Mycotoxins are invisible, odourless and cannot be detected through smell or taste. Due to the complex nature of these naturally occurring fungal contaminants and the elaborate requirements for their analysis a risk management concept has to be installed in order to reduce the risk encountered to a defined and acceptable level. While the pre- or post-harvest prevention of mycotoxin contamination is the preferred strategy for minimising their risk in food and feed, the early identification of mycotoxin-contaminated grains provide the opportunity to direct the moulds most highly contaminated grains into uses by less sensitive species or to take measures to counteract these toxins in specific and proper ways.
Mycotoxins are secondary metabolites produced by filamentous fungi that cause a toxic response (mycotoxicosis) when ingested by higher animals. Fusarium, Aspergillus, and Penicillium are the most abundant that produce these toxins and contaminate human foods and animal feeds through fungal growth prior to and during harvest or as a consequence of improper storage following harvest (Bhatnagar et al., 2004). Due to modern methods and thanks to a growing interest in this field of research more than 300 different mycotoxins can be differentiated today, although only a limited number is of relevance in animal husbandry.
Impacts of mycotoxins
The most common mycotoxins known are the aflatoxins due to the fact that they represent some of the most potential carcinogenic substances known so far, and as they are the main hepatocarcinogen in animals, although effects vary with species, age, sex, and general nutrition conditions. Fatty liver or pale bird syndrome and inhomogeneous flocks are the most typical symptoms for such a contamination in feed.
Trichothecenes are a large group of mycotoxins produced by various species of moulds, in particular, those belonging to the genus of Fusarium. Approximately 170 trichothecene mycotoxins have been identified up to date, with deoxynivalenol (DON, vomitoxin), nivalenol (NIV), 3- or 15-acetyl-deoxynivalenol (AcDON), and Fusarenon X (FUS-X), as well as T-2 toxin and HT-2 toxin as the most prevalently occurring toxins of this group.
An important issue is that some of these closely related compounds occur frequently simultaneously (Fuchs et al., 2004) and are proven to cause synergistic effects (Weidenbörner, 2001). Different types of trichothecenes vary in their toxicity though all of them are acutely toxic. They may cause haematological changes and immune suppression, reduced feed intake and skin irritations as well as diarrhoea and haemorrhages of internal tissues.
Zearalenone is also produced by Fusarium species and has strong hyperoestrogenic effects leading to impaired fertility in general.
Ochratoxin A (OTA), which is produced by Aspergillus and Penicillium species and causes renal toxicity, nephropathy and immune-suppression in several animal species, which entails reduced performance parameters. As it occurs in many commodities and there is a certain carry-over B toxins or their metabolites into products of animal origin human intake in some countries can be high.
The most recently described mycotoxins with relevance in human and animal nutrition are fumonisins, which were first reported in South Africa in 1988 and also Fusarium mycotoxins. Besides their hepatotoxicity and nephrotoxicity they affect also the immune system.
Testing for mycotoxins is a complicated process that generally consists of three steps:
(1) Sampling - i.e., selecting a representative sample of a given size from a bulk lot;
(2) Sample preparation - comprises the grinding of the sample and taking a representative sub-sample of ground material;
(3) The analytical procedure - consists of several processes where the mycotoxin is solvent extracted from the sub-sample, the solvent is purified and the mycotoxin in the solvent is quantified. The mycotoxin value, measured in the analytical step is then used to estimate the lot concentration or is compared to a maximum limit in order to classify the lot as acceptable or unacceptable.
Analytical procedures for the determination of mycotoxins have improved continuously over the past years. Chromatographic methods have been used widely, including thin-layer chromatography (TLC), gas chromatography (GC) as well as high-performance liquid chromatography (HPLC), using a large variety of detector technologies.
The most commonly used system for rapid testing is ELISA (Enzyme Linked Immunosorbent Assay) since it is the fastest and most cost effective system, in case of high sample throughput and quick result requirements. It can also come in qualitative formats like small cups, where an enzymatic colour reaction visualizes within five minutes the presence or absence of a toxin at a certain cut-off level, but does not indicate an exact contamination level.
Management practices to maximize plant performance and decrease plant stress can decrease mycotoxin contamination substantially. This includes planting adapted varieties, proper fertilization, weed control, necessary irrigation, and proper crop rotation. But even the best management strategies cannot eliminate mycotoxin contamination in years favourable for disease development.
Some fungi, like several Fusarium species, are widespread colonizers of crop residues, where the pathogen survives during winter. Thus wheat stubble, corn stalks and rice stubble can be major sources of these moulds, which produce large inocula as temperatures increase in spring. Airborne release of spores may peak during and after rainy periods, distributing the fungal sources over wide distances, and causing epidemics (CAST, 2003).
High moisture content is a significant factor for mycotoxin infestation, with the final "safe" moisture content depending on the crop and the climatic conditions where the commodity is stored, although drying to 15% moisture content or below is widely recognized as being suitable. It should be mentioned that when conditions are generally favourable for fungal contamination it is not uncommon for more than one type of fungus to be involved.
During storage, grain is often colonized by a succession of fungi, depending on temperature and moisture levels. Due to these possible interactions of several fungal species, grain may be contaminated with a number of different mycotoxins (Cast, 2003).
It is important to stress the point, that the use of mould inhibitors or preservation by acids can only reduce the amount moulds but does not influence mycotoxins contamination generated prior to treatment. If mycotoxins have been produced earlier they will not be affected in any form by mould inhibitors or acid mixtures, as they are very stable compounds. Thus these toxic compounds remain in the formerly infected commodities even if no more mould can be seen or detected. Since all mycotoxins are quite stable substances no physical or chemical treatment can be applied under practical field conditions, without altering the nutritive value of the grain or causing high cost implications.
The most commonly used strategy of reducing exposure to mycotoxins is the decrease of their bio-availability by inclusion of various mycotoxin binding agents or adsorbents, which leads to a diminishing of mycotoxin uptake and distribution to the blood and target organs. Various substance groups have been tested and used for this purpose, with aluminium silicates, in particular clay and zeolitic minerals, as the most commonly applied groups.
An important criterion for evaluation of mycotoxin adsorbers is their effectiveness at high and low pH levels since a product must work throughout the gastro-intestinal tract, thus within a broad pH range. Since the mode of action has to commence in the stomach it must be effective at least at pH level 3. Another important aspect in the evaluation of potential mycotoxin binders is the stability of the sorbent-aflatoxin bond, in order to prevent desorption of the toxin.
The elimination of other mycotoxins than aflatoxins from contaminated feedstuffs by the use of adsorbents has not lead to any satisfactory results so far, as most of the adsorbing agents bind them only weakly in vitro and are more or less ineffective in vivo. As it is known that in the case of trichothecenes the 12,13-epoxide ring is responsible for their toxic activity and removal of this epoxide group entails a significant loss of toxicity, research focused on the identification of natural processes where this reaction occurs. Biomin® researchers were the first to isolate a pure bacterial strain which is able to bio-transform the epoxide group of trichothecenes into a diene, thus detoxifying all relevant trichothecene toxins by this reaction.
The active isolate is a new species of the genus Eubacterium, named BBSH 797. For its application as feed additive, fermentation and stabilisation processes were established and optimized with respect to good and fast growth of the microbe, high biotransformation activity of the resulting product, and economic reasons. For enhancement of stability during storage and within the gastro-intestinal tract, a three-step encapsulation process was implemented. The additive's efficiency in counteracting adverse effects of feed contaminated with trichothecenes was demonstrated in feeding trials with several animal species (Binder et al., 2001). Further research by our group led to the isolation of a yeast strain, Trichosporon mycotoxinivorans (MTV), which can decompose and thus detoxify ochratoxin and zearalenone. Both, in vivo and in vitro trials have shown this additives´ high efficacy to counteract these mycotoxins under practical conditions (Schatzmayr et al., 2004).
Based on the knowledge summarized above and information about the overall contamination levels the following mycotoxin detoxification strategy can be recommended:
For any contamination where aflatoxin occurs as the only contaminant a certified binder should be used for reduction of bioavailability of aflatoxins. The adsorber´s certificate should comprise data about its efficacy, i.e., its guaranteed binding capacity at least two relevant pH levels (e.g. pH 3 and pH 6.5), as well as the absence of any potential hazard, in particular of dioxin. In case of contamination of feedstuffs with other toxins than aflatoxin, the application of proper alternative technologies, like microbial detoxicants, should be considered.
Managing mycotoxin contamination in the daily operation
Hazard analysis starts with the preparation of a list of steps in the production process, where mycotoxin or mould infestation could occur, and describes preventive measures, like purchasing of raw materials.
Many contracts do not mention mycotoxins at all and it could be an improvement to add a clause with maximum acceptable levels of mycotoxin contamination to the contract. The second step in an HACCP system is to determine the critical control points, i.e., which are the materials, products or production steps that have to be monitored for fungal contaminants. One rule of the thumb could be the ratio of tests conducted on raw materials versus tests done on finished products, which could be - for example - nine to one. A third step would be to establish critical limits, which means to determine the maximum tolerable toxin levels.
What is the internal risk profile that is acceptable within an operation? Step number four is the establishment of procedures for monitoring the critical control points. This can include procedures for sampling, sample preparation and testing itself, or the outsourcing of parts of or even the full analytical process. Step five covers the establishment of corrective actions, which would comprise the introduction of certain cleaning procedures for silos, bins, hoppers, and elevators into the maintaining plan, as repeated contamination could originate from bins containing materials like wheat bran that have never been cleaned so that contamination might originate from and spread within the same operation and not only from purchased raw materials, or feed supplementation with detoxifying products. Step six comprises the verification procedures and step seven the documentation and record keeping.
Bhatnagar, D., Payne, G.A., Cleveland, T.E., and Robens, J.F. (2004). Meeting the mycotoxin menace, pp 17-47. Wageningen Academic Publishers. ISBN 9076998280.
Binder, E.M., Heidler, D., Schatzmayr, G., Thimm, N. Fuchs, E., Schuh, M., Krska, R. and Binder, J. (2001). Proceedings of the 10th International IUPAC Symposium on Mycotoxins and Phycotoxins - 21-25 May, 2000, Brazil, 271 - 277.
CAST Report (2003) Mycotoxins: Risks in Plant, Animal, and Human Systems. Richard, J.L., Payne, G.A. (eds.). Council for Agricultural Science and Technology Task Force Report No. 139, Ames, Iowa, USA. ISBN 1-887383-22-0.
Fuchs, E., Handl, J., Binder, E.M. (2004) New Horizon of Mycotoxicology for Assuring Food Safety, pp. 225-232. Yoshizawa, T. (ed.). Japanese Association of Mycotoxicology. ISBN 4-938236-86-2.
Schatzmayr, G., Heidler, D., Fuchs, E., Täubel, M., Loibner, A.P., Braun, R., Binder, E.M. (2004). XI. International IUPAC Symposium on Mycotoxins and Phycotoxins. May 17-21, 2004, Maryland, US. Abstracts Book. p. 46.
Weidenbörner, M. (2001) Encyclopedia of Food Mycotoxins, p. 243. Springer-Verlag Berlin Heidelberg New York. ISBN 3540675566.
From Proceedings of the "17th Australian Poultry Science Symposium", New South Wales, Australia.