The Mycotoxin Challenge In Modern Feed Production
BiominÂ® Laboratory Singapore Pte. Ltd
What are mycotoxins?
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 moulds 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. Due to modern methods and thanks to a growing interest in this field of research more than 300 different mycotoxins can be differentiated today. However for our practical consideration in the feed manufacturing process a small yet potential number of toxins is of relevance.
What are the mycotoxins of relevance?
The most commonly known mycotoxins are the aflatoxins due to the fact that they represent one of the most potential carcinogenic substances known so far. They are rated as Class 1 human carcinogens by the IARC (International Agency for Research on Cancer) and recognised as the main hepatocarcinogen in animals, although effects vary with species, age, sex, and general nutrition. The 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, with approximately 170 of them being identified up to date. The most prevalent occurring mycotoxins of this group are the B-type trichothecenes as deoxynivalenol (DON, vomitoxin), nivalenol (NIV), 3- or 15-acetyl-deoxynivalenol (AcDON), and the A-type toxins T-2 toxin and HT-2 toxin. Different types of trichothecenes vary in their toxicity though all of them are highly acute toxic. An important issue is that some of these closely related compounds occur frequently simultaneously and are proven to cause synergistic effects. 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 hyperestrogenic effects, which result in impaired fertility, stillbirths in female and a reduced sperm quality in male animals.
Ochratoxin A (OTA), which is produced in moderate and colder climates and causes renal toxicity, nephropathy and immune-suppression in several animal species. As it occurs in many commodities and it is stored in fatty tissues there is a certain carry-over by products of animal origin human intake in some countries can be high. Fumonisins cause severe animal diseases like equine leukoencephalomalacia (ELEM) in horses and porcine pulmonary edema in swine (PPE). Besides their hepatotoxicity and nephrotoxicity they affect also the immune system. In addition human oesophageal cancer has been observed in distinct areas of the world where high levels of fumonisins occurred on maize and maize-based food products.
Ergot alkaloids comprise derivates of lysergic acid, isolysergic acid and the clavine alkaloids and are found in the sclerotia (i.e. ergots) of Claviceps purpurea. Ingestion of higher alkaloid levels will result in neurological and/or gangrenous disorders, i.e. tremors, staggers, conculsions, and necrosis, sloughing of the extremities, respectively.
Prevention of mycotoxins
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 favorable for disease development. Some fungi 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 get powerful inocula as temperatures increase in spring. Airborne release of spores might peak during and after rainy periods, distributing the fungal sources over wide distances, and causing epidemics. There are two routes of entry for mould infection of grain in general, and corn in particular: first, fungal spores landing on emerged silks can infect the ear by the silk channel, and second, wounds caused by birds, insects or extreme weather can provide a good opportunity for fungal invasion. During harvest it is important to prevent excess damage to kernels, which may predispose them to infection during storage. Too high moisture content is likewise a high risk 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 favorable 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).
The use of mould inhibitors or preservation by acids can reduce the amount of mould but does not influence the prior treatment generated mycotoxins. These toxic compounds remain in the formerly infected commodity even if no more mould can be seen or detected. The only way to really assess the quality of raw materials is the specific testing of mycotoxins or certain groups thereof.
Testing for mycotoxins is a complicated process that generally consists of three steps: (1) Sampling means to select a 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 step 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. This means that a very small quantity of the lot is finally used in the quantification step to estimate the mycotoxin concentration of the whole lot. Analytical procedures for the determination of mycotoxins have improved continuously over the past years. Chromatographic methods like high performance liquid chromatography (HPLC) or gas chromatography (GC) have been used widely and can be considered as the most accurate quantification systems, but also a variety of immunological methods, in particular immuno sorbent assays (ELISAs) are used frequently, as they require usually no further sample purification. ELISA test kits are well favored as high throughput assays with low sample volume requirements and proceeding times of less than an hour, some even in less than 15 minutes. However, although the antibodies have the advantage of high specifity and sensitivity to their mycotoxin target molecule, compounds with similar chemical groups would also interact with the antibodies. This so-called matrix effect is especially evident in case of high complexity of the test material, which is in particular the case with finished feed, and can lead to overestimates, underestimates, or even false negative or false positive results, so that in such cases chromatographic detection remains the method of choice.
Management of mycotoxin contamination in the daily operation.
The first and most practical approach so far has been the blending of low contaminated material with material above the limits thus lowering the average contamination levels to the accepted standards. In case that is not possible other methods need to be applied.
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 too high cost implications. Ammonia or strong oxidizing agents can reduce the contamination but reduce also the nutritive value of the feed at the same time. Based on that facts extensive research has lead to various products for the detoxification of feed.
The most commonly used strategy of reducing exposure to mycotoxins is the decrease of their bio-availability by the inclusion of various mycotoxin binding agents or adsorbents, which leads to a diminishing of mycotoxin uptake and distribution to the blood and target organs. This works in particular well with aflatoxins, as this group of toxins has a chemical structure which favors adsorption, especially by materials of mineral origin, like clay and zeolitic minerals. 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 level. Since the mode of action has to start already in the stomach it must be effective at least at a pH level of 3. An 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 (e.g. trichothecenes, zearalenone, ochratoxins or fumonisins) from contaminated feedstuffs by the use of adsorbents did not lead to any satisfactory results so far, as most of the adsorbing agents bind them only weakly in vitro and are practically 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. BiominÂ® researchers were the first to isolate a pure bacterial strain which is able to bio-transform the toxic epoxide part of trichothecenes into a biologically inactive form, thus detoxifying all relevant trichothecene toxins by the us of this microbe, which is a Eubacterium species and was named BBSH 797. For its application as feed additive fermentation and stabilization 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 proven in piglet and broiler trials (Binder et al., 2001). Further research of the same group led to the isolation of a yeast strain, namely of the strain Trichosporum mycotoxinivorans, which can decompose and thus detoxify ochratoxin and zearalenone. Both, in vivo and in vitro trials have proven 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, which means its guaranteed binding capacity at 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 trichothecene contamination of feedstuffs the addition of feed additive BBSH 797 would be a proper way to counteract the possible impacts due to this mycotoxin group, in case of positive indications of zearalenone or ochratoxins the addition of Trichosporum mycotoxinivorans should be considered.
Binder, E.M., et al. (2001). In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium. Willem J. de Koe, et al. (eds.). ISBN 90-9014801-9.
CAST Report (2003) Mycotoxins: Risks in Plant, Animal, and Human Systems. Richard, J.L., Payne, G.A. (eds.). ISBN 1-887383-22-0.
Schatzmayr, G., et al. (2004). XI. International IUPAC Symposium on Myxotoxins and Phycotoxins. May 17-21, 2004, Bethesda, Maryland. Abstracts Book. p. 46.