Mycotoxin

Mycotoxins are, in general, low molecular weight, non-antigenic fungal secondary metabolites formed by way of several metabolic pathways, e.g. the polyketide route (aflatoxins), the terpene route (trichothecenes), the amino acid 

While some mycotoxins are formed by only a limited number of fungal species others can be produced by a range of species from several genera. At least 300 potential mycotoxins have been isolated under laboratory conditions but only a relatively small number (about 20) have been shown to occur in foods and feeds at significant levels and frequency. All of these mycotoxins have been shown to have significant animal toxicity. 

It is now widely agreed that approximately 25% of the world s food crops are affected each year by variable levels of mycotoxins which can have considerable economic consequences for the crop, livestock producers, grain handlers, processors, consumers and indeed national economies (Miller, 1998). It has been conservatively estimated that annual losses in the US and Canada, as a result of mycotoxin occurrence, to the feed and livestock industries alone can be in the region of US 5 billion (Charmley et al., 1995). In developing countries, regular mycotoxin presence in the diet will also affect the human populations, causing morbidity and premature deaths. 

Direct economic losses due to the presence of mycotoxin- (especially aflatoxin-)producing fungi in agricultural crops can be detected in reduced crop yields and lower quality, reduced animal performance and reproductivity 

Mould-damaged foodstuffs Agricultural products, e.g. cereals 1 oilseeds J 

Mycotoxins are toxic secondary metabolites produced by fungi growing within or on foods. They can be a serious threat to human and animal health (Nagler el al., 2001). Table 11.4 details mycotoxins associated with soft drinks and fruit juice manufacture and raw materials. Patulin is the most common mycotoxin associated with fruit juice, particular ly apple juice (Pitt Hocking, 1997). It commonly occurs if juice is produced from stored apples. Mould growth in infected apples increases with time, raising levels of patulin. The use of windfall apples for juice is also a factor. Avoidance of windfall apples, filtration of juice and pressing quickly after harvest are all methods to reduce the incidence of patulin in juice. Patulin can be destroyed by fermentation to cider or by the addition of ascorbic acid (Marth, 1992). Within Europe, the European Union has set a limit of 50 ig/kg for patulin in both apple juice and cider. A recent survey of apple products in Chile found that 28% of samples of juice and concentrate exceeded this limit (Canas Aranda, 1996). 

Apple Penicillium expansum Patulin, citrinin, roquefortine C 

Diluted fruit/water beverages P. roqueforti Isofumigaclavine A and B 

Treated orange juice Fusarium oxysporum Oxysporone 

Fruit juices Aspergillus versicolor Geosmin sterigmatocystin 

Mycotoxins are toxic chemical metabolic products produced by microscopic mold fungi that colonize cereal crops, fruits, and vegetables and some of them exhibit carcinogenic and mutagenic properties. The allowed maximum levels in foodstuffs are very low and exhaustive exU action, preconcentration, and clean-up processes are required to attain the desired sensitivity. The alternative of ED (amperometric detection) combined with HPLC can be useful to simplify the detection of such low concentration levels. However, it has been used rarely for the analysis of these compounds in food because many of these compounds can be elecfrochemically oxidized or reduced at extreme potentials. Visconti et al [144], analyzed 

Analyte Analyzed sample HPLC mode Electrochemical detection References 

Altertoxin-I and altertoxin-ll Maize, rice, and tomatoes RP-HPLC Coulometric [144] 

Altenuene and isoaltenuene Artificially contaminated samples RP-HPLC Coulometric (ID-PCBr) [145] 

Zeranol and zearalenone metabolites Rice, corn flakes, and soybean RP-HPLC Dual amperometric-UV (SPE) [148] 

There are more than 200 mycotoxins produced under certain conditions by about 120 fungi or molds. Table 9.3 presents data on mycotoxins of particular interest to food preservation and storage. The chemical structures of these toxins are presented in Fig. 9.1. 

Most mycotoxin data are on the genera Aspergillus spp. and the aflatoxins they produce 

In the course of food monitoring between 1995 and 2002, more than 40 foods were tested for the presence of aflatoxins, deoxynivalenol, fumosins, patulin, ochratoxin A und zearalenone. Individual mycotoxins were detected in 21% of the samples pistachios were especially conspicuous. 

An assessment of the health hazards caused by mycotoxins is not meaningful when applied to the aflatoxins because these substances damage DNA, are carcinogenic and have no threshold below which no harmful effects are observed. An assessment was possible in the case of deoxynivalenol und ochratoxin A, with the reservation 

V Ochratoxin A VI trans-Zearalenone VII Fusariotoxin T2 VIII Vomitoxin 

There are over 150 forms of trichothecene mycotoxins. Most are insoluble in water and are heat resistant. For the cleanup of a unique laboratory site such as Fort Dietrich or Aberdeen, both in Maryland, a bottle of such a mycotoxin buried in some landfill could be extremely dangerous. In Laos, there were more than 6000 deaths reported, 3000 in Afghanistan, and a good number in Kampuchea from the so called yellow rain.  

The range of chemical structures and biologic activity among the broad class of fungal metabolites is large and cannot be summarized briefly. Mycotoxins do not constitute a separate chemical category, and they lack common molecular features. 

Mycotoxins of most interest are those found in human food or in the feed of domestic animals. They include the ergot alkaloids produced by Claviceps sp aflatoxins and related compounds produced by Aspergillus sp and the tricothecenes produced by several genera of fungi imperfecti, primarily Fusarium sp. 

Aflatoxins are products of species of the genus Aspergillus, particularly A flavus, a common fungus found as a contaminant of grain, maize, peanuts, and so on. First implicated in poultry diseases such as Turkey-X disease, they were subsequently shown to cause cancer in experimental animals and, from epidemiological studies, in humans. Aflatoxin Bl, the most toxic of the aflatoxins, must be activated enzymatically to exert its carcinogenic effect. 

Mycotoxins may also be used for beneficial purposes. The mycotoxin avermectin is currently generating considerable interest both as an insecticide and for the control of nematode parasites of domestic animals. 

However, although these comments are obviously of relevance, the detection of toxins is of great importance as many toxins are known carcinogens. Over two 

The determination of mycotoxins has been reviewed by Jarvis et al and van Egmond. Numerous methods have been described, including both biological and chemical assays with detection levels ranging from sensitive to relatively insensitive. 

The minicolumn methods of Holaday and Romer are ideal for developing 

Two dimensional TLC probably offers the best rapid, cheap method of detecting many toxins, especially in countries where the purchase and maintenance of expensive instruments may not be possible. It also has the major advantage in many developing countries that it is not reliant upon a source of power. 

GLC of mycotoxins is limited as most are non-volatile and derivatives need to be prepared, although GLC is of considerable use in the determination of the trichothecenes which are difficult to assay by TLC or HPLC. The preferred method of detection is electron capture, but increasing use is being made of GLC linked to mass spectrometry. 

FIGURE 6.3 Chemical structure of the different types of aflatoxins. Aflatoxins B and G occur in infested grains, whereas M aflatoxins are found in the milk of lactating animals fed contaminated feedstuffs. 

Undoubtedly, maize is the most susceptible cereal to mold infestation and myco-toxins (Krogh 1987, Krishna and Sinha 1991, Mirocha et al. 1980, Sauer 1992). This is because the cob is covered with husks creating an ideal and protective environment for molds. Research has demonstrated that fungi can penetrate the cob through previous damage caused by ear worms (Heliothis zed). The presence of aflatoxins in maize has created problems in grain elevators because in most countries the maximum level allowed is 20 ppb for humans and 200 ppb for animals. In milk, the maximum level is only 0.5 ppb. 

FIGURE 6.4 Chemical structure of most common grain mycotoxins. (a) Zeralenone. (b) Ochratoxin A or OTA. (c) Fumonisin. (d) Trichothecenes or T-2. (e) Deoxynivalenol or DON. (f) Ergotamine. 

---

Mycotoxins. The condition produced by the consumption of moldy foods containing toxic material is referred to as mycotoxicosis. Molds and fungi fall iato this category and several derive thek toxicity from the production of oxaflc acid, although the majority of mycotoxias are much more complex. 

C. W. Hesseltiue, Sixth International Symposium Mycotoxins and Phytotoxins, Pretoria, South Africa, 1985, pp. 1—18. 

This same experimental approach can be used to determine the appHcabiUty of the aDAS—AP to a competitive assay for DAS. As shown in Eigure 6, increasing amounts of free DAS were used to define the 50% inhibition level (ID q) of DAS for binding of two aDAS—AP conjugates to immobilized DAS. This approach was also used to determine the sensitivity of an EIA, as well as the specificity of the assay, as shown in Table 2. Increasing amounts of trichothecene mycotoxins closely related to DAS were added to microtiter plate wells containing a constant amount of prereacted DAS—aDAS—AP. After 30 min, excess toxin and any free toxin—aDAS—AP were washed out, and substrate was added. Quantification of the color produced was directly related to the abihty of the added toxin to displace aDAS—AP from the immobilized DAS, which is an indication that the aDAS also has an avidity for that toxin. 

Table 2. Inhibition of DAS-aDAS Binding by Various Mycotoxins...

Vitamin D deficiency in animals may be caused by the fact that the vitamin is not available to the hvestock. Modem animal husbandry subjects animals to total confinement with htde or no exposure to sunlight. This mandates that they be given vitamin D-fortified diets. The vitamin is sensitive to oxidation, heat, light, and minerals, and significant losses may occur in the fortified feed unless the product is adequately protected. Mycotoxins in feeds also interfere with utilization of vitamin D in feeds (207—209). 

Mycotoxins, toxic metaboUtes of some fungi, can be assayed by immunochemical techniques to determine concentration in animal feed and foodstuffs. Some of the analytes assayed in kits and the detection limits are Hsted in Table 4 (45). These assays are especially advantageous for routine analysis of large samples of foodstuffs (45,46). 

Table 4. Detection Limits of Immunoassays Developed for Mycotoxins and Pesticides...

Product specifications for microbial food enzymes have been estabUshed by JECEA and ECC. They limit or prescribe the absence of certain ubiquitous contaminants such as arsenic, heavy metals, lead, coliforms, E. coli and Salmonella. Furthermore, they prescribe the absence of antibacterial activity and, for fungal enzymes only, mycotoxins. 

COMPARABLE CHARACTERISTIC OF ESTIMATING MYCOTOXIN CONTENT USING HPLC AND CHROMATODENSITOMETRY METHODS... 

There have been compared the methods of mycotoxin control in food products with aflatoxin as an example, using both HPLC method with fluorescent detecting on the apparatus Thermo FL 3000 with a column BDS Hypersil C 2.1x150, as well as a chromatodensitometry method on the apparatus CAM AG TLS Scanner 3. 

UK Ministry of Agriculture Fisheries and Food and Flealth and Safety Executive, Annual Report of the Working Party on Pesticide Residues in Food (1997), MAFF Publications, London, 1997. lARC, Monographs on the Evabiation of the Carcinogenic Risk of Chemicals to Hnmans Volume 56 Some Naturally Occurring Substances Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, WFIO, Geneva, 1979, p. 397. 

R. Kfir, E. Johannsen and D. P. Botes, in Mycotoxins and Phycotoxins. Bioactive Molecules, ed. 

Compounds that Cause Kidney Damage Several drugs and some anesthetic compounds such as methoxyflurane cause kidney damage when present at high doses. Kidney-toxic compounds found in occupational environments include mycotoxins, halogenated hydrocarbons, several metals, and solvents (see Table 5.16). 

These methods are employed for the detection and determination of antibiotics and substances with similar effects, like alkaloids, insecticides, fungicides, mycotoxins, vitamins, bitter principles and saponins [14]. 

Manual transfer of the chromatographically separated substance to the detector . These include, for example, the detection of antibiotically active substances, plant and animal hormones, mycotoxins, insecticides, spice and bitter principles and alkaloids. The frequency distribution of their employment is shown in Figure 54 [295]. 

The developed chromatograms are briefly immersed in or evenly sprayed with the appropriate reagent solution. Solution I is employed for flavonoids [1, 3] and solution II for mycotoxins [5, 8, 12], phospholipids, triglycerides and cholesterol [14]. 

Reagent for Epoxides e.g. trichothecene-mycotoxins [1 — 6] valepotriates [7,17] Olefins, acetylene derivatives [8] 4-Hydroxycumarin, anthraquinone [8] Alkylating agents [9-12] NOz... 

Note Flavonoids react with the reagent even at room temperature [1] mycotoxins, steroids, purines, pyrimidines, cardiac glycosides and lipids only react on heating [2, 4-6]. Zirconyl sulfate can be used to replace the zirconyl chloride in the reagent this is reported to result in an increase in the sensitivity to certain groups of substances (e.g. cholesteryl esters, triglycerides) [4]. 

Chromatographic analysis of some mycotoxins, in particular 0-heterocycles 97KFZ(7)49. 

---