Raymond D. Coker

Analytical methods for determination of mycotoxins: An update (2009–2014)

Mycotoxins are a problematic and toxic group of small organic molecules that are produced as secondary metabolites by several fungal species that colonise crops. They lead to contamination at both the field and postharvest stages of food production with a considerable range of foodstuffs affected, from coffee and cereals, to dried fruit and spices. With wide ranging structural diversity of mycotoxins, severe toxic effects caused by these molecules and their high chemical stability the requirement for robust and effective detection methods is clear. This paper builds on our previous review and summarises the most recent advances in this field, in the years 2009-2014 inclusive. This review summarises traditional methods such as chromatographic and immunochemical techniques, as well as newer approaches such as biosensors, and optical techniques which are becoming more prevalent. A section on sampling and sample treatment has been prepared to highlight the importance of this step in the analytical methods. We close with a look at emerging technologies that will bring effective and rapid analysis out of the laboratory and into the field.

The aetiology of turkey ‘x’ disease

Abstract From a 1960s sample of groundnut cake, which had been implicated in the first record of turkey ‘X’ disease, cyclopiazonic acid has been detected at a level of 31 μg kg −1 . The presence of this mycotoxin, along with aflatoxins, explains the toxic symptoms originally recorded after the groundnut cake had been ingested by turkeys.

A comparative study of solvent extraction efficiency and the performance of immunoaffinity and solid phase columns on the determination of aflatoxin B1

Abstract The efficiency of two different immunoaffinity columns and a phenyl bonded-phase column were compared during the extraction, clean-up and quantification of aflatoxin B1 from sorghum and maize. Fluorodensitometry of high performance thin-layer chromatograms was used for quantification of aflatoxin B1. Maize had a simple matrix, and comparable precisions and accuracies were obtained for each of the methods. The sorghum matrix was complex and the bonded-phase procedure was the most accurate and precise method. There was evidence to suggest that the lower aflatoxin B1 recovery from sorghum by immunoaffinity columns is a solvent extraction problem. Better aflatoxin B1 recoveries were obtained from naturally contaminated sorghum and maize when acetonitrile was replaced with acetone as the extraction solvent.

5′-epialtenuene and neoaltenuene, dibenzo-α-pyrones from Alternaria alternata cultured on rice

Abstract From extracts of the fungus Alternaria alternata cultured on rice, five dibenzo-α-pyrones were isolated. In addition to the known compounds alternariol, alternariol monomethyl ether and altenuene, two new isomers of altenuene were characterized. From mainly NMR and MS data, these compounds were proved to be 2′,3′,4′,5′-tetrahydro-3,4′β,5′β-trihydroxy-5-methoxy-2′β-methyldibenzo-α-pyrone and 2′,3′,4′,5′-tetrahydro-3,3′α,4′β-trihydroxy-5-methoxy-6′-methyldibenzo-α-pyrone to which the trivial names 5′-epialtenuene and neoaltenuene, respectively, have been assigned.

Development of the custom polymeric materials specific for aflatoxin B1 and ochratoxin A for application with the ToxiQuant T1 sensor tool

Two polymers were computationally designed with affinity to two of the most abundant mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) for application in the ToxiQuant T1 System. The principle of quantification of AFB1 and OTA using the ToxiQuant T1 instrument comprised of a fluorimetric analysis of mycotoxins adsorbed on the polymer upon exposure to UV light. High affinity of the developed resins allowed the adsorption of both toxins as discrete bands on the top of the cartridge with detection limit as low as 1ng quantity of mycotoxins.

The validation of a solid phase clean-up procedure for the analysis of aflatoxins in groundnut cake using HPLC

A solid-phase clean-up method for the determination of aflatoxins in groundnut cake has been statistically examined. The method involves the clean-up of an acetone and water (85 + 15) extract on a bonded-phase (PH) cartridge and quantification by HPLC with fluorescence detection following post-column derivatisation with iodine. Average recoveries were calculated as 84.1, 86.1, 88.0 and 82.1% with limits of detection of 2.7, 1.6, 2.5 and 3.2 μg/kg for aflatoxins B1, B2, G1, and G2 respectively. This method was compared with the official AOAC (CB) method for its ability to determine the aflatoxin B1, and B2 contents of groundnut cake samples. The precision of the two methods was found not to be significantly different at the 5% level, but the PH method recorded significantly more aflatoxin B1. The direct extraction of aflatoxins with aqueous acetone was also compared with a slurry extraction method. It was demonstrated that the slurry technique extracted significantly more aflatoxins B1 and B2; the precision of these two extraction methods was found not to differ significantly.

Transcriptomic and phenotypic analysis of the effects of T-2 toxin on Saccharomyces cerevisiae: evidence of mitochondrial involvement

At 5 μg mL(-1) , T-2 toxin significantly upregulated the transcription of 281 genes and downregulated 86. Strongly upregulated genes included those involved in redox activity, mitochondrial functions, the response to oxidative stress, and cytoplasmic rRNA transcription and processing. Highly repressed genes have roles in mitochondrial biogenesis, and the expression and stability of cytoplasmic rRNAs. T-2 toxin inhibition of growth was greater in a medium requiring respiration, and was antagonized by antioxidants. T-2 toxin treatment induced reactive oxygen species, caused nucleolytic damage to DNA, probably mitochondrial, and externalization of phosphatidylserine. Deletion mutations causing respiratory deficiency substantially increased toxin tolerance, and deletion of some TOR (target of rapamycin) pathway genes altered T-2 toxin sensitivity. Deletion of FMS1, which plays an indirect role in cytoplasmic protein synthesis, markedly increased toxin tolerance. Overall, the findings suggest that T-2 toxin targets mitochondria, generating oxy-radicals and repressing mitochondrial biogenesis genes, thus inducing oxidative stress and redox enzyme genes, and triggering changes associated with apoptosis. The large transcriptional changes in genes needed for rRNA transcription and expression and the effects of deletion of FMS1 are also consistent with T-2 toxin damage to the cytoplasmic translational mechanism, although it is unclear how this relates to the mitochondrial effects.

Uptake of aflatoxin B1 and T-2 toxin by two mycotoxin bioassay microorganisms: Kluyveromyces marxianus and Bacillus megaterium.

Abstract. Uptake of aflatoxin B1 (AFB1) and trichothecene T-2 toxin from growth medium by mycotoxin bioassay strains of Kluyveromyces marxianus and Bacillus megaterium was assessed by incubating, washing, and sonicating the cells, extracting samples with chloroform, and analysing the extracts by a combination of high-performance thin-layer chromatography (HPTLC) and fluorescence densitometry. Using cultures of K. marxianus, the entire AFB1 dose was recovered and no AFB1 metabolites were detected. Less than 1% of the AFB1 was recovered from the cells, and AFB1 did not inhibit growth. Methanol in the incubation medium had no significant effect on the levels of AFB1 associated with K. marxianus cells. The entire dose of T-2 toxin was also recovered from K. marxianus cultures, and no metabolites were detected; again, less than 1% of T-2 toxin was cell-associated, but growth was completely inhibited. AFB1 partially inhibited the growth of B. megaterium; approximately 12% of the dose could not be recovered, and no AFB1-related metabolites were detected. Methanol increased the levels of recoverable AFB1 associated with B. megaterium cells. In the case of T-2 toxin, around 8% of the dose was not recovered, and no metabolites were detected; growth of B. megaterium was stimulated. These results suggest irreversible binding of both toxins, or derivatives of them, to the cells of B. megaterium.

A sensitive bioassay for the mycotoxin aflatoxin B1, which also responds to the mycotoxins aflatoxin G1 and T-2 toxin, using engineered baker's yeast

A novel aflatoxin B(1) bioassay was created by introducing a Lipomyces kononenkoae alpha-amylase gene into a strain of S. cerevisiae capable of expressing the human cytochrome P450 3A4 (CYP3A4), and the cognate human CYP450 reductase. This strain and a dextranase-expressing strain were used in the development of a microtitre plate mycotoxin bioassay, which employed methanol as the solvent and polymyxin B nonapeptide as a permeation enhancer. Stable co-expression of the CYP3A4 gene system and of the dextranase and amylase genes in the two bioassay strains was demonstrated. The bioassay signalled toxicity as inhibition of secreted carbohydrase activity, using sensitive fluorimetric assays. The amylase-expressing strain could detect aflatoxin B(1) at 2 ng/ml, and was more sensitive than the dextranase-expressing strain. Aflatoxin G(1) could be detected at 2 microg/ml, and the trichothecene mycotoxin T-2 toxin was detectable at 100 ng/ml.