Analysis of T-2 and HT-2 toxins in oats and other cereals by means of HPLC with fluorescence detection

Bearing in mind the high toxicity of T-2 and HT-2 toxins which occur in cereals (mainly in oats) EU plans legal limits for these mycotoxins. The occurrence data are insufficient because reliable and sensitive analysis methods are not available. A sensitive HPLC gradient method was developed which is applicable with common HPLC equipment (HPLC with fluorescence detection). After extraction of the toxins from sample matrix with methanol/water the diluted extracts were cleaned-up using immunoaffinity columns and then derivatized with 1-anthroylnitrile/DMAP. The T-2 and HT-2 toxins were separated from peaks of the cereal matrix and derivatization reagent by means of a relatively complex HPLC gradient method. The method was validated for oats, wheat, rye, barley, and maize. The recovery rates were in the range of 70–99%, the precision (RSD_R) of 3–8%. The limits of detection of T-2 and HT-2 toxins were 1 μg/kg. A total of 119 samples of cereals and cereal products was analyzed according to the optimized method. The analyses of 54 samples of dehulled oats and of 11 samples of processed oat products from food industry had a contamination frequency of 100%. The contents (sum of T-2 and HT-2 toxins) amounted to 3 to 174 μg/kg for the dehulled oats and to 4 to 48 μg/kg for the processed oat products. 29 samples of maize and maize products had a contamination frequency of 80% (2–106 μg/kg in the sum of T-2 and HT-2 toxins). In the samples of wheat and barley the toxins were detected only occasionally (contents: 1–10 μg/kg), in rye not at all.     

Vorkommen von Deoxynivalenol, Zearalenon und HT-2/T-2 Toxin in Getreide und Getreideerzeugnissen

Since February 2004 in Germany maximum limits forFusarium toxins do exist, while harmonised legislation within the EU was recently published and will come into force in July 2006. Meanwhile, the problematic nature ofFusarium mycotoxins is perceived by all participants of the processing chain of cereals. In this study the presence of deoxynivalenol and zearalenone in comparison to the rarely investigated type A-trichothecenes (HT-2, T-2 toxin) in different cereal-products is discussed. About 1000 cereal-based samples have been analysed using a recently developed multitoxin method based on HPLC-MS/MS technique. Despite, up to now no concrete limit for HT-2/T-2 toxin is discussed, the degree of contamination is of special concern for food products dedicated to be placed on the market, to avoid possible risks for consumers. The used method proved to be extremely sensitive for T-2 toxin with a LOD below 1 μg/kg, therefore a comprehensive data set was achieved.     

Evaluation of mycotoxins,mycobiota, and toxigenic fungi in selected medicinal plants of Khyber Pakhtunkhwa,Pakistan

Medicinal plants are used worldwide to treat a variety of ailments. Due to the provenance of medicinal plants, they are subjected to contamination by moulds, which may be responsible for spoilage and production of mycotoxins. The investigation was designed to throw light on mycological and mycotoxicological status of some medicinal plants from Pakistan and the result showed 30 % and 26.7 % samples were contaminated with aflatoxins and ochratoxin A, respectively. Mould contamination was present in 90 % samples, of which 70 % exceeded the permissible limits. Opium poppy, licorice root, and Indian rennet were most contaminated samples. The predominant moulds found were Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, and Penicillium spp. and 31 % of the 47 isolates tested were found to be toxigenic. The findings indicate that the contamination in the medicinal plants may contribute to adverse human health problems. This information would prove helpful for regulatory agencies to establish limits for these contaminants in medicinal plants and will explore ways for export of herbal products to countries where more stringent permissible limits of mycotoxins exist. The study is first of its kind in the country reporting natural occurrence of mycotoxins in medicinal plants in Pakistan.     

Binding rather than metabolism may explain the interaction of two food-Grade Lactobacillus strains with zearalenone and its derivative (')alpha-earalenol

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative (')alpha-zearalenol ((')alpha-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 microg ml(-1)) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and (')alpha-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or (')alpha-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with (')alpha-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.     

Fusarium mycotoxins: a review of global implications for animal health, welfare and productivity

Trichothecenes, zearalenone (ZEN) and fumonisins are the major Fusarium mycotoxins occurring on a worldwide basis in cereal grains, animal feeds and forages. Other important Fusarium mycotoxins include moniliformin and fusaric acid. Spontaneous outbreaks of Fusarium mycotoxicoses have been recorded in Europe, Asia, New Zealand and South America and, in addition, chronic exposure occurs on a regular and more widespread scale. The metabolism and adverse effects of the Fusarium mycotoxins are considered in this review with particular reference to recent data on specific and proposed syndromes and to interactions among co-occurring mycotoxins. Within the trichothecene group, deoxynivalenol (DON) is associated with emesis, feed refusal and depressed feed intake in pigs, while T-2 toxin and diacetoxyscirpenol (DAS) are now clearly linked with oral lesions in poultry. The gut microflora of farm livestock are able to transform DON to a de-epoxy derivative. In contrast, the ovine metabolism of ZEN results in the production of five metabolites and relatively high levels of these forms may be excreted in the urine as glucuronides. There is now undisputed evidence that ZEN and its metabolites possess estrogenic activity in pigs, cattle and sheep, but T-2 toxin has also been implicated in reproductive disorders in farm livestock. Fumonisins are positively linked with pulmonary edema in pigs, leukoencephalomalacia in equines and with deranged sphingolipid metabolism in these animals. Fusarium mycotoxins have also been provisionally implicated in ovine ill-thrift, acute mortality of poultry and in duodenitis/proximal jejunitis of horses. Several Fusarium mycotoxins may co-occur in a particular feed ingredient or in compound feedingstuffs. In general, combinations of Fusarium mycotoxins result in additive effects, but synergistic and/or potentiating interactions have been observed and are of greater concern in livestock health and productivity. Synergistic effects have been reported between DON and fusaric acid; DON and fumonisin B₁ (FB₁); and DAS and the Aspergillus-derived aflatoxins. Limited evidence of potentiation between FB₁ and DON or T-2 toxin has also emerged recently. Additive and synergistic effects between known and unidentified mycotoxins may account for enhanced adverse effects observed on feeding Fusarium-contaminated diets. The potential for transmission of DON into eggs and of ZEN into porcine kidney and liver has been demonstrated. However, lactational carry-over of FB₁ appears not to occur, at least in cows and sows. It is concluded that livestock health, welfare and productivity may be severely compromised by consumption of DON, T-2 toxin, DAS, ZEN and fumonisins and by interactions among these mycotoxins. Safety of some animal products may also be at risk. Furthermore, in view of the limited options available for remediation, it is concluded that exploitation of crops resistant to Fusarium infection offers the most viable strategy for reducing mycotoxin contamination of grain and animal feed.     

Isolation of deoxynivalenol-transforming bacteria from the chicken intestines using the approach of PCR-DGGE guided microbial selection

Background  

Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON.     

Deoxynivalenol in Lebensmitteln

Within a joint research project entitled “Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer”, supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.     

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A,and their combination in cultured Vero cells

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food‐borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl‐2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose‐dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl‐2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:42–50, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20310     

Glucosylation and Other Biotransformations of T-2 Toxin by Yeasts of the Trichomonascus Clade

Trichothecenes are sesquiterpenoid toxins produced by Fusarium species. Since these mycotoxins are very stable, there is interest in microbial transformations that can remove toxins from contaminated grain or cereal products. Twenty-three yeast species assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including four Trichomonascus species and 19 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert the trichothecene T-2 toxin to less-toxic products. These species gave three types of biotransformations: acetylation to 3-acetyl T-2 toxin, glycosylation to T-2 toxin 3-glucoside, and removal of the isovaleryl group to form neosolaniol. Some species gave more than one type of biotransformation. Three Blastobotrys species converted T-2 toxin into T-2 toxin 3-glucoside, a compound that has been identified as a masked mycotoxin in Fusarium-infected grain. This is the first report of a microbial whole-cell method for producing trichothecene glycosides, and the potential large-scale availability of T-2 toxin 3-glucoside will facilitate toxicity testing and development of methods for detection of this compound in agricultural and other products.     

Biosynthesis of sphinganine-analog mycotoxins

Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.     

The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts

The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth. When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls. However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture. As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied.     

Mycotoxins in indoor environments

Exposure to mycotoxins produced by toxigenic molds growing in damp indoor spaces has been difficult to assess. Monitoring methods limit the characterization of inhalation exposure of any bioaerosol, especially that of mycotoxins. Biomarkers promise better ability to determine mycotoxin exposures 1.) through direct measures of toxins and their products in human tissues, 2.) through immunochemical methods, and 3.) measures of effect through novel approaches,e.g., proteomics or genomics. This paper summarizes both the problems inherent in measuring exposures and some of the promising methods that could help to resolve the current impasse. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005.     

Methods of detecting and determining nitrogen-containing mycotoxins]

The influence of various nitrogen functional groups used for extraction cleanup and determination of N-containing mycotoxins (NM) in feeds and foodstuffs have been considered. TLC and LC are the most common techniques for detection and determination of nitrogen-containing mycotoxins. Gas chromatography has been used for determination (with or without derivatization) of several nitrogen-containing mycotoxins and/or their degradation products. Immunochemical techniques, in particular ELISA are available for only a very limited number of NM (e.g. ochratoxin A). Numerous methods for determination of ochratoxin A in feeds, grains, animal products and other foodstuffs have been developed. Methods for which recoveries have been carried out on spiked samples are also available for several other NM.     

Mycotoxins and food contamination

The contamination of food and feed by fungi and their toxins (mycotoxins) has to be considered as a serious hazard in the daily live. Mycotoxins are natural contaminants in foods and could induce several syndromes. Many mycotoxicosis are described. The control and surveillance of mycotoxins involve the carrying out of surveys, toxicological studies and institution of regulations.     

Mycotoxicoses of animals

Mycotoxicoses are intoxications caused by ingestion of foodstuffs contaminated with mycotoxins, i.e. toxic secondary metabolites of microscopic filamentous fungi (moulds). By field observations and by experimental testing, toxins or toxic strains of more than 100 species of fungi have been encountered. However, causal associations have so far only been established for a small number of mycotoxicoses in farm animals, and the more important mycotoxicoses are aflatoxicosis, facial eczema, mycotoxic nephropathy, and estrogenic syndrome. A full assessment of the impact of mycotoxins on the health of farm animals can hardly be made at present, because the amount of surveillance data from mycotoxicoses is very limited, due mainly to inadequate diagnostic criteria. A new set of criteria for the diagnosis of mycotoxicosis is proposed, which has been successfully applied in the causative study of mycotoxic porcine nephropathy.     

Natural occurrence of alternariol and alternariol monomethyl ether in soya beans

The natural occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in soya beans harvested in Argentina was evaluated. Both toxins were simultaneously detected by using HPLC analysis coupled with a solid phase extraction column clean-up. Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soya bean samples. Out of 50 soya bean samples, 60% showed contamination with the mycotoxins analyzed; among them, 16% were only contaminated with AOH and 14% just with AME. Fifteen of the positive samples showed co-occurrence of both mycotoxins analyzed. AOH was detected in concentrations ranging from 25 to 211?ng/g, whereas AME was found in concentrations ranging from 62 to 1,153?ng/g. Although a limited number of samples were evaluated, this is the first report on the natural occurrence of Alternaria toxins in soya beans and is relevant from the point of view of animal public health.     

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.     

Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ¯α-zearalenol (¯α-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml⁻¹) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ¯α-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ¯α-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ¯α-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.     

Development,validation and application of a multi-mycotoxin method for the analysis of whole wheat plants

Mycotoxins are known to affect the health of humans and husbandry animals. In contrast to wheat grains used for food and feed, whole wheat plants are rarely analysed for mycotoxins, although contaminated straw could additionally expose animals to these toxic compounds. Since the entire wheat plant may also act as source of mycotoxins emitted into the environment, an analytical method was developed, optimised and validated for the analysis of 28 different mycotoxins in above-ground material from whole wheat plants. The method comprises solid-liquid extraction and a clean-up step using a Varian Bond Elut Mycotoxin^® cartridge, followed by liquid chromatography with electrospray ionisation and triple quadrupole mass spectrometry. Total method recoveries for 26 out of 28 compounds were between 69 and 122% and showed limits of detection from 1 to 26 ng/g_{dry weight (dw)}. The overall repeatability for all validated compounds was on average 7%, and their mean ion suppression 65%. Those rather high matrix effects made it necessary to use matrix-matched calibrations to quantify mycotoxins within whole wheat plants. The applicability of this method is illustrated with data from a winter wheat test field to examine the risks of environmental contamination by toxins following artificial inoculation separately with four different Fusarium species. The selected data originate from samples of a part of the field which was inoculated with Fusarium crookwellense. In the wheat samples, various trichothecenes (3-acetyl-deoxynivalenol, deoxynivalenol, diacetoxyscirpenol, fusarenone-X, nivalenol, HT-2 toxin, and T-2 toxin) as well as beauvericin and zearalenone were identified with concentrations ranging from 32 ng/g_dw to 12 × 10³ ng/g_dw.     

Stability of Aflatoxin B1 and Ochratoxin A in Brewing

The stability of aflatoxin B₁ and ochratoxin A in brewing was investigated by adding the purified toxins to the raw materials at 1 and 10 μg/g levels during mashing in a conventional micro-brewing process. The results indicate that both toxins are stable to heat and are insensitive to cooker mash treatment. Both mycotoxins were partially removed in the mashing and brewing processes. About 14 to 18% and 27 to 28% of the added toxins were found in the final beers brewed from starting materials containing 1 and 10 μg, respectively, of either toxin per g. The possible route of transmission of mycotoxins into beer is discussed.