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.     

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.     

Destruction of aflatoxins in peanut protein isolates by sodium hypochlorite

Sodium hypochlorite has been tested for destruction of aflatoxins during the preparation of peanut protein isolates from raw peanuts and defatted peanut meal. The treatments were evaluated by determination of the aflatoxins in the products by thin layer chromatography. Effects of sodium hypochlorite concentration, reaction pH, temperature, and time were studied. Results show that both the sodium hypochlorite concentration and pH are important factors in reducing the concentration of aflatoxins in the protein isolates to nondetectable levels. The treatment with 0.4% sodium hypochlorite at pH 8 produced protein isolates with trace amounts of aflatoxins B₁ and B₂ from ground raw peanuts containing 725 ppb aflatoxin B₁ and 148 ppb aflatoxin B₂, whereas untreated protein isolates contained 384 ppb aflatoxin B₁ and 76 ppb aflatoxin B₂. At pH 9, 0.3% sodium hypochlorite reduced the aflatoxin B₁ content in the protein isolates from 300 ppb to below detectable quantities and the aflatoxin B₂ content from 52 ppb to 2 ppb. Similar results were obtained at pH 10 for 0.3% sodium hypochlorite concentration. In the case of defatted peanut meal which contained 136 ppb aflatoxin B₁ and 36 ppb aflatoxin B₂, 0.25% sodium hypochlorite concentration at pH 8 (0.20% at pH 9; 0.15% at pH 10) reduced both the aflatoxin B₁ and B₂ contents to below detectable quantities in protein isolates as compared to aflatoxin levels of ca. 75 ppb B₁ and 17 ppb B₂ in the untreated protein isolates. Reaction temperature and time did not affect the destruction of aflatoxins significantly.     

Preparation of14C-labeled aflatoxins and incorporation of unlabeled aflatoxins in a blocked versicolorin-A-accumulating mutant ofaspergillus parasiticus

We have compared 2 methods for preparing radiolabeled aflatoxins from [¹⁴C] acetate for use in biosynthetic studies at the end of the aflatoxin pathway. The Salhab-Edwards method (SE) used a 3-day-old mycelium and a defined medium containing MnCl₂ with incubation at 28 C. The Lee-Bennett method (LB) used a 2-day-old mycelium and a defined medium containing low levels of Mn with incubation at 30 C. Generally, the LB method produced lower quantities of aflatoxin but the product had higher specific activities (sp act). The SE method produced 0.157μmol of aflatoxin B₁ and 0.028μmol of G₁ compared to the LB method with 0.058μmol of aflatoxin B₁ and 0.001μmol of G₁. The sp act (inμCi/μmol) for aflatoxin produced by the LB method were: B₁ = 1.379; B₂ = 0.130; G₁ = 5.0 and G₂ - 0.063. The sp act of aflatoxin produced by the SE method were: B₁ = 0.267; B₂ = 0.014; G₁ = 0.178; and G₂ =0.133. Unlabeled aflatoxins were presented to resting cell cultures of the versicolorin-A-accumulating mutant ofAspergillus parasiticus. No conversion of aflatoxin B, was noted. However, when aflatoxins B₂ or G₁ were presented low levels of aflatoxins B₁ and G₂ were recovered. Aflatoxins B₂ and G₁ were recovered when aflatoxin G₂ was presented. Similar low levels of recovery were obtained in experiments using autoclaved mycelia. Thus we conclude that these minor quantities of aflatoxins recovered were not produced enzymatically.     

Reduction of aflatoxin levels in cottonseed and peanut meals by ozonization

Cottonseed and peanut meals were treated with ozone to destroy or eliminate aflatoxins. High meal moistures (cottonseed 22%, peanut 30%), high temperature (100C), and longer treatment times favored inactivation as measured by thin-layer chromatography. Aflatoxins B₁ and G₁ were readily destroyed by the ozone processes whereas aflatoxin B₂ appeared relatively resistant. In cottonseed meal, 91% of the total aflatoxins was destroyed in 2 hr, a decrease from 214 to 20 ppb; in peanut meal, 78% was destroyed in 1 hr, a decrease from 82 to 18 ppb. In both meals, aflatoxin B₁ was totally inactivated within the times specified. E. Util. Res. Dev. Div., ARS, USDA.     

Determination of aflatoxins in peanut and cottonseed soapstocks

An accurate and sensitive procedure is proposed for estimating aflatoxins in both alkaline and acidulated soapstocks. Sample suspensions in aqueous acetone are adjusted to pH 3 with hydrochloric acid, extracted in a high speed blender, treated with lead acetate and partitioned into chloroform. After silica gel cleanup, aflatoxins in purifie extracts are estimated by thin layer chromatography. The use of acetone and lead acetate together apparently catalyzes the relactonization of flatoxins B₁ nd G₁ and leads to essentially quantitative recovery of aflatoxin B₁ and somewhat lower recovery of G₁ added to alkaline or acidulated soapstock. Presented at the AOCS Meeting, San Francisco, April 1969.     

Soybean phytoalexin,glyceollin, prevents accumulation of aflatoxin B1 in cultures ofAspergillus flavus

The soybean phytoalexin, glyceollin, suppresses the accumulation of aflatoxin B₁ in cultures ofAspergillus flavus. At concentrations of 6.25g/ ml and 62.5g/ml, glyceollin causes 70% and 95% decreases in the maximum observed levels of aflatoxin B₁, respectively. In contrast to the dramatic effect on aflatoxin B₁ levels, these concentrations have little effect on fungal growth. For example, at 62.5g/ml in liquid culture, glyceollin causes a barely discernible lag in the beginning of growth and a 11.5% decrease in maximum fungal mass. When the same concentration of glyceollin is added to the colony margin on semisolid medium, an inhibition zone is formed and then overgrown in one day. Glyceollin appears to act by inhibiting aflatoxin B₁ synthesis, since the rate of aflatoxin B₁ breakdown is not increased in fungal cultures that have been grown in the presence of glyceollin. Glyceollin does accumulate in viable soybean seeds that have been infected withAspergillus flavus. Such seeds accumulate aflatoxin B₁ at one-third the rate of non-glyceollin-producing, nonviable seeds. These results suggest that the synthesis of glyceollin in infected seeds may explain, at least in part, why aflatoxin contamination of soybeans is not a common problem.     

Extraction partition and column chromatographic separation of aflatoxins B1 and M1

Extraction, partition and chromatographic elution characteristics of aflatoxin were studied. The novel use of aqueous dimethylsulfoxide or aqueous dimethylformamide for extraction from agricultural products was tested and found effective. Partition studies suggest advantages in analytical work of using solvent pairs in which benzene rather than (the usually employed) chloroform is used to transfer aflatoxin B₁ from primary extracts (aqueous phases). Tests of elution characteristics of aflatoxins B₁ and M₁ on silica gel columns with different developing solvents provided a basis for a procedure in which aflatoxins B₁ and M₁ of a test sample are recovered in separate eluates in which substances which interfere with TLC separation are minimized. Presented at the AOCS Spring Meeting, San Francisco, April 1969. W. Utiliz. Res. Dev. Div., ARS, USDA.     

An improved separation of aflatoxins

Crude aflatoxin from a chloroform extract ofAspergillus flavus cultures on rice was precipitated with Skelly Solve B and chromatographed on 100–200 mesh silica gel columns, using ethyl acetate as eluant. On this column there was no separation of aflatoxins from each other, but most of the brown, oily material was removed. The next step in the purification was chromatography on 100–200 mesh silica gel columns with chloroform and 5% methanol/chloroform as eluants. A large part of the B₁ was purified, but B₂, G₁ and G₂ did not separate, and M₁ had a brown oil that prevented crystallization. The M₁ was purified by chromatography on Sephadex LH-20 with chloroform; the brown material was retained while the M₁ passed through. The separation of aflatoxin B₂, G₁, and G₂ was achieved by column chromatography on Silica Gel H for TLC. In addition, aspertoxin was separated and identified. The purity and identity of the compounds were established by 100 MHz NMR. Presented at the AOCS-AACC Joint Meeting, 1968, Washington, D.C. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Effects of ammoniation on aflatoxins in rations fed lactating cows

A multifaceted cooperative research program involving industry, government and universities was initiated to determine the effects of feeding lactating dairy cows rations containing various levels of cotton-seed and cottonseed meal that had been naturally contaminated with aflatoxins. Evidence is presented that ammoniation of aflatoxin-contaminated cottonseed and cottonseed meal eliminates the aflatoxins, producing a product safe for feeding to ruminants. The aflatoxin M₁ content of milk samples of individual cows receiving rations containing (a) prime cottonseed meal, (b) aflatoxin contaminated meal, and (c) aflatoxin contaminated meal that had ammoniation treatment is reported. Data comparing results with (d) prime cottonseed, (e) aflatoxin contaminated seed, and (f) aflatoxin-contaminated seed that had ammoniation treatment are also reported. None of the milk samples from cows fed ammoniated rations contained any detectable M₁ by the modified Jacobson et al. methodology used. The sensitibity of the method in this laboratory is 0.1 μg M₁/liter of milk. Under the conditions of this study, aflatoxin M₁ levels are related to the levels of aflatoxin B₁ consumed in the diet of the cows. Conversion ratios are reported. Aflatoxin M₁ levels in the milk, relative to the time of the cows’ initial ingestion of aflatoxin B₁, the persistence of M₁ in the milk after discontinuing ingestion of B₁, and disappearance of M₁ under the conditions of the analytical methodology used relative to storage time and temperatures, are reported for liquid milk and for frozen milk. Milk containing the highest level of aflatoxin M₁ was treated with rennet. An 80:20 partion of aflatoxin M₁ was observed between curd and whey, respectively.     

Elaboration of aflatoxin on cottonseed products byAspergillus flavus

In controlled laboratory experiments heat sterilized and unautoclaved glanded and glandless whole cottonseed or decorticated kernels and sterilized cottonseed meals were found to be utilized as substrates by an aflatoxin elaborating strain ofA. flavus with the production of high levels of aflatoxins B₁, B₂, G₁ and G₂. Gossypol pigments in cottonseed products are apparently not a barrier to either mold invasion or aflatoxin production. Cottonseed hulls, lint cotton, and cottonseed linters were found to be poorly utilized as substrates for either mold growth or aflatoxin production. So. Utiliz. Res. Dev. Div., ARS. USDA.     

Effects of varied substrates on aflatoxin production byA. parasiticus

Sterilized and nonsterilized wheat kernels, soybean seeds, sesame seeds, peanut and faba bean were infected byA. parasiticus. The chemical composition, aflatoxin content and fatty acid patterns of the seeds were determined. The aflatoxins B₁, B₂, G₁ and G₂ were detected, and the amounts of the unsaturated toxins (B₁ and G₁) were greater than the respective dihydro derivatives (B₂ and G₂). Sterilized seeds infected by the fungus contained greater amounts of aflatoxins than those infected without previous sterilization. the highest and lowest toxicity indices were recorded for sterilized wheat and soybeans, respectively. Sesame, peanut and soybean exhibited intermediate toxicity indices. The toxicity of the aflatoxins produced was related significantly in every instance to the carbohydrate and lipid:protein ratio, and not to the polyunsaturated fatty acids of the seeds.     

Crystal structure and thermal expansion of ammonium pentaborate NH<Subscript>4</Subscript>B<Subscript>5</Subscript>O<Subscript>8</Subscript>

Anhydrous ammonium pentaborate NH₄B₅O₈ has been synthesized by thermal dehydration of larderellite NH₄[B₅O₇(OH)₂] · H₂O at a temperature of 290°C for 7 h. The crystal structure has been determined from the X-ray powder diffraction data: a = 7.58667(5) Å, b = 12.00354(8) Å, c = 14.71199(8) Å, R _p = 6.23, R _wp = 7.98, R _B = 12.7, R _F = 8.95, and β-KB₅O₈ structure type. The double interpenetrating framework is formed by pentaborate groups, each consisting of a boron-oxygen tetrahedron and four triangles, in which all oxygen atoms are bridging. The thermal behavior of the NH₄B₅O₈ compound has been investigated using thermal X-ray diffraction. As for other pentaborates of this type, the thermal expansion of the NH₄B₅O₈ compound is anisotropic and reaches a maximum along the a axis. The thermal expansion coefficients are as follows: α _a = 39 × 10^?6, α _b = 6 × 10^?6, α _c = 20 × 10^?6, and α _V = 65 × 10^?6 °C^?1.     

Separation and purification of aflatoxins B1, B2, G1 and G2, and comparison of semi-synthetic aflatoxins B2 and G2 with naturally-occurring aflatoxins B2 and G2

A method is described for the isolation of highly purified aflatoxins B₁, B₂, G₁ and G₂ from extracts ofAspergillus flavus. The four aflatoxins, isolated from background impurities by rapid passage of the extracts through an acid alumina column, are separated from each other by chromatography on a silica gel column. Aflatoxins B₂ and G₂ are prepared by hydrogenation of the mixture of aflatoxins B₁, B₂, G₁ and G₂ and then separated by elution from a silica gel column with chloroform containing 0.7% ethanol. A comparison of semi-synthetic aflatoxins B₂ and G₂ with naturally-occurring aflatoxins B₂ and G₂ shows no significant difference in physical properties. Presented at the AOCS-AACC Meeting, Washington, D.C. April, 1968.     

Facile preparation of SiO2 hybrid nanoparticles via Cu2+-amine redox-initiated radical polymerization

The CuSO₄-catalyzed surface-initiated conventional radical graft polymerization of electron-deficient monomers such as N,N-dimethylacryamide (DMAAm) was carried out from amino-functionalized SiO₂ nanoparticles (SiO₂–NH₂) to directly prepare hybrid SiO₂ nanoparticles (SiNPs) chemically grafted with functional poly(N,N-dimethylacryamide) (SiO₂-g-PDMAAm) in aqueous media with trimethyltetradecylammonium chloride (TTAC) added as a surfactant. The optimum reaction conditions were as follows: weight feed ratio CuSO₄·5H₂O:DMAAm:H₂O:SiO₂–NH₂:TTAC of 1:500:5,000:1,000:100 and reaction temperature at 80 °C. Spectroscopy and microscopy confirmed the successful graft polymerization and the formation of hybrid SiNPs with PDMAAm chains. Thermogravimetric analysis indicated the grafting yield could be up to 15.39 wt%. As the active radicals only formed on the SiNP surfaces, there was no free polymer in the reacting mixture. Because of the presence of PDMMAm chains, the hybrid SiO₂-g-PDMAAm nanoparticles exhibited an enhanced dispersion stability in aqueous media over the pristine SiO₂–NH₂, and a thermoresponsive settlement behavior. The parallel experiments with other electron-deficient monomers suggest that the current strategy requires less post-treatment, mild conditions and affords universal applicability.     

A method for determining kernel moisture content and aflatoxin concentrations in peanuts

A method was developed to determine kernel moisture content (KMC) and aflatoxin concentration in discrete peanut samples. Shelled peanuts were weighed to the nearest 0.01 g, and a water slurry was made by blending the peanuts for 2 min with 2.2 ml of water per g of peanuts. The slurry (10 g) was withdrawn and dried at 130°C for 3 h to determine KMC. Methanol was added to the remaining slurry and blended for an additional 1 min, and aflatoxins were quantitated with high-performance liquid chromatography. Comparison of the slurry method with an official peanut moisture method showed good agreement between the two over a range of moisture levels. Recovery of aflatoxin B₁ from spiked samples averaged 97% with an average coefficient of variation of 3.6%. The method enables determination of both KMC and aflatoxin content in peanut samples without degradation of aflatoxin that would occur when using the official moisture method.     

A new method for determining aflatoxins in groundnut and groundnut cake using fourier transform infrared spectroscopy with attenuated total reflectance

A new analytical method was developed for the determination of aflatoxins in groundnut and groundnut cakes by Fourier transform infrared (FTIR) spectroscopy using horizontal attenuated total reflectance technique. Groundnut and groundnut cake samples were used in this study. The wave-lengths were selected for the four types of aflatoxins—B₁, B₂, G₁, and G₂—and the standards prepared for earch by spiking some clean sample with the aflatoxins in concentrations of 0–1200 parts per billion. A partial least square regression was used to derive the calibration models for each toxin. The coefficients of determination (R ²) of the calibration model were computed for the FTIR spectroscopy predicted values vs. actual values of aflatoxins in parts per billion. The R ² was found to be 0.9911, 0.9859, 0.9986, and 0.9789 for aflatoxins B₁, B₂, G₁ and G₂, respectively. Standard errors of calibration for groundnut samples were found to be 1.80, 2.03, 1.42, and 2.05 for aflatoxins B₁, B₂, G₁, and G₂, respectively. Calibration models were validated with an independent set of samples. The R ² of validation models were computed. The SD of the difference for repeatability of the FTIR method was found to be better than that of the chemical method. Based on the results obtained, FTIR spectroscopy can be a useful instrumental method for determining aflatoxins in oilseeds and oilseed cakes. With its speed and ease of data manipulation by computer software, it is a possible alternative to the standard wet chemical methods for a rapid and accurate routine determination of aflatoxin levels in food and feed.     

Kinetic study of acid-catalyzed conversion of aflatoxins B1 and G1 to B2a and G2a

Adjusting dilute aqueous solutions of aflatoxins B₁ and G₁ to pH 1, 2 and 3, and heating over a range of 40–100 C resulted in the conversion of B₁ to B_2a and G₁ to G_2a as major products. Both B_2a and G_2a were identified by co-thin layer chromatography with authentic B_2a and G_2a and M₁ on silica gel plates developed in two different solvents. The rate of disappearance of B₁ or G₁ at given temperature and at constant pH was found to be first order with respect to each aflatoxin. At given temperature the conversion is strongly pH dependent, a 10-fold increase in H⁺ ion (1 pH unit) producing about a 9-fold increase in the reaction rate, indicating first order dependent of the rate on H⁺ ion concentration. S. Market. Nutr. Res. Div., ARS, USDA.     

Chemical studies on corn embryos infected by various fungi

The occurrence of various fungi in corn kernels obtained from eight localities in Egypt in two successive years was studied. Values for refractive index, color, acid value, saponification value, iodine value, peroxide value and unsaponifiable matter content of oils extracted from corn embryos that were deliberately infected by various fungi were compared to those for oil extracted from healthy embryos. Spectrometric analyses (UV, visible and IR) were done to deduce differences in the functional groups of the oils. Corn oil extracted from embryos infected with various fungi contained the same lipid classes as the oil extracted from healthy embryos. Contents of mono- and diglycerides and free fatty acids were much smaller for the oil extracted from healthy embryos. The fatty acid and unsaponifiable compositions of oils were studied by gas liquid chromatography. The fatty acid composition of corn oil extracted from infected embryos showed that some new and short-chain fatty acids had appeared and that some of the 18:2 was converted to 18:0. Analysis of the hydrocarbon fraction of the unsaponifiables showed also that some new compounds had appeared and others disappeared. The sterols were greatly influenced by the fungi and the ratio between different sterols might be used to characterize the effect of fungi. Aflatoxin B₁ content of oil extracted from corn embryos infected byA. flavus was 300 μg/kg.     

Aflatoxin formation on whole and ground cumin and anise seeds

The purpose of this study was to evaluate the potential productivity and growth ofAspergillus parasiticus (NRRL 2999) and the resulting toxin production on natural and autoclaved (cooked) cumin and anise spice seed substrates. Both whole and ground seeds were used. Mycelia and sporulation were also noted in this 17-day experiment. Cumin and anise seeds are capable of supporting mycelial growth, sporulation and toxin production when the seeds are moist and maintained at room temperature. Toxin yields were higher on ground sterile seed substrates. Of the commercial samples tested, neither the resulting cultures of natural flora nor dry whole seeds were found to contain aflatoxin or aflatoxin-like producing organisms. The anise substrates were more conducive to mycelial growth, sporulation and aflatoxin production than the cumin. Toxin levels in the various anise substrates ranged from 0.83 to 6.5μg/g total for the 4 aflatoxins, B₁, B₂, G₁ and G₂. Cumin seed substrates usually showed only B, and G, at total levels ranging from 0.23 to 0.63μg/g- Both spice seeds had mycelial growth and sporulation to occur at some time during the experimental period. Both substrates could be considered as low-level-producer substrates for aflatoxins. Anise seeds should be monitored occasionally for aflatoxin contamination when the commodities are purchased and used in large quantities.