Aflatoxins B1, B2, G1, and G2: Separation and purification

Aflatoxins B₁, B₂, G₁, and G₂ have been separated on a series of chromatographic columns. Chromatography of crude products isolated from molded wheat and rice on silicic acid with washed chloroform:ethanol (99:1) gave relatively pure B₁. The rest of the column fractions containing predominantly G₁, along with B₁, B₂, and G₂, were pooled and fractionated on a Silica Gel G column. The mobile phase was washed chloroform:acetone:ethanol (97.3:2.0:0.75). Thin-layer chromatography was used to follow column development. Each of the aflatoxins was treated with either decolorizing carbon or copper carbonate to remove colored pigments, and rechromatographed on Silica Gel G. Crystalline aflatoxins were prepared from chloroform solutions by addition ofn-hexane, methanol, or ethanol. Presented at the AOCS Meeting, New Orleans, May 1967. No. 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.     

The determination of aflatoxins in cottonseed products

A rapid and simplified procedure is proposed for the determination of aflatoxins B₁, B₂, G₁, and G₂ in cottonseed products. The method involves extraction of aflatoxins essentially free of lipid contamination by equilibrium extraction with 70% acetone. Interfering gossypol pigments are removed from the aqueous acetone extract by precipitation as insoluble lead salts. Aflatoxins in the centrifugate are quantitatively separated from excess lead salts, residual pigments and carbohydrates by extraction into chloroform to yield final extracts for thin-layer chromatographic (TLC) analysis on silica gel which are low in total solids and pigmentation. The procedure is sensitive to about 1 ppb in cottonseed meats and 4 ppb in cottonseed meal. Honorable mention, Bond Award competition. Presented at the American Oil Chemists’ Society, Chicago, October 11–14, 1964. A laboratory of the So. Utiliz. Res. and Dev. Div., ARS, USDA.     

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.     

Aflatoxins M1 and M2: Preparation and purification

A crude product containing aflatoxins M₁ and M₂, as well as large quantities of aflatoxins B₁ and B₂, obtained by fermentation of rice withAspergillus flavus NRRL 3251 was chromatographed on a silicic acid column. Almost all the B₁ and B₂ were separated from M₁ and M₂. Aflatoxins M₁ and M₂ were eluted together with ethanol-chloroform (5∶95 v/v). The combined M₁ and M₂ fraction was placed on a Merck silica gel (0.05–0.2 mm) column to be washed with hexane-chloroform (1∶1 v/v) and chloroform to remove traces of B₁ and B₂ and eluted with ethanol-chloroform (1.5∶98.5 v/v) to obtain aflatoxin M₁ and mixtures of M₁ and M₂. Rechromatography of M₁ on another silica gel column gave pure M₁ which was crystallized from acetonitrile. Aflatoxin M₂ was prepared by hydrogenation of M₁ and crystallized from acetonitrile. Presented at the AOCS Meeting, New Orleans, April 1970. No. Utiliz. 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.     

Conditions and techniques for thin layer chromatography of aflatoxins

Satisfactory resolution of the four common aflatoxins, B₁, B₂, G₁ and G₂, on thin layer chromatograms has been a recurring problem. The most frequently observed cause of poor resolution and tailing of spots in the chromatograms was the variable properties of the commercial silica gel-calcium sulfate adsorbent preparations. Variations in quality were observed even from one container to the next within single lots produced by individual manufacturers. Other variables which affected the chromatography to some degree included adsorbent particle size, concentration and nature of the calcium sulfate binder, silica gel layer thickness and moisture content, vapor phase composition in the developing chamber and the solvent used for development.     

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.     

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.     

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.     

Fluorometric measurement of aflatoxins

An accurate, precise and sensitive fluorometric method for quantifying purified aflatoxins in solution is proposed. After purification, aflatoxin samples are read in a fluorometer with a primary filter of 365 nm and a secondary filter of 435 nm for B₁ and B₂ and 465 nm for G₁ and G₂. The method is precise at as little as 0.0002 μg/ml B₁ and G₁ and 0.00006 μg/ml B₂ and G₂, and accurate at concentrations of 0.002 μg/ml B₁ and G₁ and 0.0006 μg/ml B₂ and G₂. University of Georgia College of Agriculture Experiment stations, Journal Series Paper No. 810, College Station, Athens, Georgia 30601.     

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.     

Application of TLC chemical confirmatory tests to minicolumn chromatography of aflatoxins

The minicolumn (MC) proposed by Holaday and Lansden was developed with standard aflatoxin solution and also with the extracts of corn, rice, wheat, cottonseed, peanut cake and black pepper; each having different levels of aflatoxins. One-half mL each of 2,4-dinitrophenylhydrazine,p-anisaldehyde, 20% H₂SO₄, 20% HCl and trifluoroacetic acid (TFA) with 25% HNO₃, which were used for confirming aflatoxins on TLC, were applied to the developed column. Among these, all the 3 acid reagents changed the blue fluorescence of aflatoxins to yellow and thus were found to be satisfactory confirmatory tests. The TFA with 25% HNO₃ had the lowest detection limit-5 ppb.     

Tolerance to in vitro accumulation of aflatoxins in pecan meal as affected by factors associated with yield (carya illinoensis [wangenh.] K. Koch)

Unautoclaved pecan (Carya illinoensis [Wangenh.] K. Koch) meal from selected trees, 10 each with high or low nut yields, were inoculated with a spore suspension of Aspergillus parasiticus. Significantly greater concentrations of aflatoxins (B₂ + B₂ + G₁ + G₂) occurred in substrates from high-yielding trees. The data suggest physiological differences associated with yield resulted in tolerance to accumulation of aflatoxins.     

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.     

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.     

Thin layer chromatographic analysis of possible aflatoxins within grain dusts

The National Institute for Occupational Safety and Health is interested in assessing the hazards to grain workers associated with respirable grain dusts of all types. One of these hazards could involve the occurrence of mycotoxin producing fungi either upon or within grains. Aflatoxins, types of mycotoxins produced by bothAspergillus flavus andA. parasiticus, are hepatocarcinogens, mutagens, teratogens and toxins. Here, we report an attempt to determine whether settled and/or airborne dusts from barley, corn, flax, oats and Durum as well as spring wheats contain aflatoxins. These dusts were collected at port grain terminals in the Superior-Duluth regions of the United States. The dusts were extracted with and chromatographed upon thin layer plates in a variety of solvents which have been approved for the separation of aflatoxins. Two acceptable aflatoxin B₁ confirmatory tests were employed to verify suspected aflatoxin B₁ within the extracts. Each dust contained a chloroform-soluble, blue fluorescent compound(s) which possessed an Rf similar to that of aflatoxin B₁ upon chromatography of chloroform extracts in chloroform/95% methanol. Methylene chloride/H₂O) extracted a blue fluorescent compound(s) from each dust, and the compound(s) possessed Rf intermediate between those of aflatoxin B₁ and B₂ upon chromatography in acetone/methylene chloride. The methylene chloride/H₂O extracted compounds failed to turn yellow upon spraying with 25% sulphuric acid in methanol and subsequent viewing with an ultraviolet source. Our results confirm those of Sorenson et al., who reported that aflatoxins were absent from airborne grain dusts collected from the Superior-Duluth areas of the United States in the fall of 1977. In conclusion, we stress the need for extracting, detecting, and identifying aflatoxins by a variety of analytical procedures including thin layer and high performance liquid chromatography and “approved” confirmatory tests.     

Objective fluorometric measurement of alfatoxins on TLC plates

Measurement of the solid state fluorescence of aflatoxins on silica gel-coated TLC plates on a densitometer equipped for fluorescence measurements showed a linear relationship between peak areas and concentration over a range of at least 2 to 105×10⁻⁴ μg of aflatoxins per spot. Response of individual aflatoxins was in order of B₂>G₂>B₁>G₁. Aflatoxins can be measured with a precision of ±2–4%. Presented at the AOCS Meeting, Los Angeles, April 1966. So. Utiliz. Res. Dev. Div., ARS, USDA.     

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.     

CFD Modeling of a Bubble Column Reactor Carrying out a Consecutive A → B → C Reaction

In this paper, we develop a CFD model for describing a bubble column reactor for carrying out a consecutive first‐order reaction sequence A → B → C. Three reactor configurations, all operating in the homogeneous bubbly regime, were investigated: (I) column diameter D_T = 0.1 m, column height H_T = 1.1 m, (II) D_T = 0.1 m, H_T = 2 m, and (III) D_T = 1 m, H_T = 5 m. Eulerian simulations were carried out for superficial gas velocities U_G in the range of 0.005–0.04 m/s, assuming cylindrical axisymmetry. Additionally, for configurations I and III fully three‐dimensional transient simulations were carried out for checking the assumption of cylindrical axisymmetry. For the 0.1 m diameter column (configuration I), 2‐D axisymmetric and 3‐D transient simulations yield nearly the same results for gas holdup ?_G, centerline liquid velocity V_L(0), conversion of A, χ_A, and selectivity to B, S_B. In sharp contrast, for the 1 m diameter column (configuration III), there are significant differences in the CFD predictions of ?_G, V_L(0), χ_A, and S_B using 2‐D and 3‐D simulations; the 2‐D strategies tend to exaggerate V_L(0), and underpredict ?_G, χ_A, and S_B. The transient 3‐D simulation results appear to be more realistic. The CFD simulation results for χ_A and S_B are also compared with a simple analytic model, often employed in practice, in which the gas phase is assumed to be in plug flow and the liquid phase is well mixed. For the smaller diameter columns (configurations I and II) the CFD simulation results for χ_A are in excellent agreement with the analytic model, but for the larger diameter column the analytic model is somewhat optimistic. There are two reasons for this deviation. Firstly, the gas phase is not in perfect plug flow and secondly, the liquid phase is not perfectly mixed. The computational results obtained in this paper demonstrate the power of CFD for predicting the performance of bubble column reactors. Of particular use is the ability of CFD to describe scale effects.