Robert D. Stubblefield

The rapid determination of aflatoxin M1 in dairy products

A method for aflatoxin M1 in dairy products is presented. It decreases the analysis time compared to currently accepted methods. Samples are extracted in a blender or separatory funnel for 1 min with chloroform and saturated sodium chloride solution. The chloroform extracts are partially purified on a small silica gel column (2 g), and M1 is determined by thin layer chromatography (TLC) and densitometry. Recoveries of M1 added to milk are about 80%. Recovery of M1 from cheeses is variable depending on the type of cheese. The method gave results for a naturally contaminated powdered milk comparable to analyses by accepted methods. The determination limit of the method is about 0.1 μg/kg.

Aflatoxin M1 removal from aqueous solutions by Flavobacterium aurantiacum

In liquid cultures growing and stationary phase cells ofFlavobacterium aurantiacum NRRL B-184 eliminated aflatoxin M1. Toxin concentrations of 15µg/ml and 37.5µg/ml interfered with bacterial growth, and at the higher level 4.4µg M1 was removed from the growth medium by a milligram (dry weight) of bacteria. Toxin was completely removed from the liquid medium by incubating 5 × 1010 resting cells per milliliter with 8µg/ml of aflatoxin M1 for 4 h. Attempted recovery of M1 from cells following incubation of the bacteria with the toxin demonstrated that the M1 was essentially nonextractable. Bacterial cells also removed aflatoxin M1 from toxin-contaminated milk.

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.

Organic acids in the haemolymph of healthy and diseased Popillia japonica (Newman) larvae

Abstract Organic acid constituents in the haemolymph of healthy and diseased Popillia japonica (Newman) larvae were determined by partition chromatography, enzymatic analyses, and colorimetric procedures. Haemolymph from larvae infected with Bacillus popilliae Dutky, compared with that from healthy larvae, contained higher concentrations of malic, glycolic, tartaric pyruvic, and glyoxylic acids. No change in concentrations of butyric, propionic, acetic, formic, succinic, lactic, citric, and α-ketoglutaric acids occurred when larvae were infected. None of the acids decreased during the course of the milky disease. Gluconic and oxaloacetic acids were detected in the haemolymph but not determined. Organic acids account for most of the anion titre in the haemolymph.

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.

Amino acids in the haemolymph of diseased Popillia japonica (Newman) larvae.

Abstract Free amino acid constituents in the haemolymph from diseased Popillia japonica (Newman) larvae were determined by ion-exchange chromatography, paper chromatography, enzymatic analysis, and amperometric titration. Haemolymph from larvae infected with Bacillus popilliae, compared with that from healthy larvae, contained higher concentrations of glutamic acid, β-alanine, aspartic acid, phenylalanine, threonine, serine, and lysine and lower concentrations of glycine, tyrosine, and histidine. Differences in the amino acid composition depend on the infective organism. The only amino acid to increase during infection with B. lentimorbus was glutamic acid. Amounts of histidine, proline, glycine, alanine, valine, isoleucine, tyrosine, and arginine decreased. The level of protein material in the haemolymph as well as the composition did not change markedly during infection with either B. popilliae or B. lentimorbus. Haemolymph contains little peptide of low molecular weight. Several additional amino acids were determined in haemolymph from healthy and diseased larvae. Milky disease apparently has no effect on the concentration of tryptophan, cystine, cysteine, glutamine, asparagine, and lanthionine.

Determination of micro quantities of rubber by a modified bromination method

Abstract A method is described for determination of as little as 200 μg rubber with an accuracy of 96–97%. cis -Polyisoprene with average molecular weight as low as 41,000 was accurately determined employing a modification of a gravimetric bromide precipitation method.

Aflatoxins in swine tissues during drought conditions : an epidemiologic study

The purpose of this joint Agricultural Research Service/Food Safety and Inspection Service (FSIS) project was to determine the presence of aflatoxins in swine tissues in the United States during a drought year (1988). A worst-case sampling plan for aflatoxin from swine slaughtered in FSIS inspected plants was conducted during the drought. Swine tissues were screened for aflatoxins by high pressure liquid chromatography and confirmed by two-dimensional thin layer chromatography. Results indicate that swine effectively metabolize aflatoxins present in feed. Eight of 160 (5%) liver samples had confirmed aflatoxin, with only 4 of these 8 exceeding 0.1 ppb. Only 1 of 160 liver samples had total aflatoxin B1 and M1 in excess of the milk enforcement level of 0.5 ppb for M1 alone. In severe drought conditions, the presence of aflatoxins in animal feed does not result in a significant frequency or magnitude of tissue residues in swine even in worst-case type biased sampling. Since swine appear to be the most sensitive species insofar as tissue concentration of aflatoxins, it is logical to conclude that residues in other food-producing species would be significantly lower.

Transmission and distribution of aflatoxin in contaminated beef liver and other tissues

A 160-kg Holstein steer was fed 52 mg1, equivalents/day (orally) for 5 consecutive days and then slaughtered. The liver, kidneys, spleen, heart and skeletal muscle (round) were retained and assayed for aflatoxin. The liver was cut into 14 samples and each was analyzed to determine the distribution of aflatoxin in liver. Afla-toxin levels in samples from the edges of the liver were 44% lower than averages of all the remaining samples, which were 25.0 ng B,, 15.4 ng M1 and 47.1 ng total aflatoxin/g; however, cross-sectional samples would give representative assay results. Lower total aflatoxin concentrations (16.0 ng, 18.5 ng and 12.9 ng/g) were found for heart, spleen and muscle, respectively. The kidneys had the highest level of aflatoxin (145.9 ng/g) with M1 levels (105.5 ng/g) 3.6 times the B, concentration (29.3 ng/g). In the other tissues, B1 concentrations were 1.6-2.9 times greater than aflatoxin M1.