Mycotoxins in food: An update for 2010

Since biblical times, the toxic response caused by ingestion of mycotoxins, the secondary metabolites of moulds, has had a significant impact on the health and welfare of human and animal populations. Since the early 1960’s, a wealth of knowledge about mycotoxigenic fungi, such as Aspergillus, Penicillium and Fusarium, and their associated toxins has become available. Scientific journals, the traditional source of information on mycotoxins, are complemented nowadays by web-based information sources, such as the European Mycotoxin Awareness Network (EMAN, www.mycotoxins.org) and even a dedicated Mycotoxin Channel on YouTube presenting video clips from well-respected mycotoxin researchers.

In recent years, mycotoxins have been the hazard category with the majority of notifications from RASFF (the Rapid Alert System for Food and Feed in the European Union). In 2008, a total of 931 notifications were on mycotoxins (29.7 per cent of all notifications), of which 902 concerned aflatoxins1. This was an increase on previous years and these findings are discussed in the RASFF Annual Report for 2008, which considers that the detection of significant levels of aflatoxins in rice the previous year contributed to the increase in notification as levels of control in the EU increased.

Much is now known about a few highly toxic and, in some cases, carcinogenic myco – toxins, and legislative limits have been set for a number of mycotoxins in over 100 different countries and states around the world. For example, since 2007, in the European Union there have been maximum levels for approximately 70 toxin-matrix combinations2. There are nearly 500 recognised mycotoxins, and moulds from the genera Aspergillus, Penicillium, Fusarium and Claviceps infect crops around the globe both in the field and in storage. In addition, there have been instances in recent years of previously unrecognised combinations of toxins and matrices and, owing to the nature of spoilage moulds, it is not beyond the bounds of possibility in the future for there to be new or emerging mycotoxins or toxin/matrix combinations. The most recent discovery was reported in November 20093,4; some strains of Aspergillus niger, occurring on wine grapes, have the potential to produce fumonisins. This work was followed by another report, published in March 2010, in which 77 samples of wine from 13 countries were analysed and 23 per cent of samples were found to contain fumonisin B2 at low levels, which were too low to be a food safety concern5.

A large proportion of the world’s population can be affected by mycotoxins in their diet – mycotoxins are a world-wide problem. In the developed world, high standards of food quality generally protect consumers, but less developed countries are at greater risk from these contaminants. The effects of mycotoxins can be economic, for example, rejection of contaminated produce at point of export, or can impact on animal productivity and even human health. A number of episodes of aflatoxinpoisoning outbreaks have been reported around the world in the past, and the cooccurrence of aflatoxins and fumonisins in maize and their synergistic action is a cause for concern in those parts of the world where maize is a staple. In some parts of the world, the link between mycotoxins and food security is significant, particularly considering the stability of most of these toxins to food processing, such as cooking or freezing.

Fungal growth, prior to and during harvest, as well as improper storage following harvest, can lead to mycotoxin contamination of agricultural produce. In the field, climate conditions are critical for the growth of mycotoxic fungi, as was witnessed in the grain-belt of the US during the 2009 harvest that increased incidences of mouldy grain and trichothecene contamination6.

As we progress towards an effective food production chain with minimal or no mycotoxin contamination, various strategies have been employed, and further novel techniques are in development, to either prevent fungal infestation or check the production of mycotoxins.

Preharvest control

Mycotoxin contamination levels may be reduced by optimisation of agriculture practices (Good Agricultural Practices). For instance, drought stress is recognised as a trigger for aflatoxin contamination in groundnuts, so irrigation of the crop should prevent drought stress and remove this trigger. Insect damage of crops is also well recognised as causing increased levels of mould growth and subsequent mycotoxin contamination. Crops that are protected against insect damage, such as the transgenic Bt maize, appear to be insusceptible to contamination with mycotoxigenic fungi7.

Transgenics

Potential future strategies include traditional plant breeding to produce plant lines (in particular maize) with resistance to fungal infection. Genetic modification also offers a number of possible solutions7.

Postharvest control

The environmental conditions of storage have been shown to be critical in the control of mycotoxin contamination of agricultural products. Simply maintaining optimum temperature, humidity and air flow during storage can control toxin levels, but in many parts of the world, this is not possible and hand sorting and removal of obviously damaged or diseased produce is often the only treatment possible8.

Biological control (biocompetition)

Certain bacteria and yeasts show promise in controlling the growth of toxigenic fungi, either by biocompetition or by detoxifying the mycotoxin. For example, the application of atoxigenic (non-mycotoxin-producing) strains of Aspergillus flavus has been studied in various crops. Trials involving the inoculation of wheat, cotton and peanuts with these strains have demonstrated a significant reduction in levels of aflatoxin contamination. Similar studies have also been reported on biocontrol agents for fumonisins and deoxynivalenol, but with less success to-date7. Scientists at the Agricultutal Research Service in the US have patented the use of a yeast for reducing aflatoxin contamination by the inhibition of growth of Aspergillus flavus. This was trialled in a pistachio orchard, but it is recommended for tree nuts and corn and the yeast can be sprayed on trees and plants before harvest, or applied to harvested and stored crop9.

Genomics and proteomics

The elucidation of the complete genome and proteins of mycotoxigenic fungi will lead to an understanding of the ecology of the mould, as well as the effect of environmental factors on toxin production. The results of proteomic studies may also be used to develop novel or enhance conventional techniques for the detection of mycotoxins.

Animal feeds

An indirect means of reducing mycotoxins in the human food chain is by controlling the mycotoxins consumed by food-producing animals, significantly dairy cows (aflatoxin M1) and pigs (ochratoxin A). Mycotoxin contamination in animal feeds can also lead to economic losses due to poor feed conversion, diminished body-weight gain, feed refusal and increased incidence of disease. In 2009, a new regulation appeared in the Official Journal of the European Commission which is an amendment to the EU Feed Additive Regulation (EC 1831/2003 for feed additives)10. This has introduced procedures for the authorisation and labelling of various feed additives, including binders. Commercially available products work by means of biotransformation (detoxification of the mycotoxin), adsorption (elimination of the toxin) and bioprotection (elimination of the toxic effects) and are claimed to be effective in controlling aflatoxins, ochratoxin A, zearalenone, certain trichothecenes and fumonisins.

Surveillance and monitoring

There are also challenges for analytical chemists in the development of methods and techniques for the rapid and sensitive detection of mycotoxins. This is important, owing to the natural occurrence of chemically diverse mycotoxins in materials, such as cereals at different levels.

Many novel techniques are based on immunochemical methods. Based on either monoclonal or polyclonal antibodies, immunoassays have been in use for some time and allow the sensitive and selective detection of a single mycotoxin. In addition to traditional microwell formats or immunoaffinity clean-up columns, a range of novel assay formats have been developed. Microbead-based immunaffinity columns, tandem column immunoassays and flow injection immunoassays are available. Membrane-based methods include lateral-flow assays, and electrode-based assays exist as miniaturised immunoassays with electro – chemical detectors. Fluorescence polarisation immunoassay has been developed for the determination of aflatoxins, ochratoxin A, fumonisins, type B trichothecenes (in particular deoxynivalenol) and zearalenone11. Dipstick technology for the determination of some fusarium toxins (fumonisin B1, T-2 toxin, HT-2 toxin, deoxynivalenol and zearalenone) is in development for the rapid and simple screening of cereals12.

Protein engineering and recombinant DNA technologies have led to the design of small molecular affinity proteins (iMaBs or industrial Molecular Affinity Bodies), which are similar to antibodies with specific binding properties but are considerably smaller. The cost of manu facturing these is also much lower than conventional monoclonal antibodies, and they can be designed to bind industrial molecules, including mycotoxins, at the nano-level, with detection by mass spectrometric, NMR, circular dichroism or fluorescence techniques13.

Surface plasmon resonance (SPR) has been used in the development of biosensors for the detection of small, low molecular weight compounds, such as mycotoxins, and techniques involving electrochemical sensors show promise as a sensitive, cost-effective and userfriendly means of analysis of trichothecenes. This forms part of the work of Biocop, an EC FP6 integrated project (Food Quality and Safety priority). This project, which was established to develop new technologies to screen multiple contaminants in foods, has also reported on transcriptomics for the detection of certain trichothecene mycotoxins by the development of a microarray platform14.

A number of these novel strategies (for both prevention of mycotoxin contamination and their analysis and detection) have been developed, or are in the development process, in research laboratories in Europe. Several of these organisations are partners in the European Mycotoxin Awareness Network (EMAN) which is coordinated by Leatherhead Food Research. EMAN was originally launched in 2001 with financial support from the European Commission (fourth framework) and the main aim of the project at the time was to provide quality scientific information on mycotoxins to all stakeholders (academia, industry and consumers). The mission statement of EMAN, which can be accessed through www.mycotoxins.org, now reads ‘Free and easy access to mycotoxin information for food producers and consumers around the world’. Despite what the name might suggest, EMAN does not report exclusively from European activities, as regular reports from international universities or organisations feature on the EMAN website. A unique facility of EMAN is a new free quarterly bulletin, which includes the results of a search of Leatherhead Food Research’s Foodline Science database15.

References

1. The Rapid Alert System for Food and Feed (RASFF) Annual Report 2008. http://ec.europa.eu/food/food/rapidalert/ report2008_en.pdf

2. Commission Regulation 1881/2006 setting maximum residue levels for certain contaminants in foodstuffs http://eurlex.europa.eu/LexUriServ/site/en/oj/ 2006/l_364/l_36420061220en00050024.pdf

3. Production of fumonisin B2 and B4 by Aspergillus niger on grapes and raisins. J.M. Mogensen, J.F. Frisvad, U. Thrane, K.F. Nielsen, 2009 J. Agric Food Chem. 58 (2) 954-958.

4. Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must. A. Logrieco, R. Ferracane, M. Haidukowsky, G. Cozzi, A. Visconti, A. Ritieni, 2009, Food Additives and Contaminants 26 (11), 1495-1500.

5. Widespread occurrence of the mycotoxin fumonisin B2 in wine. J.M. Mogensen, T.O. Larsen, K.F.Nielsen, 2010 J. Agric. Food Chem. 58 (8) 4853-4857.

6. Molds and Mycotoxins show up in corn. University of Illinois College of Agriculture, Consumer and Environmental Sciences (ACES) newsletter, October 29 2009. http://www.aces.uiuc.edu/news/stories/ news4933.htm

7. Meeting the mycotoxin menace. D. Barug, H. van Egmond, R. Lopez-Garcia, T. van Osenbruggen, A. Visconti., 2004

8. Food mycotoxins: an update. P.A. Murphy, S. Hendrich, C. Landgren, C.M. Bryant, 2006, Journal of Food Science 71 (5), R51-R65.

9. Aflatoxin Control in pistachios, almonds and figs: biocontrol using atoxigenic strains. ARS Research Project, Food and Feed Safety Research. http://www.ars.usda.gov/research/projects/ projects.htm?accn_no=412279

10. 48th meeting of the Advisory Committee on Animal Feedingstuffs, Food Standards Agency, UK, December 3 2009. http://www.food.gov.uk/multimedia/pdfs/ committee/acaf0917.pdf

11. Fluorescence polarization immunoassay of mycotoxins: a review. C.M. Maragos, December 10 2009, Toxins 1:196-207.

12. Fusarium toxins: extraction procedure and test strip detection. http://www.conffidence.eu/archive/ newsletter.php?id=12

13. Development of new technology for the detection of toxins and pathogens. http://www.pri.wur.nl/UK/newsagenda/ archive/news/2005/Plant_Research_ International_and_CatchMabs_join_forces.htm

14. Work Package 3: Biosensors of the Biocop project. http://www.biocop.org/theproject_ packages.html

15. FoodlineWeb. http://www.foodlineweb.com/ foodline/index.aspx

About the Author

Catherine Entwisle

Catherine has worked at Leatherhead Food Research since 1988 in analytical chemistry sections, initially working on nutritional testing and contaminants before specialising in mycotoxin analysis and research.

Leatherhead Food Research

Leatherhead Food Research, based near London, UK, is an independent organisation delivering innovative research, scientific consultancy and regulatory guidance and interpretation. Leatherhead’s industry-leading scientific, regulatory and market research capabilities create a unique combination of food industry acumen and scientific expertise. Leatherhead offers clients practical advice, tailored consultancy, research and businessoriented support closely aligned to both their strategic and everyday demands. These services are built around five key platforms; Regulatory, Food Innovation, Food Safety, Nutrition Research and Knowledge Transfer, each representing a core area of our expertise.