Limiting temperature and relative humidity for aflatoxin production byAspergillus flavus in stored peanuts

Sound mature kernels, broken mature kernels, immature kernels and unshelled cured Early Runner peanuts were inoculated with spores ofAspergillus flavus and incubated up to 84 days in controlled environment cabinets. In a series of experiments temperatures ranged from 8 to 49 C in combination with 98±1% relative humidity (RH); in others RH was varied from 70% to 99% at 30±1/2 C and from 83% to 99% at 20±1/2 C. Samples were removed after 7, 21, 42 and 84 days of incubation and assayed for aflatoxin, free fatty acids and peanut kernel moisture. Aflatoxin was formed in sound mature kernels at 40 C and 14 C and in broken mature kernels at 13 C, but none was formed at 41 C after 21 days or at 12 C after 84 days in 98±1% RH. The limiting temperatures for aflatoxin formation in peanut kernels with intact shells were 41 C for 21 days and 16 C for 84 days of incubation. The limiting RH at 30 C for aflatoxin production in sound mature kernels was 84%, whereas in broken mature and immature kernels it was 83% and in kernels from unshelled peanuts the limiting RH was 86% for 84 days of incubation. The limiting RH at 20 C for sound and broken mature kernels was 83%, whereas it was 86% RH for immature kernels and 92% for kernels from unshelled peanuts. Free fatty acid formation was correlated with visible growth of fungi rather than with aflatoxin production. Aflatoxin formation was generally correlated with kernel moisture contents of 10% or higher.     

Aflatoxin in Arizona cottonseed: Field inoculation of bolls byAspergillus flavus spores in wind-driven soil

Conditions typical of an Arizona monsoon were mimicked in the field to inoculate cotton plants withAspergillus flavus. Spores, mixed with autoclaved local soil, were blown onto cotton plants having bolls at all stages of maturity, using a modified commercial leaf blower. Half the plants were sprayed with water following inoculation. After one month, plants were pulled and the position of bolls mapped. All bolls were examined for bright-green-yellow fluorescence (BGYF) of lint, and ginned seeds from each boll were assayed for aflatoxin. Control non-wetted, non-inoculated bolls had no BGYF lint and no aflatoxin-containing seed. In contrast, 15% of the bolls from wetted, inoculated plants exhibited BGYF; 18% of these BGYF bolls had toxin. Only 3% of the non-wetted bolls had BGYF lint and none contained toxin. Lower bolls (fully fluffed at inoculation) were not infected, nor were upper bolls (flower stage at inoculation). Infection occurred only in bolls that had opened during the 30 days following inoculation. While the position of BGYF bolls on naturally contaminated plants was the same as for the inoculated, the ratio of toxic bolls to BGYF bolls was different. All BGYF bolls from plants naturally contaminated withA. flavus contained aflatoxin.     

Seed viability and aflatoxin production in individual cottonseed naturally contaminated withAspergillus flavus

Individual cottonseed naturally contaminated withAspergillus flavus Link were tested for viability and assayed for aflatoxin B₁. Of the 55 infected seeds tested, less than half (21) germinated normally, a rate much lower than expected for uninfected cottonseed. Aflatoxin levels in the seeds varied from below 300 ng/g to over 100,000 ng/g of seed. Statistical analyses indicate that the presence of aflatoxin is correlated with poor germination in cottonseed naturally contaminated withA. flavus.     

Distribution of ammonia-related aflatoxin reaction products in cottonseed meal

The fate of aflatoxin during ammoniation of contaminated cottonseed meal was studied under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to 27.7 kg meal (14% moisture) that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with 4% ammonia at 40 psi, 100 C for 30 min. This treatment reduced the chemically detected aflatoxin B₁ to less than 4 μg/kg. In control nonammoniated meals, 90% of the radiolabeled material was accounted for in the methylene chloride extract. Duplicate 2-kg samples of the ammoniated meal were fractionated and the distribution of radioactivity was determined. Ca. 86% of the radioactivity was detected in the meal after initial air-drying. Ca. 25% of the added radioactivity was extracted from the air-dried meal with methylene chloride and another ca. 5% was extracted from this residue with methanol. Weak acid released 3% of the added radioactivity from the residue after methanol extraction, bicarbonate released 1% and Pronase digestion, including methylene chloride extraction of the residue, accounted for nearly 19% of the total added radioactivity. Only 37% of the added radioactivity remained in the meal matrix following solvent extractions and chemical and enzymic treatments.     

Factors affecting aflatoxin contamination of cottonseed I. Contamination of cottonseed withAspergillus flavus at harvest and during storage

Examination of cottonseed production has shown that boll weevils (Anthonomus grandis), boll rots, and improper handling and storage conditions are critical factors inAspergillus flavus contamination of cotton-seed.A. flavus cultures were isolated from both field-collected and laboratory emerged boll weevils, as well as from boll weevil emergence holes. Diseased cotton bolls have been found to containA. flavus conidia. Infection byA. flavus was limited to the surface of cottonseeds collected from gin and from the gin blower. Seeds improperty stored outside the gin were infected internally withA. flavus. Observation of bright greenish-yellow fluorescence was not useful as a diagnostic procedure to detect contaminated seeds. Aflatoxin-producing potential ofA. flavus isolates is being investigated. WhenA. flavus conidia were artifically inoculated onto the surface of the seeds, 87% of the seeds from Athens, GA, were internally infected whereas only 29% of the seeds from Macon, GA, were internally infected.A. flavus invade the cottonseed embryo through the chalazal region, the micropylar region, or cracks developed in the seed coat during ginning. Invasion of cottonseed byA. flavus was predominant at 28, 30, and 37 C while at 15 and 20 C other fungi dominated in surface and internal invasions of cottonseed. At relative humidities of 75% and 80%,Chaetomium spp. successfully competed in growth on the surface, while at a relative humidity of 90% and above, fungi belonging to the orderMucorales outgrewA. flavus.     

Esterification of fatty acids byAspergillus flavus

Aspergillus flavus, grown on soybean oil fatty acids as the sole carbon source, produced triglycerides. While most of the triglycerides were intracellular, considerable amounts also were found extracellularly. The latter originated most likely from esterification of fatty acids by a cell-bound lipase. Although the fatty acids of these fungal triglycerides were the same as those of soybean oil fatty acids, polyunsaturated acid content was greater than expected from the added substrate. Part of a presentation of the American Oil Chemists' Society Annual Meeting in Phoenix, AZ, in May 1988.     

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to cottonseed meal that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B₁ was mutagenic at a concentration of ca. 0.005 μg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 μg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 μg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.     

Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids byAspergillus flavus in sterile peanuts

Sound mature kernels, broken mature kernels, immature kernels, and unshelled Early Runner peanuts were heat-treated in controlled environment cabinets and inoculated with spores ofAspergillus flavus. Treatments were incubated at 97-99% relative humidity at different temperatures ranging from 5 to 55C and also at 30C with relative humidities ranging from 55 to 99%. Samples were removed after 7 and 21 days and assayed for aflatoxin, free fatty acids, and peanut kernel moisture. The limiting relative humidity for aflatoxin production byA. flavus was 85ŷ1% relative humidity for 21 days at 30C. The limiting low temperature for visible growth and aflatoxin production by the fungus was 13ŷ1C for 21 days at 97-99% relative humidity. Damaged kernels, however, developed some afllatoxin in 21 days at 12C. The maximum temperature for aflatoxin production was 41.5ŷ1.5C for 21 days at 97-99% relative humidity. Fungus growth and sporulation at 43C were equal to that at 40C, but no aflatoxin was produced. Moisture content of immature kernels was higher at equilibrium with the same relative humidity than the moisture content of sound mature kernels, damaged kernels, or kernels from unshelled peanuts. There appeared to be no proportional quantitative correlation between synthesis of aflatoxin and production of free fatty acids in nonliving peanuts, but no aflatoxin was produced without a simultaneous increase in free fatty acids.     

Use of zinc acetate in extract purification for aflatoxin assay of cottonseed products

The official Association of Official Analytical Chemists’ and American Oil Chemists’ Society’s methods for aflatoxin assay of cottonseed products utilize 20% lead acetate solution to remove gossypol, fatty acids, and traces of lipids as insoluble lead derivatives. The substitution of a 20% zinc acetate solution containing a trace of a trivalent metal chloride to precipitate zinc derivatives yields, in some cases, superior cleanup of interfering compounds of primary extracts.     

Evaluation of cottonseed aflatoxin testing programs

A computer model was developed to simulate cottonseed aflatoxin testing programs. By use of the model, probabilities of obtaining certain aflatoxin test results for various lot concentrations and sample sizes were determined. Also, the effects of sample size and the definition of good and bad sample quality on the probability of lot acceptance were demonstrated. Paper number 5247 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Sampling cottonseed lots for aflatoxin contamination

Large samples called “sublots” were drawn from 41 commercial lots of contaminated cottonseed. Each sublot was subdivided into twenty 5 lb samples which were analyzed for aflatoxin. The mean, median, variance, coefficient of variation, and the estimated range among the sample concentrations were computed. The results indicated that: (A) the variance among sample concentrations was large and was found to be a function of sample concentration and (B) the distribution of sample concentrations was skewed; the density of sample values was greater below the sublot concentration.     

Note on the hygroscopic equilibrium of cottonseed and cottonseed products

The hygroscopic equilibrium of a sample of the Stoneville 2B variety of cottonseed and its derived products has been determined over the relative humidity range of 11 to 93%. In a previous publication (1) the hygroscopic equilibrium of a sample of the D and PL variety of cottonseed was determined over the range of 31 to 93% relative humidity: Comparison of the previous and present results show that the whole seed and kernels of both varieties exhibit the same hygroscopic equilibrium behavior. A comparison of the aforementioned results with those reported by Simpson (3) on Stoneville 2B variety, and by Franco (4) on the 1A 7387 variety grown in São Paulo, Brazil, show that for intact cottonseed the hygroscopic equilibrium behavior is the same. On the basis of the curves given in Figure 1 and on the assumption that the cottonseed used was representative of cottonseed in general, it is possible to calculate the equilibrium moisture content of the whole cottonseed or any component thereof. For example, cottonseed consisting of 12.1% linters, 87.9% delintered seed, 56.0% kernels, and 31.9% lint-free hulls, and containing 12% moisture on a whole seed basis, will yield linters, delintered seed, kernels, and lint-free hulls having 9.4, 12.7, 10.8, and 15.4% moisture, respectively.     

Variability associated with testing cottonseed for aflatoxin

The sampling, subsampling, and analytical variance associated with testing cottonseed for aflatoxin were estimated by use of 4.54 kg samples, 100 g subsamples, and the Velasco method of analysis. Regression analysis indicated that each of the above variance components is a function of the concentration of aflatoxin in the populations tested. Functional relationships are presented to determine the sampling, subsampling, and analytical variance for any size sample, subsample, and number of analyses. Paper 4821 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Effects of oilseed storage proteins on aflatoxin production by aspergillus flavus

Potential involvement of seed storage proteins in susceptibility to aflatoxin contamination was assessed with in vitro tests. Initially, two oilseed storage proteins [cottonseed storage protein (CSP) and zein] were compared with bovine serum albumin (BSA) and collagen. Supplementation of a complete defined medium with either oilseed storage protein resulted in significantly more aflatoxin production by Aspergillus flavus than supplementation with either BSA or collagen. Little or no aflatoxin was produced when either BSA, CSP, or zein was employed (at 0.5%) as both the sole carbon and the sole nitrogen source. Media with collagen (0.5%) as the sole nitrogen and carbon source supported aflatoxin production similar to the complete defined medium. Although lower than levels observed with defined medium, aflatoxin production increased with both increasing CSP concentration (0 to 2.0%) and increasing zein concentration (0 to 6.0%) when these proteins served as both the sole carbon and sole nitrogen source. Denaturing polyacrylamide gel electrophoresis and protease activity assays indicated that fungal acquisition of protein carbon was probably via hydrolysis mediated by the 35 kD metalloprotease of A. flavus. Media lacking nitrogen but containing sucrose (5.0%) and supplemented with either zein (1.7%) or CSP (2.0%) supported three- to eightfold more aflatoxin production than the complete defined medium. The results suggest seed storage proteins, when present with an accessible carbon source, may predispose oilseed crops to support production of high levels of aflatoxins by A. flavus during seed infection.     

Effects of oilseed storage proteins on aflatoxin production by Aspergillus flavus

Potential involvement of seed storage proteins in susceptibility to aflatoxin contamination was assessed with in vitro tests. Initially, two oilseed storage proteins [cottonseed storage protein (CSP) and zein] were compared with bovine serum albumin (BSA) and collagen. Supplementation of a complete defined medium with either oilseed storage protein resulted in significantly more aflatoxin production by Aspergillus flavus than supplementation with either BSA or collagen. Little or no aflatoxin was produced when either BSA, CSP, or zein was employed (at 0.5%) as both the sole carbon and the sole nitrogen source. Media with collagen (0.5%) as the sole nitrogen and carbon source supported aflatoxin production similar to the complete defined medium. Although lower than levels observed with defined medium, aflatoxin production increased with both increasing CSP concentration (0 to 2.0%) and increasing zein concentration (0 to 6.0%) when these proteins served as both the sole carbon and sole nitrogen source. Denaturing polyacrylamide gel electrophoresis and protease activity assays indicated that fungal acquisition of protein carbon was probably via hydrolysis mediated by the 35 kD metalloprotease of A. flavus. Media lacking nitrogen but containing sucrose (5.0%) and supplemented with either zein (1.7%) or CSP (2.0%) supported three- to eightfold more aflatoxin production than the complete defined medium. The results suggest seed storage proteins, when present with an accessible carbon source, may predispose oilseed crops to support production of high levels of aflatoxins by A. flavus during seed infection.     

Drying moist cottonseed protein products

A practical procedure is described together with data obtained during its experimental application, for drying moist oilseed protein products. Specific data are given for drying defatted, raw cottonseed meats, containing as much as 30% of water, to a granular, free-flowing meal with a moisture content of 10% or less at temperatures below 150 F. This is accomplished by adjustment of pH of the mixture during agitative drying to control its rheological properties so as to keep power requirements within a practical range. So. Utiliz. Res. Dev. Div., ARS, USDA. Presented at the AOCS Meeting, New York, October, 1968.