Stability of the Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in ground maize under typical cooking environments

The effects of moisture, pH and heat on the stability of nivalenol (NIV), deoxynivalenol (DON) and zearalenone (ZEN) present as natural contaminants of ground maize were measured for different periods. Standard solution tests were also performed to measure pH, salt and temperature effects on NIV and DON. The solution tests showed NIV and DON to be relatively stable in buffer solutions over the pH range 1-10. Quite harsh conditions (pH 12, high salt concentration, 80°C, prolonged exposure) were needed to give substantial breakdown. In the ground maize substrate, these toxins were further stabilized relative to the solution tests. NIV and DON were both reduced (range 60-100%) by treatment with aqueous bicarbonate solution at 10, 20 or 50% of the ground maize dry weight, and subsequent heating at 80 or 110°C for 2 and 12 days. There was no measurable reduction at lower test temperatures (20, 40°C). NIV (but not DON) also showed some reduction following addition of water and heating at 80 or 110°C for 12 days. ZEN content was not reduced even by 12 days of heating at 110°C after treatment with a sodium bicarbonate solution.     

Occurrence of deoxynivalenol and T-2 toxin in bread and pasta commercialised in Spain

Deoxynivalenol and T-2 toxin were extracted from wheat-based bread (n = 75) and pasta (n = 75) samples using a mixture of acetonitrile:water (86:14 v/v); for analysis, gas chromatography/mass spectrometry after derivatisation with trifluoroacetic anhydride was utilised. The recovery of deoxynivalenol and T-2 toxin from both food matrixes ranged from 90.1 to 94.0%. The occurrence of these mycotoxins in bread was 28.0% and 2.6% for deoxynivalenol and T-2 toxin, respectively, whereas in pasta, the occurrence of both mycotoxins was higher, varying from 9.3 to 62.7%. The mean content of deoxynivalenol (42.5 μg/kg) in bread was lower than the content of T-2 toxin (68.37 μg/kg), while in pasta the content of deoxynivalenol (137.1 μg/kg) was superior. The estimated daily intake of deoxynivalenol and T-2 toxin from the consumption of these products represents 8.4% and 0.2% of the tolerable daily intake, respectively. These results back up the necessity to take a vigilant attitude in order to prevent human intake of trichothecenes. This information is necessary and of high priority in order to protect the consumer’s health from the risk of exposure to these toxins.     

Fate of deoxynivalenol and nivalenol during storage of organic whole-grain wheat flour

This study was carried out to determine the level of retention of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) during 120 days of storage (aging) of flours produced from organic wheat grain naturally infected with Fusarium fungi. Three types of flour (standard white flour prepared by a roller-grinder mill - IRG, whole-grain flour produced by a hammer-crusher mill - IHC and whole-grain flour prepared by a millstone - OMS) were packaged in food-grade paper or polypropylene plastic bags and stored at two different storage temperatures (constant 10 °C or 25 °C). The concentrations of DON and NIV were measured prior to and after storage by means of HPLC-UV detection methods. After 120 days of storage, the concentrations of DON and NIV decreased between 0% and 29% compared to the initial measurements, depending on the combination of experimental factors. The greatest decrease in mycotoxin concentration was observed in the IHC and OMS flours packaged in paper bags and stored at 25 °C. The smallest decrease in mycotoxin concentration was observed in the IRG flours packaged in sealed plastic bags and stored at 10 °C. Statistical analysis showed that the level of retention of DON and NIV depended significantly on the type of packaging material, but did not depend on the type of flour or the storage temperature.     

Development and application of a method for the analysis of two trichothecenes: deoxynivalenol and T-2 toxin in meat in China by HPLC-MS/MS

A reliable and sensitive method was developed and successfully applied for the determination of deoxynivalenol and T-2 toxin simultaneously in pig dorsal muscle, pig back fat and chicken muscle by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis. Limit of detection of deoxynivalenol and T-2 was 0.02 μg/kg and 0.007 μg/kg, and limit of quantification of deoxynivalenol and T-2 was 0.07 μg/kg and 0.02 μg/kg, respectively. Sixty-six meat samples were analyzed and deoxynivalenol was detected in the samples of pig back fat, with concentrations lower than 0.5 μg/kg, and T-2 toxin was detected in the samples of pig dorsal muscle, pig back fat and chicken muscle, with concentrations lower than 0.5 μg/kg. The results of sample analysis show that only trace residues of deoxynivalenol and T-2 toxin were detected in the samples analyzed.     

Thermal stability of T-2 and HT-2 toxins during biscuit- and crunchy muesli-making and roasting

ABSTRACT

The mycotoxins T-2 and HT-2 toxin are frequently occurring food contaminants which are produced by Fusarium species. Humans and animals are mainly exposed to these substances by the consumption of contaminated oats, maize and wheat. For the production of crunchy muesli, bread and bakery products, these cereals undergo multiple processing steps, including baking, roasting and extrusion cooking. However, the influence of food processing on T-2 and HT-2 toxin levels is to date poorly understood. Thus, the effects of baking and roasting on both mycotoxins were evaluated during biscuit-, crunchy muesli- and toasted oat flakes-production under precise variation of various parameters: heating time and temperature as well as recipe formulation were varied in the range they are applied in the food processing industry. Therefore, oatmeal or flaked oats were artificially contaminated individually with both toxins and processed at the laboratory scale. T-2 toxin generally showed a higher degradation rate than HT-2 toxin. During biscuit-making up to 45% of T-2 toxin and 20% of HT-2 toxin were thermally degraded, showing a dependency on water content, baking time and temperature. The preparation of crunchy muesli yielded no significant toxin degradation which is probably due to the low temperatures applied. Roasting led to a degradation of 32% of T-2 toxin and 24% of HT-2 toxin. Taken together, both mycotoxins are partially degraded during thermal food processing; the degradation rates are influenced by the food composition and processing parameters.     

The effects of T-2 toxin exposure on liver drug metabolizing enzymes in rabbit

High doses of T-2 toxin are known to decrease protein synthesis and mono-oxygenase activities in rat liver. The purpose of this study was to investigate whether exposure at a low dose could alter the normal metabolism of the xenobiotic by the liver. Three doses of T-2 toxin, dissolved in olive oil, were orally and daily administered to New Zealand white rabbits for five days. At 0.5mg/kg, three of the five animals died, whereas only a weak decrease in body weight gain and moderate signs of toxicity occurred in rabbits receiving 0.25mg/kg/day, and the body weight increased without signs of toxicity at 0.1mg/kg/day. At 0.25mg/kg/day, total liver microsomal P450 content, and the activities of aminopyrine and benzphetamine N-demethylases, pentoxyresorufin O-depentylase, glutathione S-transferases accepting 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, were decreased. By contrast, ethylmorphine and erythromycin N-demethylases, ethoxyresorufin and methoxyresorufin O-dealkylases, aniline hydroxylase, and UDP-glucuronyltransferase accepting p-nitrophenol as substrate, were unaffected. The expression of P450 1A1, 1A2, 2A1, and 2B4, but not P450 2C3 and 3A6, were also decreased, whereas microsomal conjugated dienes, fluorescent substances, and malondialdehyde contents were increased. At 0.1mg/kg/day, neither significant effects on drug metabolizing enzymes nor microsomal oxidative damages were obtained. Taken together, these results suggest that a short exposure time to the mycotoxin would not be associated with significant changes in the normal metabolism of xenobiotics by the liver.     

T-2毒素的高灵敏时间分辨荧光免疫分析

以时间分辨荧光免疫分析(TRFIA)方法建立快速灵敏的T-2毒素的全自动检测方法. 采用T-2-BSA包被96孔板作为固相抗原,与游离的T-2竞争有限的抗T-2单克隆抗体,以Eu3+标记的羊抗鼠抗体示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立T-2-TRFIA.同时对这一方法的稳定性、灵敏度、回收率和特异性进行考核.此方法灵敏度为0.2 μg/L,测量范围0.2～500 μg/L,批内变异为7.5%,批间变异为12.7%,平均回收率为89.6%.与赭曲霉毒素A、黄曲霉毒素B1、牛血清白蛋白无交叉反应.10条不同时间进行的间接竞争T-2-TRFIA的效应点均值ED80、ED50和ED20分别为2.66,6.97,29.11 μg/L.同时,用TRFIA和ELISA试剂盒同时检测T-2毒素,在ELISA和TRFIA产品的共同可测范围之内,两者的相关系数为0.926.     

Fate of deoxynivalenol,T-2 and HT-2 toxins and their glucoside conjugates from flour to bread: an investigation by high-performance liquid chromatography high-resolution mass spectrometry

Deoxynivalenol, T-2 and HT-2 toxins are mycotoxins frequently occurring in cereals and cereal-based products along with their conjugated forms. In this paper, we provide insights into the fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside derivatives during bread making, using naturally contaminated wheat flour. High-resolution mass spectrometry was used to assess the extent of degradation of the three mycotoxins during bread baking and to identify some glucoside conjugates, namely deoxynivalenol, T-2 and HT-2 mono-glucosides, detected both in the flour and in the respective breads. Our findings show deoxynivalenol's levels markedly increased upon baking, whereas those of HT-2 and T-2 toxins were decreased in the final bread with special regard to the T-2 toxin.     

Natural co-occurrence of fumonisins, deoxynivalenol, zearalenone and aflatoxins in field trial corn in Argentina

Corn samples collected from the main production area in Argentina in 1995 were surveyed for the natural occurrence of Fusarium mycotoxins and aflatoxins. Fumonisins B₁, B₂ and B₃ and zearalenone were found in all samples. A positive relationship was found between fumonisins B₁, B₂ and B₃, B₁ and B₃, and B₂ and B . Deoxynivalenol and aflatoxins were not de3 tected. Mycological survey has also revealed the predominance of Fusarium moniliforme . This is the first report on the simultaneous occurrence of fumonisins and zearalenone in corn from the main production area in Argentina.     

Survey of the presence of the Fusarium mycotoxins nivalenol, deoxynivalenol and T-2 toxin in Korean cereals of the 1989 harvest.

Twenty eight samples of rice, barley, millet, corn and Indian millet harvested in Korea in 1989 were subjected to assay for contamination of nivalenol (NIV), deoxynivalenol (DON) and T-2 toxin by using gas chromatography and gas chromatography-mass spectrometry. Seven samples were found to be positive for NIV and DON in the ranges of 189-624 micrograms/kg and 168-506 micrograms/kg, respectively. Of the contaminated samples, three samples, one barley, one Indian millet and one corn sample were contaminated simultaneously with both NIV and DON. T-2 toxin was not detected in any samples.     

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a_w at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED₅₀ and ED₉₀ values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a_w and the influence of temperature on growth rates was 25 > 20 > 15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED₅₀ and ED₉₀ values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a_w, type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a_w T-2 levels increased in cultures containing fenpropimorph while at 0.96 a_w the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a_w and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a_w. This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.     

Fate of the fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone, during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

Temperature and water activity effects on production of T-2 and HT-2 by Fusarium langsethiae strains from north European countries

This study has examined the effect of ecophysiological factors, water activity (a_w, 0.995-0.90) and temperature (10-37 °C), on the T-2 and HT-2 toxins production by Fusarium langsethiae. Two dimensional profiles for optimum and marginal conditions have been built for two strains from each of four northern European countries (UK, Norway, Sweden, Finland) on an oat-based medium. This showed that the optimum a_w and temperature conditions for T-2 + HT-2 production was between 0.98-0.995, and 20-30 °C respectively. Kruskal-Wallis analysis of ranks showed a statistically significant differences between the different a_w levels examined (P < 0.001) but no significant effect of the temperatures examined. The ratio of HT-2/T-2 was investigated and non-uniform distribution of HT-2 toxin was found under different ecological conditions. No statistically significant differences were found for the mean toxin production between strains from the different countries. Intra-strain differences in toxin production was only found for those from Finland (P-value = 0.0247). The growth/no growth and toxin/no toxin conditions in relation to a_w x temperature have been constructed for the first time. This knowledge will be useful in developing prevention strategies to minimise T-2 and HT-2 toxin contamination by strains of F. langsethiae on important small grain cereals.     

Validation of a gas chromatography-electron capture detection of T-2 and HT-2 toxins in Chinese herbal medicines and related products after immunoaffinity column clean-up and pre-column derivatization

A sensitive, reproducible and accurate gas chromatography-electron capture detection (GC-ECD) method was developed for simultaneous determination of T-2 and HT-2 toxins in Chinese herbal medicines (CHMs) and related products after immunoaffinity column (IAC) clean-up and pre-column derivatization with N-heptafluoro-butyryl imidazole (HFBI). Then, gas chromatography-spectrometry spectrometer (GC-MS) was applied to confirm the positive results and interfering peaks. The limits of detection (LODs) for T-2 and HT-2 toxins were 1.88 and 0.47 ng/g, and the recoveries for different CHMs ranged from 89.2% to 99.1% with relative standard deviation (RSD) <6.0% for T-2 and from 85.9% to 99.0% with RSD <8.8% for HT-2 toxin, respectively. The validated method was successfully applied for the determination of T-2 and HT-2 toxins in 89 Chinese herbal medicines and 10 related products from various sources, where it was found that T-2 and HT-2 toxins were not detected in any of the tested samples. These results were reliable by confirmation using GC-MS. Some unknown peaks were interfering peaks not the target toxins.     

Development of a sensitive monoclonal-based enzyme-linked immunosorbent assay for monitoring T-2 toxin in food and feed

The consumption of food or feed contaminated with high levels of T-2 toxin may cause adverse health effects in humans and other animals. In this study, to monitor T-2 toxin rapidly in food and feed, a sensitive and specific monoclonal antibody (mAb) against T-2 toxin was generated and a simple and rapid indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) developed. T-2 toxin was first converted to T-2-hemisuccinate (T-2HS) and T-2-hemiglutarate (T-2HG), which were then conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare an immunogen and coating antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, one cell line, 4D8, with the IgG1 isotype was obtained. The 4D8 antibody exhibited the ability specifically to recognise T-2 toxin with IC₅₀ 1.46 µg l^?1. Based on this 4D8 mAb, an optimised ic-ELISA protocol was developed using only methanol–water (7:3, v/v) in feed and cereal samples and ethyl acetate in muscle samples. The limits of detection of T-2 toxin in various sample matrices varied from 0.07 to 15.8 µg kg^?1; the recoveries ranged from 50.3% to 113.6%; and the CVs were less than 19.0%. These results suggest that the prepared mAb and the developed ic-ELISA method will be a useful tool for detecting T-2 toxin in foods and feeds.     

Fusarium langsethiae: Storage environment influences dry matter losses and T2 and HT-2 toxin contamination of oats

The aim of this study was to determine the environmental conditions over which Fusarium langsethiae species can (a) grow, (b) produce T-2 and HT-2 toxins in oats, and (c) to determine the relationship between dry matter losses (DMLs) and mycotoxin accumulation in stored oats for the first time. Oats were stored under different combinations of water activity (0.89–0.97 a_w) and temperature (15–30 °C) and the amount of CO₂ produced was measured on a daily basis by gas chromatography (GC). These data were used to quantify the effect of storage on dry matter losses. Results showed that the optimum conditions for colonisation of oats and T-2 and HT-2 toxin production by F. langsethiae were at 0.97 a_w and 25 °C. T-2 and HT-2 contamination exceeded 4 mg/kg and 0.75 mg/kg respectively after 10 days storage at these conditions and the calculated DMLs were also higher, exceeding 2%. At intermediate (0.945) and suboptimal (0.89) a_w levels for growth of F. langsethiae, DMLs were higher at 30 °C than 25 °C. Models were developed to relate quality losses and toxin production to the different storage conditions. This allows the identification of high and low risk conditions for storage of oats. A good positive correlation was obtained between combined T-2 and HT-2 toxin production and DMLs. This suggests that in situ measurements of CO₂ production during storage may be useful in predicting toxin accumulation in stored oats.     

对虾中T-2毒素对小鼠免疫功能和血清生化指标的影响

研究了对虾中T-2毒素对小鼠免疫学和血清学方面的毒性效应。分别用低、中、高剂量T-2毒素注射染毒对虾的不同组织灌胃小鼠1周,检测小鼠免疫功能指标(免疫器官指数、淋巴细胞转化能力、碳廓清能力、腹腔巨噬细胞吞噬能力)及血清生化指标的变化,进行概率统计和多维尺度分析。结果表明,与对照组相比,随着染毒剂量的递增,出现显著性差异的概率增大。低剂量组间作用点距离短,为一类聚点,而中、高剂量组间的作用点相对距离较远。其中以高剂量肌肉组对免疫功能的影响最大,以巨噬细胞的吞噬功能指标变化最为明显。血清指标中,中、高剂量组血尿素氮含量显著下降(P0.05),说明对虾中T-2毒素主要降低小鼠的免疫功能,同时对肾脏产生一定毒性效应。随着染毒剂量的递增,不同组织中T-2毒素的存在形式和毒性效应有差异,以肌肉中的T-2毒素毒性最强。     

Occurrence of T-2 toxin in processed cereals and pulses in Turkey determined by HPLC and TLC

The purpose of this study was to investigate the T-2 toxin level of contaminated cereal and pulse products in Turkey. T-2 toxin was detected using high performance liquid chromatography (HPLC) with UV detection at 208 nm and thin layer chromatography (TLC) was used for confirmation of the T-2 toxin-contaminated samples (> or = 1 ppm). The recovery was 93 +/- 3.3% (SD 3.29, n = 5) for chickpea spiked with a known amount of T-2 toxin (1 ppm). The detection limits for T-2 toxin for HPLC and TLC were 25 ng per injection and 50 ng per spot, respectively. A total of 50 commercially available cereal and pulse product samples, collected from markets and street bazaars, were analysed. Incidences of T-2 toxin detected in cereal and pulse products were 23.5% and 31.2%, respectively and the maximum detected amount was 1.9 ppm in a sample of dried beans. The incidence of toxin-contaminated specimens is not so low relative to the volume of specimens produced.     

Natural co-occurrence of deoxynivalenol and fumonisins B1 and B2 in Italian marketed foodstuffs

A survey was performed to obtain the frequency and levels of contamination by deoxynivalenol (DON) and fumonisins B₁ and B₂ (FB1, FB2) mycotoxins in Italian marketed foods. Of 202 samples investigated, including raw materials and processed cereal foods (bread, pasta, breakfast cereals, biscuits, baby and infant foods), 84% were contaminated with DON at levels from 0.007 to 0.930 μg g^-1 (median 0.065 μg g^-1); 26% contained FB1 ranging from 0.010 to 2.870 μg g^-1 (0.070 μg g^-1); 35% contained FB2 at 0.010-0.790 μg g^-1 (0.080 μg g^-1). The highest levels of DON and FB1 were detected in raw cereals and wholemeal flours. The highest levels of FB2 were detected in durum wheat pasta. A widespread DON contamination was found in baby and infant foods at levels varying from 0.007 to 0.166 μg g^-1.     

Development and in-house validation of an LC-MS/MS method for the quantification of the mycotoxins deoxynivalenol,zearalenone, T-2 and HT-2 toxin,ochratoxin A and fumonisin B1 and B2 in vegetable animal feed

Animal feed can be contaminated with various mycotoxins. To ensure animal health and safe food and feed production, the European Commission has recommended increased monitoring of the co-occurrence of deoxynivalenol, zearalenone, ochratoxin A, fumonisin B₁ and B₂, T-2 and HT-2 toxin in feed. Thus, there is a need for an analytical method that enables their simultaneous detection and quantification. This paper describes the development and in-house validation of such a method, in which the mycotoxins were extracted from spiked and naturally contaminated cereal-based compound feed, corn and wheat. The extracts were divided into two aliquots where one was diluted and then analysed directly and the other was cleaned by using MultiSep^®226 and then diluted and analysed. Separation and detection was achieved with LC-ESI-MS/MS by using a triple quadrupole instrument in the SRM mode. The precision (in terms of intra-day repeatability and inter-day reproducibility), accuracy, linearity, apparent recovery and expanded measurement uncertainty in feed, corn and wheat were evaluated. The LODs ranged from 1.0 to 72?μg/kg, and the LOQs ranged from 2.5 to 115?μg/kg. The apparent recovery was higher than 86% for all the mycotoxins, and the precision was better than that defined by the Horwitz equation for all concentrations. Proficiency test materials were analysed to assess the accuracy of the method, and the results were satisfactory for all seven mycotoxins. The method will be used to monitor the occurrence of these mycotoxins in products intended for animal feeding in Sweden.