The mycotoxin research in foodstuffs in the Czech Republic in the 90th Years

Mycotoxins are naturally occurring secondary metabolites produced by several toxigenic microscopic fungi on a variety of crops, especially cereal grains and further foodstuffs. Series of experimental research projects on the determination of mycotoxins (aflatoxins, cyclopiazonic acid, ochratoxin A, patulin, deoxynivalenol, fumonisin B₁, T-2 toxin, zearalenone, sterigmatocystin, alternaria toxins) in several foods were realized in the National Reference Centre for Microscopic Fungi and Mycotoxins in the 90^th years. The aim of our work was an estimation of dietary exposure to mycotoxins and risk assessment. The method of a solid phase extraction (SPE), liquid — liquid extraction and immunoaffinity chromatography (f. e. R-Biopharm, VICAM) were used to elaborate for sample analyses of mycotoxins in our projects. The mycotoxins were detected most frequently by chromatographic methods (HPTLC, HPLC, GC) and immunochemical methods (ELISA). Average dietary exposure has been calculated by multiplying of concentration data for specific foods with their consumption rates per 1 kg of b. w. per day. The estimation of the dietary exposure dose of mycotoxins for the Czech population is presented.     

Monoclonal antibody technology for mycotoxins

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.     

Analysis of mycotoxins in cereals and animal feed using a multi-toxin gel permeation chromatography clean-up method

Gel permeation chromatography has been used to clean up extracts from cereals and animal feeds containing a range of mycotoxins. A mixture of dichloromethane: IM hydrochloric acid, 10:1 by volume is used as the extraction solvent and clean-up is carried out on a Bio — Beads S-X3 column using dichloromethane: ethyl acetate containing a small amount of formic acid as the elution solvent. Chromatographic separation and detection was by HPLC with fluorescence or ultraviolet detection although the choice of detection method is left to the user. The method has been tested for 14 mycotoxins and results are presented for cereals fortified with mycotoxins and for samples naturally contaminated with aflatoxins, citrinin, zearalenone, and ochratoxin A.     

Decontamination of ochratoxin A by yeasts: possible approaches and factors leading to toxin removal in wine

Biological decontamination of mycotoxins using microorganisms is one of the well-known strategies for the management of mycotoxins in foods and feeds. Yeasts are an efficient biosorbant, used in winemaking to reduce the concentration of harmful substances from the must which affect alcoholic fermentation (medium-chain fatty acids) or which affect wine quality in a negative way (ethyl phenols and sulphur products). In recent years, several studies have demonstrated the ability of yeasts to remove ochratoxin A (OTA) by live cells, cell walls and cell wall extracts, yeast lees. In spite of the physical and chemical methods applied to remove the toxin, the biological removal is considered a promising solution, since it is possible to attain the decontamination without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food. In addition, adsorption is recognized as economically viable, technically feasible and socially acceptable. This paper intends to review the current achievements of OTA removal mediated by yeasts, the recent updates in the selection of strains acting at the same time as starters and as biological tools to remove OTA and the factors affecting the removal process.     

Study on distribution of mycotoxins in cocoa beans

Mycotoxins are not homogeneously distributed in foods which come in naturally small units, such as pistachios and peanuts, and may instead be extremely inhomogeneously distributed due to the occurrence of so-called hot spots. Tests conducted on pistachios, for example, show that a mouldy kernel can be so strongly contaminated with mycotoxins that it has a significant impact on the contamination profile of several thousand kernels. This makes a representative sampling of such foodstuffs very important but also a very difficult task. Whether cocoa beans also have a tendency to form so-called mycotoxin hot spots is hitherto unknown. A miniaturised analysis method was used in tests made on several independent batches of cocoa beans and although these tests showed that the mycotoxins ochratoxin A and the aflatoxins are not homogeneously distributed in cocoa, the tested batches revealed no real hot spots. Presented at the 27^th Mykotoxin-Workshop, Dortmund. Germany, June 13–15, 2005     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

Immunochemical identification of fungi using polyclonal antibodies raised in rabbits to exoantigens from Aspergillus ochraceus

Aspergillus ochraceus Wilhelm is a producer of highly toxic mycotoxin, ochratoxin A, and is known to contribute to the damage of grain on storage. Rabbit antibodies were produced against the exoantigens of A. ochraceus , and ELISA and immunoblotting procedures were used to characterize their reactivity towards fungal exoantigens of four Aspergillus , seven Penicillium , three Fusarium , one Mucor and one Alternaria species. The antibodies were specific for A. ochraceus and essentially cross-reacted neither with other fungi nor with the water-soluble grain components. These antibodies, therefore, have the potential of being utilized for the discriminative monitoring of A. ochraceus in foodstuffs and feeds.     

Mycotoxins: food contamination, mechanism, carcinogenic potential and preventive measures

Mycotoxins constitute a large number of naturally occurring fungal secondary metabolites with very diversified toxic effects in humans and animals. Among many mycotoxins discovered, aflatoxins, ochratoxin A, sterigmatocystin and several others are identified as carcinogens; several others were found to be mutagenic. Nevertheless, aflatoxin B1 has been found to be one of the most potent carcinogens and contamination of aflatoxins in the food supply is still a major concern. Whereas extensive studies have been made on aflatoxins, little is known about the mode of action of other carcinogenic and mutagenic mycotoxins. Recent progress on research for the carcinogenic and mutagenic mycotoxins is presented in this review with emphasis on their contamination in foods, their carcinogenic potential to humans, and the mode of action as well as possible preventive measures.     

Mycotoxin contamination of food in Europe: Early detection and prevention strategies

This paper reviews the early detection and prevention strategies which have been employed in Europe for the control of mycotoxin contamination of food in the context of a hazard analysis critical control point (HACCP) framework. The critical control points (CCPs) in the whole food chain where mycotoxins such as trichothecenes and ochratoxins are important have been identified. Ecological studies on the effect of environmental factors which are marginal for growth and mycotoxin production have been identified for Fusarium culmorum and F. graminearum (deoxynivlenol production), and for Penicillium verrucosum and Aspergillus ochraceus (ochratoxin production) in relation to cereal production and for A. carbonarius in relation to grapes and wine production (ochratoxin formation). To minimise the entry of these mycotoxins into the food chain, effective and rapid diagnostic tools are required to monitor the CCPs effectively. To this end the potential use of molecular imprinted polymers, lateral flow devices and molecular-based techniques for the rapid detection and quantification of the mycotoxigenic moulds or their toxins have also been developed.     

Mycotoxins in food systems in Sub Saharan Africa: A review

Mycotoxins, toxic secondary metabolites of fungi are now recognised as major cause of food intoxications in Sub Saharan Africa (SSA). Aflatoxins, the most important of the group have been implicated in acute aflatoxicoses, carcinogenicity, growth retardation, neonatal jaundice and immunological suppression in SSA. The hot and humid tropical climate provides ideal condition for growth of toxigenicAspergillus spp, making food contamination to be widespread in SSA, with maize and groundnuts being the most contaminated. The available data suggests that cassava products (the most important African food) are not prone to aflatoxin contamination. Recent data on ochratoxin A produced by species ofAspergillus on grains have indicated the necessity for it to be monitored in SSA. Fumonisins represent the most importantFusarium mycotoxins in SSA, and surveillance data indicate very high contamination rates of almost 100% in maize samples from West Africa. Limited information exists on the occurrence of trichothecenes, while the data currently available suggest that zearalenone contamination seems not to be a problem in SSA. The strategies under investigation to mitigate the mycotoxin problem in SSA include education of the people on the danger of consuming mouldy foods, pre and post harvest management strategies with emphasis on biological control, use of plant products to arrest fungal growth during storage, enterosorbent clay technology, and the search for traditional techniques that could reduce/detoxify mycotoxins during food processing.     

Determination of ochratoxin A in liquorice products using HPLC-based analytical methods. Part II: harmonised method and method validation study

On the basis of the outcome of an European proficiency test series conducted on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A, a new harmonised method was developed for the analysis of ochratoxin A in liquorice extracts. This method works without the use of halogenated solvents because, as the proficiency test showed, an aqueous extraction solution can be used instead of, for example, chloroform, whose use is restricted in the EU. The main objective of this method validation study was to check the performance of this harmonised method. To carry out the method validation study, a set of three different test samples (one liquorice powder and two liquorice pastes) and a liquorice powder sample with an indicated range of ochratoxin A (a so-called sunshine sample) was distributed to 21 laboratories in ten countries throughout Europe and to one laboratory in the USA. The study was evaluated according to internationally recognised guidelines. In terms of its repeatability and reproducibility for determining ochratoxin A in liquorice extracts with a relative standard deviation for repeatability (RSD_r) of between 6.68 and 19.95 and a relative standard deviation for reproducibility (RSD_R) of between 17.39 and 29.08 the performance of the harmonised method was found to be in the accepted range of the EU directive for the analysis of mycotoxins in several foodstuffs.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Mycotoxins as harmful indoor air contaminants

Fungal metabolites (mycotoxins) that pose a health hazard to humans and animals have long been known to be associated with mold-contaminated food and feed. In recent times, concerns have been raised about exposures to mycotoxin-producing fungi in indoor environments, e.g., damp homes and buildings. The principal mycotoxins that contaminate food and feed (alfatoxins, fumonisins, ochratoxin A, deoxynivalenol, zearalenone) are rarely if ever found in indoor environments, but their toxicological properties provide an insight into the difficulties of assessing the health effects of related mycotoxins produced by indoor molds. Although the Penicillium and Aspergillus genera of fungi are major contaminants of both food and feed products and damp buildings, the particular species and hence the array of mycotoxins are quite different in these environments. The mycotoxins of these indoor species and less common mycotoxins from Stachybotrys and Chaetomium fungi are discussed in terms of their health effects and the need for relevant biomarkers and long-term chronic exposure studies.     

Investigation of different yeast strains for the detoxification of ochratoxin A

High concentrations of ochratoxin A (OTA) in feed lead to growth depression in animals. It has been reported that binders can be used for deactivating aflatoxins but not for other mycotoxins without negatively influencing the animals health. In this study a strain from the genus ofTrichosporon with the ability to cleave ochratoxin A very selectively into phenylalanine and the non-toxic ochratoxin α (OTα) could be isolated. This strain was selected from a pool of OTA detoxifying microorganism by carrying out several investigations.Trichosporon sp. nov. can be fermented and stabilized. In a feeding trial with broilers lyophilizedTrichosporon-cells could compensate performance losses caused by OTA. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Mycotoxicoses of domestic animals and their diagnosis

Mycotoxins have been named "agents in search of a disease," and the considerable progress in analytical methodology over the last 10 years has not changed this very much. The following are factors that contribute to the difficulty of making a diagnosis: (1) nonspecificity of lesions; (2) masking of mycotoxic effects by secondary effects, e.g., through immunosuppression; (3) late appearance of a lesion, e.g., bone marrow damage or neoplasia; (4) interaction of several mycotoxins or presence of other toxicants or deficiency states; (5) species variation in the response to the mycotoxin(s); (6) difficulty of linking a late appearing effect with a demonstrable cause; (7) low doses of mycotoxins may cause stimulating effects; and (8) not being aware of the potential of a mycotoxin as a causative factor in disease. The mycotoxins of major importance in Canada are trichothecenes, ochratoxin, zearalenone, and ergot. It is concluded that the significance of mycotoxins for animals in Canada is likely generally underestimated.     

Fumonisin and ochratoxin production in industrial Aspergillus niger strains

Aspergillus niger is perhaps the most important fungus used in biotechnology, and is also one of the most commonly encountered fungi contaminating foods and feedstuffs, and occurring in soil and indoor environments. Many of its industrial applications have been given GRAS status (generally regarded as safe). However, A. niger has the potential to produce two groups of potentially carcinogenic mycotoxins: fumonisins and ochratoxins. In this study all available industrial and many non-industrial strains of A. niger (180 strains) as well as 228 strains from 17 related black Aspergillus species were examined for mycotoxin production. None of the related 17 species of black Aspergilli produced fumonisins. Fumonisins (B(2), B(4), and B(6)) were detected in 81% of A. niger, and ochratoxin A in 17%, while 10% of the strains produced both mycotoxins. Among the industrial strains the same ratios were 83%, 33% and 26% respectively. Some of the most frequently used strains in industry NRRL 337, 3112 and 3122 produced both toxins and several strains used for citric acid production were among the best producers of fumonisins in pure agar culture. Most strains used for other biotechnological processes also produced fumonisins. Strains optimized through random mutagenesis usually maintained their mycotoxin production capability. Toxigenic strains were also able to produce the toxins on media suggested for citric acid production with most of the toxins found in the biomass, thereby questioning the use of the remaining biomass as animal feed. In conclusion it is recommended to use strains of A. niger with inactive or inactivated gene clusters for fumonisins and ochratoxins, or to choose isolates for biotechnological uses in related non-toxigenic species such as A. tubingensis, A. brasiliensis, A vadensis or A. acidus, which neither produce fumonisins nor ochratoxins.     

The emerging <Emphasis Type="Italic">Fusarium</Emphasis> toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC₂₀ and IC₅₀ values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 μM and 4 μM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC₂₀ 0.7 μM, IC₅₀ of 0.8 μM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations.     

Einsatz einer kombinierten Immunoaffinitätssäule zum Nachweis von Aflatoxinen (B<Subscript>1</Subscript>, G<Subscript>1</Subscript>, B<Subscript>2</Subscript>, G<Subscript>2</Subscript>), Ochratoxin und Zearalenon

A method for the combined determination of the mycotoxins aflatoxin B₁, G₁, B₂, G₂, ochratoxin A and zearalenone in cereals and feed is described. After extraction with acetonitrile/water or methanol/water the cleaning takes place with new combined immunoaffinity clean-up column “AflaOchraZea” by VICAM. When the mycotoxins are determined in different cereals with this new type of clean-up column low detection limits and high recovery rates can be reached similar to those obtained by using separate immunoaffinity clean-up colums for the said mycotoxins.     

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.