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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Esterified alkan-1-ols and alkan-2-ols in barley epicuticular wax

The epicuticular wax on all barley organs is characterized by the presence of long chain esters which have alkan-1-ols as their alcohol moiety (mainly C₃₈−C₄₈). A second type of long chain ester in which alkan-2-ols serve as the alcohol moiety has been identified in the wax from all organs except the awns and leaf blades (mainly C₃₃−C₃₅). Utilizing Silica Gel H column chromatography, a 95% separation of the two ester types was achieved. Unlike alkan-1-ols, the alkan-2-ols (primarily C₁₃ and C₁₅) do not occur free in the wax. Esters isolated from spike minus awn wax consisted of 62% alkan-2-ol and 38% alkan-1-ol containing esters. The isomeric composition of each ester was determined with the aid of gas liquid chromatography-mass spectrometry. In the mass spectra of the alkan-2-ol containing esters, mass ions were absent and the relative intensities of the RCO₂H₂ ⁺, RCO₂H⁺, [R′-1]⁺, and RCO⁺ ions were markedly different from those characteristic for alkan-1-ol containing esters. Since esterified alkan-2-ols occur only in those waxes having β-diketones, a close biosynthetic relationship between these two lipid classes is suggested.     

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.     

EDTA and pH‐sensitive crosslinking polymerization of acrylic acid, 2‐acrylamidoglycolic acid,and 2‐acrylamide‐2‐methyl‐1‐propanesulfonic acid

Network formation was monitored by shear storage modulus (G′) during free radical crosslinking polymerization to investigate the effects of pH and ethylenediaminetetraacetic acid (EDTA; a complex agent). Three types of acrylic monomers, acrylic acid (AAc), 2‐acrylamidoglycolic acid (AmGc), and 2‐acrylamido‐2‐methyl propanesulfonic acid (AmPS), were polymerized in the presence of a crosslinking agent. The ratio of crosslinking agent (methylene bis‐acrylamide; MBAAm) to monomer was varied as: 0.583 × 10^?3, 1.169 × 10^?3, 1.753 × 10^?3, and 2.338 × 10^?3. G′ of the hydrogel in crosslinking polymerizations of AAc and AmPS was effectively increased by addition of EDTA, which was not the case for the crosslinking polymerization of AmGc. The order of magnitude of G′ differed based on the acidity of monomer. The maximum values of G′ in crosslinking polymerizations of AAc, AmGc, and AmPS were ～20,000 Pa, 6000 Pa, and 400 Pa, respectively. G′ varied linearly with the molecular weight between crosslinks (M_wc). pH and EDTA‐complex affected the rate of intramolecular propagation during crosslinking polymerization. Our results indicated that G′ was primarily affected by the following factors in the order: (1) acidity of monomer, (2) M_wc, and (3) physical interactions induced by pH and EDTA. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41026.