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.     

食品添加剂对脱氧雪腐镰刀菌烯醇和T-2毒素 体外去除作用研究

目的为了探索食品中单端孢霉烯族毒素残留的控制方法,研究了多种食品添加剂对脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和T-2毒素的体外去除作用。方法通过食品添加剂处理DON和T-2毒素,研究它们对DON和T-2毒素的体外去除作用。结果去除效果最显著是磷酸三钠、焦磷酸钠、碳酸钠,对DON的去除率分别为76.60%±3.70%、33.30%±3.10%、45.90%±3.30%,对T-2毒素的去除率分别为99.98%±0.01%、95.20%±2.10%、99.98%±0.01%,处理后毒素溶液中不含有已知产物,可能将DON、T-2毒素转化为新的未知产物。结论多种受试食品添加剂对DON、T-2毒素具有不同程度的去除作用。     

青海省兴海县大骨节病病户主食中T-2毒素的检测

为了解大骨节病重病区唐乃亥乡上鹿圈村、下鹿圈村病户主食中T 2毒素的污染状况 ,在两村分别选择病户 19名 ,每户收集小麦及面粉样品各 1份 ,用ELISA法检测样品中T 2毒素的含量。上鹿圈村面粉、小麦中T 2毒素阳性率均为 89 5 % (17 19) ,平均含量分别为 70 7μg kg(8～2 0 1μg kg)、2 37 1μg kg(5～ 5 43μg kg) ;下鹿圈村面粉、小麦中T 2毒素阳性率均为 6 8 4 % (13 19) ,平均含量分别为 13 0 μg kg(9～ 197μg kg)、4 0 0 μg kg(19～ 333μg kg)。其中 2个采样点T 2毒素的阳性率和含量差异均存在显著性 ,2种样品T 2毒素的含量差异也存在显著性 (P <0 0 5 )。小麦中T 2毒素的污染情况比面粉严重 ,上鹿圈村主食中T 2毒素的污染情况比下鹿圈村严重 ,具体原因有待进一步研究。     

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毒素的产生、毒性及脱毒研究进展

T-2毒素是由多种镰刀菌代谢产生的一种单端孢霉烯族类毒素,广泛存在于田间作物及贮藏加工过程的谷物中,对经济造成了很大程度的损失。产毒菌株的菌种及侵染时间、外界环境、宿主均可影响T-2毒素的产生。该毒素是倍半萜类化合物,环氧环、C9-C10间双键、羟基、乙酰氧基为其毒性官能团,其毒性主要表现为细胞毒性和免疫系统毒性,具有致畸、致癌、致突变的"三致"作用,对人畜健康具有潜在的致癌威胁。目前脱除方法有物理法、化学法及生物法,生物法因其独特优势成为当前研究热点,该方法包括源头的预防控制以及侵染后毒素本身的降解,前者主要通过对前期菌株侵染、生长与产毒之间关系的研究来实现脱除;后者主要是利用自然界中动植物天然提取物或某些微生物菌体进行直接降解。该文重点阐述脱毒方法的最新研究进展,旨在为T-2毒素的脱除研究提供理论依据。     

T-2毒素检测方法研究进展

T-2毒素主要是由镰刀菌属霉菌（Fusarium）产生的一类单端孢霉烯族类（Trichothecenes，Ts）真菌毒素，具有毒性强、污染范围广的特点，常见于受污染的小麦、玉米、大麦、燕麦等粮谷及其制品和相关动物饲料中，对粮食安全及人体健康构成了极大的威胁，已逐渐成为粮食行业和畜牧行业监测和检测关注的重点。欧盟已针对谷物原料及其加工产品分别制定了相应的限量标准，我国虽然还没有制定相应的限量标准，但于2016年发布了T-2毒素食品安全国家标准《食品中T-2毒素的测定》。本文简要介绍了T-2毒素危害及其限量标准，重点就近年来色谱法、免疫化学检测技术、适配体生物传感器检测技术等方法在T-2毒素检测方面的应用进行了综述，并对未来发展方向进行了展望，以期为粮谷产品T-2毒素污染防控和质量监测提供借鉴。     

低剂量T-2/HT-2毒素生物转化产物的遗传毒性研究

本文为了研究低剂量T-2和HT-2毒素经弯曲假单胞菌(Pseudomonas geniculata)生物转化至无游离态毒素检出时,降解产物是否具有遗传毒性。通过连续7 d给小鼠灌胃不同剂量T-2/HT-2毒素降解产物,检测小鼠骨髓嗜多染红细胞微核率和精子致畸率。结果表明雄性小鼠产生的微核率均高于雌性小鼠,各剂量组的微核率与阴性组相比差异显著(P0.05),10/20 ng/m L和25/50 ng/m L的雄性小鼠微核率与阳性组相比差异不显著(P0.05)。2.5/5 ng/m L T-2/HT-2组的小鼠精子畸形与阴性组相比差异不显著(P0.05),畸形精子主要表现为胖头和折尾,随着T-2、HT-2剂量的增加,小鼠精子畸形率呈线性增加。说明经过弯曲假单胞菌(Pseudomonas geniculata)转化的T-2和HT-2毒素仍具有遗传毒性,而且雄性比雌性易感性更强,同时为T-2和HT-2毒素生物转化物中存在结合态的T-2毒素提供佐证。     

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.     

T-2 and HT-2 toxins in cereals and cereal-based products in South Korea

A total of 214 samples, consisting of brown rice, barley, mixed grains, corn, wheat and wheat flour were analysed for T-2 and HT-2 toxins using high-performance liquid chromatography with fluorescence detection. Recovery and repeatability were 79.9%–107.5% and 4.9%–14.5% for T-2, and 74.0%–106.1% and 5.0%–17.9% for HT-2, respectively. T-2 toxin was detected in 11 (5.1%) of all samples. The highest incidence was found in corn (21.7%) followed by mixed grains and brown rice. Mean of all samples was 1.5–4.1?µg?kg^?1, the maximum level being 41.5?µg?kg^?1 in corn. HT-2 toxin was detected in 126 (58.9%) of all samples, and the mean values were 26.4–59.2?µg?kg^?1. The estimated daily intakes for the sum of T-2 and HT-2 toxins were 2.56, 3.22, 2.53, 0.03, 0.01 and 2.45?ng (kg?bw)^?1?day^?1 in brown rice, barley, mixed grains, corn, wheat and wheat flour, respectively.     

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.     

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.     

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.     

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.     

高效液相色谱-串联质谱法测定3种水产品中的T-2毒素与HT-2毒素

建立高效液相色谱-串联质谱定量快速检测罗非鱼、南美白对虾和黄金贝中的T-2毒素与HT-2毒素方法。以10 mL乙酸乙酯作为提取溶剂,振荡提取,无水硫酸钠除水,定量移取5 mL提取液氮气吹干后用1 mL含有0.1%甲酸的甲醇-5 mmol/L乙酸铵溶液（3∶7,V/V）复溶,正己烷脱脂净化,基质匹配法外标定量。3 种水产品中T-2毒素和HT-2毒素的检出限分别为2 μg/kg和4 μg/kg。T-2毒素质量浓度为2～100 ng/mL,HT-2毒素质量浓度为4～200 ng/mL,范围内线性良好。在3 种样本中进行3 个水平添加实验（n=6）,T-2毒素回收率为84.3%～109.9%,HT-2毒素的回收率为90.9%～103.2%。T-2毒素的相对标准偏差为2.0%～8.7%,HT-2毒素的相对标准偏差为2.6%～10.6%。本方法简便快速、准确度好、精密度高,适用于3 种代表性水产品中T-2与HT-2毒素的同时检测。     

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.