aflatoxin contamination depends on the type of Aspergillus fungus infecting the crops. In this study, Aspergillus species were isolated from households’ staple foods and were characterized for different aflatoxin chemotypes. The non-aflatoxigenic chemotypes were evaluated for their ability to reduce aflatoxin levels produced by aflatoxigenic A. flavus strains on maize grains. Aspergillus flavus (63%), A. tamarii (14%), and A. niger (23%) were the main species present. The A. flavus species included isolates that predominantly produced aflatoxins B1 and B2, with most isolates producing a high amount (>20 ug/µL) of aflatoxin B1 (AFB1), and a marginal proportion of them also producing G aflatoxins with a higher level of aflatoxin G1 (AFG1) than AFB1. Some non-aflatoxigenic A. tamarii demonstrated a strong ability to reduce the level of AFB1 by more than 95% when co-inoculated with aflatoxigenic A. flavus. Therefore, field evaluation of both non-aflatoxigenic A. flavus and A. tamarii would be an important step toward developing biocontrol agents for mitigating field contamination of crops with aflatoxins in Uganda.     

The effect on performance and biochemical parameters when soil was added to aflatoxin-contaminated poultry rations.

The effects of silty clay loam soil on the performance and biochemical parameters of chicks were investigated when the soil was added to aflatoxin B1 (AFB1)-contaminated diets. One hundred 14-d-old White Leghorn chicks were fed a control ration (clean corn), a low aflatoxin-contaminated ration (120 ng AFB1/g), a high aflatoxin-contaminated ration (700 ng AFB1/g), or high aflatoxin-contaminated rations (700 ng AFB1/g) +10% or 25% soil. Body weight, feed consumption and blood samples were monitored weekly. Decreased feed consumption, body weight gain and efficiency of feed utilization, increased SGOT and LDH activities, and cholesterol and triglyceride concentrations, and decreased uric acid concentrations and ALP activity were observed in the chicks fed the high aflatoxin-contaminated ration without soil. Hepatomegaly was prominent in chicks fed the high aflatoxin-contaminated ration without soil, and some livers had extensive hepatocyte vacuolation, hepatocellular swelling, fatty change and hydropic degeneration, and stained positive for fat accumulation. Addition of soil reduced the detrimental effects of AFB1 for some parameters, although the reduction was less when 10% soil was fed compared with the 25% soil feeding.     

Effects of long-term feeding of ammoniated, aflatoxin-contaminated corn to Fischer 344 rats

The effectiveness of ammonia treatment in reducing the chronic toxicity of aflatoxin-contaminated corn was determined. Fischer 344 rats were fed semi-purified rations containing 20% w/w corn that was either free of aflatoxin or naturally contaminated with 880 micrograms/kg total aflatoxin and was either treated with ammonia gas or was not treated. Therefore the rats that were fed the aflatoxin-contaminated diet received 176 ppb total aflatoxins. Body weight and food consumption were recorded throughout the study; hematological measurements were made after 87 weeks of feeding; and after 91 weeks the rats were killed and histopathological abnormalities were noted. Signs of chronic toxicosis in rats fed aflatoxin-contaminated corn included increased mortality, decreased hematocrit and hemoglobin levels, elevated serum alkaline phosphatase activities, and a 100% incidence of liver neoplasia. These signs did not occur in rats in the other dietary treatment groups, including those fed ammoniated, aflatoxin-contaminated corn. The results provide further evidence that the atmospheric ammoniation process effectively reduces the toxicity of aflatoxin-contaminated corn.     

The occurrence of mycotoxins in key commodities in Bangladesh: surveillance results from 1993 to 1995

A three-year surveillance program assessed the extent of mycotoxin contamination of key foods and feeds grown in Bangladesh. The study also included groundnuts utilized as snack food. In the first two phases of the program the samples collected were analyzed only for aflatoxins, but in the third phase, as well as for aflatoxins, samples were tested for the presence of fumonisin B1, ochratoxin A, zearalenone, deoxynivalenol, and T-2 toxin. Of the foods and feeds tested, the incidence of aflatoxin contamination varied from low (rice collected from farmers' stores, 8%) to high (maize, 67%). However, both the average total aflatoxin contents (< 1.0 microg/kg) and the maximum aflatoxin B1 contents (< or = 5.0 microg/kg) recorded for pulses, rice and its various products, and wheat were low. On the other hand, the levels of contamination of maize, roasted and raw groundnuts, and poultry feed were considerably higher, with average total aflatoxin B1 contents of 33, 13, 65, and 7 microg/kg, respectively, and maximum aflatoxin B1 contents of 245, 79, 480, and 160 microg/kg, respectively. Fumonisin B1, ochratoxin A, zearalenone, deoxynivalenol, and T-2 toxin were found, to any significant extent, only in some of the maize samples tested, always accompanied by aflatoxins. One sample of maize contained five mycotoxins, namely, the aflatoxins, fumonisin B1, deoxynivalenol, zearalenone, and ochratoxin A. In a limited trial using hospital staff in Dhaka, the analysis of the aflatoxin-albumin adduct in serum showed that approximately half of the test group had been recently exposed to low levels of aflatoxins.     

Aflatoxin-producing fungi associated with Nigerian maize

Maize samples were obtained from different locations—namely Aba, Abakaliki, Afikpo, Okigwe, and Owerri—all in southeast Nigeria. Twelve mold species of the genera Aspergillus, Penicillium, Cladosporium, Alternaria, Fusarium, and Acremonium (Cephalosporium) were isolated. The presence of aflatoxin B₁ was detected in 80% of the samples by the characteristic blue fluorescence that appeared on silica gel coated thin layer chromatography plates when viewed with a long-wave ultraviolet radiation source alongside an aflatoxin standard. Eight isolates of the Aspergillus flavus group obtained from the maize samples were tested for aflatoxin production. Natural medium (maize) at 26°C and moisture content adjusted to no less than 20% was used. Aflatoxin was produced to some degree by 87.5% of the isolates. There was no aflatoxin production at a market moisture content of 5.4% and temperature of 25, 30, and 35°C. However, at 26°C and increased moisture contents of 22.3–24.9%, varying amounts of aflatoxin were produced. © 1994 by John Wiley & Sons, Inc..