Enniatin A1 Research Articles

Development of a high-throughput UHPLC-MS/MS method for the analysis of Fusarium and Alternaria toxins in cereals and cereal-based food.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004µg/kg for enniatin A1 (ENN A1) and 3.16µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded.

Associated factors with mycotoxin exposure in Spanish population

Human exposure to mycotoxins is a global concern since filamentous fungi can contaminate food and feed from crops to ready-to-eat meals. Human urine biomonitoring is a widely used technique to evaluate mycotoxins exposure, as an alternative to food correlation studies. The aim of this study is to describe human exposure to mycotoxins and to investigate the associated sociodemographic, lifestyle and dietary variables. Participants were 540 women from the Valencia (Spain) cohort of the Spanish Childhood and Environment Project (INMA). A validated multi-mycotoxin method using HPLC-Q-TOF-MS was applied to determine the concentration of ten selected mycotoxins: Enniatin A, Enniatin B, Enniatin A1, Enniatin B1, Beauvericine, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 and Ochratoxin A. A simultaneous untargeted screening of mycotoxins and their metabolites has been performed. Mycotoxins associations were assessed by bivariate and multivariate regression models using participants' sociodemographic, lifestyle and dietary data collected through questionnaires. Mycotoxins were detected in 81% of urine samples. The method quantified mycotoxins concentrations in up to 151 samples. Most quantified mycotoxins were: Enniatin B [% of detection (concentration range)] = 26% (1.0–39.7 ng/mg) and Enniatin B1 = 7% (0.5–14.4 ng/mg). Besides the ten-targeted mycotoxins, other mycotoxins and metabolites were studied, and higher incidence was observed for Deepoxy-deoxynivalenol (45%), Ochratoxin B (18%) and Ochratoxin α (17%). Higher mycotoxins concentrations were associated with rural areas as well as with participants belonged to lower social class, beer, light sodas and fruit juice consumers. On the contrary, higher processed meat intake was related to lower mycotoxins’ levels. Studies are required to better evaluate the exposure to mycotoxins from food and their environmental relationships.

Determination of beauvercin and enniatins in rice flour and wheat flour by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

To develop a method for the determination of beauvercin(BEA), enniatin A(ENNA), enniatin A1(ENNA1), enniatin B(ENNB) and enniatin B1(ENNB1) in rice flour and wheat flour by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). Samples were extracted by acetonitrile-water, purified by Oasis Prime HLB solid-phase extraction column. The sample solution was separated by waters BEH C_(18) column(2.1 mm×100 mm, 1.8 μm). The detection was performed in the electrospray positive ionization(ESI+) under multiple reaction monitoring(MRM) mode. The internal standard method and the matrix-matched calibrations were used for quantification. The linear relationships of BEA and 4 kinds of enniatins(ENNs) were good in the range of 0.1-50.0 ng/mL(r>0.999). The average recoveries of BEA and ENNs in rice flour and wheat flour were 96.4%-105.4% and 99.1%-109.2%, with the relative standard deviations(RSD) of 1.01%-7.42% and 1.09%-9.69%(n=6). The detection limits(LOD) of BEA and ENNs were 0.03 μg/kg. The quantitative limits(LOQ) of BEA and ENNs were 0.1μg/kg. The matrix induced suppression or enhancement effect were 72.7%-99.3% and 60.8%-100.4%, respectively. The levels of emerging BEA and ENNs in wheat flour were higher than rice flour. The detection rate of enniatin B was highest in wheat flour and rice flour, the contents were 0.03-9.57 μg/kg and 0.03-0.56 μg/kg, the positive percentage were 98.5% and 36.4%. The method is quick, easy, accurate and sensitive, which is suitable for the determination of BEA and 4 kinds of ENNs in rice flour and wheat flour.

Occurrence of pre- and postharvest multi-mycotoxins in durum wheat grains collected in 2020 and 2021 in two climatic regions of Tunisia

ABSTRACT Twenty two mycotoxins in 136 durum wheat collected from Tunisia in 2020 and 2021 were investigated. Mycotoxins were analyzed by UHPLCMS/MS. In 2020, 60.9% of the samples were contaminated with Aflatoxin B1 (AFB1) and/or enniatin. Whereas, in 2021, 34.4% were contaminated by enniatins. AFB1 was detected only in 2020, in the continental region (6/46) and all samples exceeded limits. AFB1 was detected in stored wheat (24-37.8 µg/kg) but also in pre-stored wheat (17-28.4 µg/kg) and in one sample collected in the field (21 µg/kg). Enniatin A1, enniatin B and enniatin B1 were detected in wheat collected in the field (30-7684 µg/kg), pre-storage (42-1266 µg/kg) and storage (65.8-498.2 µg/kg) from the continental region also, in sample collected in pre-storage (31.3-1410 µg/kg) and at harvest (48- 1060 µg/kg). Samples had a water activity less than 0.7 and moisture content ranged between 09-14%. AFB1 level represent a health risk to the Tunisian consumers.

Occurrence of Fusarium mycotoxins in freshly harvested highland barley (qingke) grains from Tibet, China.

Highland barley, also called "qingke" in Tibetan, is mainly cultivated in the Tibetan Plateau of China and has been used as a major staple food for Tibetans. Recently, Fusarium head blight (FHB) of qingke was frequently observed around the Brahmaputra River in Tibet. Considering the importance of qingke for Tibetans, the assessment of Fusarium mycotoxin contamination is essential for food safety. In this study, a total of 150 freshly harvested qingke grain samples were obtained from three regions around the Brahmaputra River in Tibet (China) in 2020. The samples were investigated for the occurrence of 20 Fusarium mycotoxins using high-performance liquid chromatography-tandem mass spectrometry (HPLC‒MS/MS). The most frequently occurring mycotoxin was enniatin B (ENB) (46%), followed by enniatin B1 (ENB1) (14.7%), zearalenone (ZEN) (6.0%), enniatin A1 (ENA1) (3.3%), enniatin A (ENA) (1.3%), beauvericin (BEA) (0.7%), and nivalenol (NIV) (0.7%). Due to the increase in altitude, the cumulative precipitation level and average temperature decreased from the downstream to the upstream of the Brahmaputra River; this directly correlated to the contamination level of ENB in qingke, which gradually decreased from downstream to upstream. In addition, the level of ENB in qingke obtained from qingke-rape rotation was significantly lower than that from qingke-wheat and qingke-qingke rotations (p < 0.05). These results disseminated the occurrence of Fusarium mycotoxins and provided further understanding of the effect of environmental factors and crop rotation on Fusarium mycotoxins.

Aggressiveness and mycotoxin profile of Fusarium avenaceum isolates causing Fusarium seedling blight and Fusarium head blight in UK malting barley.

Fusarium avenaceum causing Fusarium seedling blight (FSB) and Fusarium head blight (FHB) on barley is associated with economic losses of crop yield and quality, and the accumulation of mycotoxins including the enniatins (ENNs) A, A1, B and B1. Although F. avenaceum is the main producer of ENNs, studies on the ability of isolates to cause severe Fusarium diseases or produce mycotoxins in barley are limited. In this work, we investigated the aggressiveness of nine isolates of F. avenaceum to two cultivars of malting barley, Moonshine and Quench, and defined their ENN mycotoxin profiles in in vitro and in planta experiments. We assessed and compared the severity of FSB and FHB caused by these isolates to disease severity by F. graminearum, F. tricinctum and F. poae. Quantitative real-time polymerase chain reaction and Liquid Chromatography Tandem Mass Spectrometry assays were used to quantify pathogen DNA and mycotoxin accumulation, respectively, in barley heads. Isolates of F. avenaceum were equally aggressive to barley stems and heads and caused the most severe FSB symptoms resulting in up to 55% reductions of stem and root length. Fusarium graminearum caused the most severe FHB disease, followed by the isolates of F. avenaceum with the most aggressive F. avenaceum isolates capable of causing similar bleaching of barley heads as F. avenaceum. Fusarium avenaceum isolates produced ENN B as the predominant mycotoxin, followed by ENN B1 and A1 in vitro. However, only the most aggressive isolates produced ENN A1 in planta and none produced ENN A or beauvericin (BEA) either in planta or in vitro. The capacity of F. avenaceum isolates to produce ENNs was related to the accumulation of pathogen DNA in barley heads, whilst FHB severity was related to the synthesis and accumulation of ENN A1 in planta. Cv. Moonshine was significantly more resistant than Quench to FSB or FHB, caused by any Fusarium isolate, and to the accumulation of pathogen DNA, ENNs or BEA. In conclusion, aggressive F. avenaceum isolates are potent ENN producers causing severe FSB and FHB with ENN A1 requiring further investigation as potential virulence factor for F. avenaceum in cereals.

Secondary Metabolites of Pathogenic Fungi in Triticum durum Grain Protected with Debaryomyces hansenii in Two Different Locations in Poland

Durum wheat grain can accumulate mycotoxins because it is highly sensitive to infections caused by pathogens of the genera Fusarium and Alternaria. Reduced fungicide use increases the demand for biological methods of pathogen control. The aim of the experiment was to evaluate the efficacy of Debaryomyces hansenii (Dh) yeast in reducing the content of secondary fungal metabolites present in the spikes of five durum wheat cultivars grown in southern and northern Poland. A total of 27 Fusarium metabolites and nine metabolites produced by other fungi were identified in the grain. The application of the Dh yeast strain decreased deoxynivalenol concentration in all samples relative to control treatments (by 14–100%) and treatments inoculated with F. graminearum (by 23–100%). In northern Poland, the biological treatment also led to a considerable reduction in the content of culmorin (by 83.2–100%) and enniatins A1 and B (by 9.5–65.3% and 6.7–70%, respectively) in the grain. An analysis of multiple fungal metabolites is a highly useful tool for determining grain quality and its suitability for consumption. When applied in the flowering stage, yeasts can partly complete fungicides in reducing Fusarium head blight.

Determination of Regulated and Emerging Mycotoxins in Organic and Conventional Gluten-Free Flours by LC-MS/MS.

Gluten-free cereal products have grown in popularity in recent years as they are perceived as "healthier" alternatives and can be safely consumed by celiac patients, and people with gluten intolerance or wheat allergies. Molds that produce mycotoxins contaminate cereal crops, posing a threat to global food security. Maximum levels have been set for certain mycotoxins in cereal flours; however, little is known about the levels of emerging mycotoxins in these flours. The aim of this study was to develop an efficient, sensitive, and selective method for the detection of four emerging (beauvericin and enniatins A1, B, and B1) and three regulated (aflatoxin B1, zearalenone, and deoxynivalenol) mycotoxins in gluten-free flours. Ultrasound-assisted matrix solid-phase dispersion was used in the extraction of these mycotoxins from flour samples. The validated method was utilized for the LC-MS/MS analysis of conventional and organic wholegrain oat and rice flours. Six of the seven target mycotoxins were detected in these samples. Multi-mycotoxin contamination was found in all flour types, particularly in conventional wholegrain oat flour. Despite the low detection frequency in rice flour, one sample was found to contain zearalenone at a concentration of 83.2 μg/kg, which was higher than the level set by the European Commission for cereal flours. The emerging mycotoxins had the highest detection frequencies; enniatin B was present in 53% of the samples at a maximum concentration of 56 μg/kg, followed by enniatin B1 and beauvericin, which were detected in 46% of the samples, and at levels reaching 21 μg/kg and 10 μg/kg, respectively. These results highlight the need to improve the current knowledge and regulations on the presence of mycotoxins, particularly emerging ones, in gluten-free flours and cereal-based products.

Enniatins A1 and B1 alter calcium homeostasis of neuronal cells leading to apoptotic death.

Enniatins (ENNs) A1 and B1 are non-regulated mycotoxins produced by Fusarium spp. that commonly occur in different types of food. These toxins are cytotoxic in several cell lines, but their mechanism of action is unclear. In this study, the cytotoxic effects of ENNs A1 and B1 in SH-SY5Y human neuroblastoma cells were analysed. Moreover, to better understand their mechanism of action, mitochondrial function, reactive oxygen species (ROS) levels and calcium fluxes were monitored. ENNs A1 and B1 reduced cell viability, presenting IC50 values of 2.0 and 2.7μM, respectively. Both toxins induced caspase-dependent apoptosis, but only ENN A1 increased ROS production. Apoptotic cell death seems to be triggered by the increase in cytosolic calcium produced by both ENNs, since the toxins altered Ca2+ homeostasis by depleting intracellular reservoirs. Finally, binary combinations of ENN A1, ENN B1, ENN A and ENN B were tested. All mixtures resulted in an antagonistic effect, with the exception of ENN A and ENN B1 combination, which produced an additive effect. The results presented in this study provide the first evidence of ENNs A1 and B1 effects on calcium fluxes, providing new insights into the mechanism of action of these mycotoxins.

Non-aqueous capillary electrophoresis–time of flight mass spectrometry method to determine emerging mycotoxins

Enniatins (ENN) and beauvericin (BEA) are emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)–MS/MS have been reported so far. Due to their non-polar nature, in this work, a non-aqueous CE (NACE) method coupled to quadrupole time-of-flight–MS is proposed for the first time to identify and quantify these mycotoxins. Determination was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) acetonitrile-methanol (buffer), 30 kV (voltage), 80 cm (capillary length), 20 °C (capillary temperature) and 50 mbar × 30 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M + CH3CH2NH3]+. “All Ions” acquisition mode was selected as it allows the quantification of the usual ENNs, as well as the identity confirmation of less common ENNs.The method was validated for wheat samples, obtaining limits of quantification from 4.0 to 8.3 μg/kg depending on the emerging mycotoxin, recovery values higher than 87.4%, and intra- and inter-day precision values (RSDs) lower than 15.1% in all cases. Finally, 29 wheat samples were analyzed, finding 26 samples with concentrations of enniatin B higher than the limit of quantification (7.5–1480 μg/kg), 20 for enniatin B1 (5.2–550 μg/kg), 7 for enniatin A (10–55 μg/kg), 4 for enniatin A1 (12.6–77 μg/kg) and 5 for BEA (9.2–16.4 μg/kg). Moreover, two other ENNs were tentatively identified.

A validated analytical method for biomonitoring the mycotoxins exposure in urine

BACKGROUND AND AIM Mycotoxin exposure from food may produce adverse effects in human health. Most mycotoxins are excreted in urine, but a reliable detection method is required considering the low levels present in this biological matrix. The aim of this work is to validate an analytical method for detecting and quantifying in urine different mycotoxins usually present in the food chain. METHODS HPLC-QTOF-MS method was used. The targeted mycotoxins were: Enniatin A (ENNA), Enniatin B (ENNB), Enniatin A1 (ENNA1), Enniatin B1 (ENNB1), Beauvericine, Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2) and Ochratoxin A. Different extraction procedures, liquid-liquid extraction, dilute and shoot, and Quick, Easy, Cheap, Effective, Rugged and Save (QuEChERS) methods were assessed followed by HPLC-QTOF-MS and different combinations of solvent extraction and mobile phases were tested. QuEChERS extraction was selected because better recoveries were achieved (&amp;#x3e;70%) for all mycotoxins. The method was applied to be validated in 150 samples of urine of women participating in the INMA (Environment and Childhood) Spanish project. RESULTS For all mycotoxins, matrix-matched calibration curves showed values of regression determination coefficients ranging from 0.998 to 0.999. The LOD and LOQ values ranged from 0.1 to 5.0 ng/ml and from 0.3 to 15.0 ng/ml, respectively. Assessed mycotoxins were detected in the 95% of samples and 8 out of 10 of them were quantified. Mostly quantified mycotoxins were: ENNB [% of detection (concentration range)] = 94% (0.54 – 2.83 ng/ml), ENNA = 47%, (0.30 – 0.68 ng/ml), ENNB1 = 35% (0.45 – 0.89 ng/ml), and AFB2 (18% (0.51 – 8.97 ng/ml). CONCLUSIONS The proposed analytical method quantified 8 out of 10 assessed mycotoxins (ENNA, ENNB, ENNA1, ENNB1, AFB1, AFB2, AFG1 and AFG2). These results support the methodology and provide a tool for identifying exposures in susceptible populations. KEYWORDS biomonitoring, mycotoxins, urine

The application of antagonistic yeasts and bacteria: An assessment of in vivo and under field conditions pattern of Fusarium mycotoxins in winter wheat grain

Bacteria of the genus Sphingomonas, Aureobasidium pullulans yeast-like fungus and yeast Debaryomyces hansenii naturally colonize wheat grain. Selected isolates of these microorganisms, antagonistic against Fusarium spp., can be applied to wheat spikes to complement chemical control methods. The aim of this study was to determine the content of Fusarium mycotoxins in winter wheat grain in vivo and under field conditions, and to analyze the interactions between antagonistic microorganisms and F. culmorum and F. avenaceum pathogens in dual cultures. Seventeen toxic metabolites produced by Fusarium fungi were identified in grain: deoxynivalenol, deoxynivalenol-3-glucoside, culmorin, 15-hydroxyculmorin, 5-hydroxyculmorin, HT-2 toxin, nivalenol, nivalenol-3-glucoside, aurofusarin, enniatins, zearalenone, moniliformin, equisetin, and apicidin. Integrated fungicide and biological treatments decreased deoxynivalenol levels in grain in the field conditions from 141.36 to 72.76 μg/kg. Deoxynivalenol was not detected in grain treated with D. hansenii suspension, and culmorin content was 38 times lower than in unprotected grain. Fungicides and integrated treatments involving D. hansenii also decreased enniatin A1 content by 76.61 and 48.17%. In vitro yeasts considerably reduced moniliformin and enniatin concentrations by 7.67–92.87%. Antagonistic microorganisms, which inhibit the growth of Fusarium fungi through antibiosis and competition for nutrients, can effectively reduce the production of selected Fusarium toxins when combined with fungicides in the integrated approach.

Cytochrome P450 isoforms contribution, plasma protein binding, toxicokinetics of enniatin A in rats and in vivo clearance prediction in humans

Emerging mycotoxins, such as enniatin A (ENNA), are becoming a worldwide concern owing to their presence in different types of food and feed. However, comprehensive toxicokinetic data that links intake, exposure and toxicological effects of ENNA has not been elucidated yet. Therefore, the present study investigated the in vitro (rat and human) and in vivo (rat) toxicokinetic properties of ENNA. Towards this, an easily applicable and sensitive bioanalytical method was developed and validated for the estimation of ENNA in rat plasma. ENNA exhibited high plasma protein binding (99%), high hepatic clearance and mainly underwent metabolism via CYP3A4 (74%). The in-house predicted hepatic clearance (54 mL/min/kg) and observed in vivo rat clearance (55 mL/min/kg) were comparable. The predicted in vivo human hepatic clearance was 18 mL/min/kg. ENNA underwent slow absorption (Tmax = 4 h) and rapid elimination following oral administration to rats. The absolute oral bioavailability was 47%. The toxicokinetic findings for ENNA from this study will help in designing and interpreting toxicological studies in rats. Besides, these findings could be used in physiologically based toxicokinetic (PBTK) model development for exposure predictions and risk assessment for ENNA in humans.

Mycotoxins in cereal-based infant foods marketed in China: Occurrence and risk assessment

Infants are considered as the most vulnerable subgroup susceptible to mycotoxins in food. A total of 820 cereal-based infant foods marketed in China were screened for 20 mycotoxins using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Low levels of 12 mycotoxins were detected including deoxynivalenol (DON), tenuazonic acid (TeA), enniatin A (ENA), zearalenone (ZEN), enniatin B1 (ENB1), alternariol (AOH), enniatin B (ENB), alternariol nonomethyl ether (AME), enniatin A1 (ENA1), fumonisin B1 (FB1), ochratoxin A (OTA), and fumonisin B2 (FB2) with the incidence rate of 55.7, 45.1, 13.9, 8.2, 6.6, 5.6, 4.8, 4.3, 3.9, 3.7, 1.1, and 0.7% of samples, respectively. One or more mycotoxins were found in 73.7% of all samples, 71.2% of processed infant foods (infant cereal, infant cracker, and infant noodle), and 82.3% in common cereals (wheat flour, rice, and millet) for infant consumption. Wheat-based infant foods were contaminated with more varieties of mycotoxins than those rice-based infant foods. A deterministic approach was used to evaluate dietary risk assessment through cereal-based food consumption for the average and heavy infant consumers in China. Overall, dietary exposure of DON, ZEN, FB1, FB2, OTA, and TeA by cereal-based infant food intakes was considered to be acceptable (Hazard quotient <1). However, the estimated dietary exposure of AME and AOH exceeded the threshold of toxicological concern of 0.0025 μg/kg b.w./day, which confirmed the need for further study to assess the potential health risks for infants.

High Pressure Processing Impact on Emerging Mycotoxins (ENNA, ENNA1, ENNB, ENNB1) Mitigation in Different Juice and Juice-Milk Matrices.

The aim of the present study was to investigate the potential of high-pressure processing (HPP) (600 MPa during 5 min) on emerging mycotoxins, enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1) reduction in different juice/milk models, and to compare it with the effect of a traditional thermal treatment (HT) (90 °C during 21 s). For this purpose, different juice models (orange juice, orange juice/milk beverage, strawberry juice, strawberry juice/milk beverage, grape juice and grape juice/milk beverage) were prepared and spiked individually with ENNA, ENNA1, ENNB and ENNB1 at a concentration of 100 µg/L. After HPP and HT treatments, ENNs were extracted from treated samples and controls employing dispersive liquid-liquid microextraction methodology (DLLME) and determined by liquid chromatography coupled to ion-trap tandem mass spectrometry (HPLC-MS/MS-IT). The results obtained revealed higher reduction percentages (11% to 75.4%) when the samples were treated under HPP technology. Thermal treatment allowed reduction percentages varying from 2.6% to 24.3%, at best, being ENNA1 the only enniatin that was reduced in all juice models. In general, no significant differences (p > 0.05) were observed when the reductions obtained for each enniatin were evaluated according to the kind of juice model, so no matrix effects were observed for most cases. HPP technology can constitute an effective tool in mycotoxins removal from juices.

Simultaneous determination of beauvericin and four enniatins in eggs by ultra-performance liquid chromatography-tandem mass spectrometry coupled with cold-induced liquid-liquid extraction and dispersive solid phase extraction

新型生物毒素白僵菌素(BEA)和恩镰孢菌素(ENNs)是由镰刀菌种产生的有毒代谢产物,主要污染谷物及其制品,会威胁人类健康,因此受到人们越来越多的关注。该工作建立了冷诱导液液萃取-分散固相萃取净化-超高效液相色谱-串联质谱法(CI-LLE-DSPE-UPLC-MS/MS)同时测定鸡蛋中白僵菌素和4种恩镰孢菌素残留的分析方法。以乙腈-水-乙酸(79:20:1, v/v/v)为提取溶剂,采用冷诱导液液萃取与分散固相萃取净化相结合的方法进行样品处理,同时,对影响待测物提取与净化效率的提取溶剂、冷冻萃取温度与时间、净化剂用量等因素和色谱条件进行了优化。样品经20 mL提取液涡旋提取20 min,放入-40 ℃冰箱静置30 min后,取2 mL上层溶液经70 mg C18粉末净化,离心,上清液于40 ℃浓缩至近干,残留物用1 mL 80%(v/v)乙腈水溶液溶解,进样分析。以乙腈与5 mmol/L甲酸铵溶液作为流动相进行梯度洗脱,经ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm, 1.7 μm)分离,采用ESI+电离,在多反应监测模式下采集,白僵菌素采用稳定同位素内标法定量,4种恩镰孢菌素采用基质匹配曲线外标法定量。结果表明,5种待测物在0.1~50.0 μg/L范围内线性关系良好,相关系数(r2)为0.9983~0.9997,该方法的检出限(LOD)为0.05~0.15 μg/kg,定量限(LOQ)为0.20~0.50 μg/kg。以阴性鸡蛋样品为基质,在低、中、高3个浓度水平(0.5、5.0、25.0 μg/kg)下进行加标试验考察方法的准确度与精密度,各待测物的平均回收率为81.1%~106%,相对标准偏差(RSD)为0.27%~9.79%。采用所建立的方法对农村散养鸡蛋与市售鸡蛋进行检测,结果表明,BEA在散养鸡蛋的检出率为30.4%, 4种ENNs均未被检出。该方法灵敏度高,稳定性好,回收率高,定量准确,简单易操作,适用于禽蛋食品中白僵菌素与恩镰孢菌素的同时快速测定。

Relationship between Mycotoxin Content in Winter Wheat Grain and Aspirated Dust Collected during Harvest and after Storage.

A total of 323 paired grain and grain dust samples (particle size <1650 μm) were collected from combines at harvest (56%), on-farm bins (28%), and experimental minibins seeded with an ochratoxin A (OTA)/Penicillium verrucosum hot spots (15%) of which >98% were soft red winter wheat. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to detect 21 mycotoxins, including deoxynivalenol (DON) and its plant-conjugated form, deoxynivalenol 3-β-d-glucoside (DON 3-Glc). Except for DON 3-Glc, all mycotoxin concentrations found in grain dust were higher than in grain (p < 0.0030). Pearson correlation coefficients and two-variable regression show a significant (p < 0.0001) linear relationship between the mycotoxin content in grain and that in grain dust with 19 toxins. In only five mycotoxins (DON, OTA, ochratoxin B, citrinin, and enniatin A1), more than 82% of the variation in the data is explained by the two-variable regression model. Because of its higher mean concentration and detection frequency, only DON produced a strong relationship (p < 0.0001, r2 = 0.949) with low root-mean-square error (RMSE) (293.41 ng/g). The results suggest that modeling levels in grain based upon levels in grain dust can be used to estimate DON in grain bulk.

Further data on the levels of emerging Fusarium mycotoxins in cereals collected from Tianjin, China

ABSTRACT A number of 344 samples were collected from Tianjin, China and were analysed for the occurrence of emerging Fusarium mycotoxins including enniatin A (ENN A), enniatin A1 (ENN A1), enniatin B (ENN B), enniatin B1 (ENN B1) and beauvericin (BEA) by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The frequencies of mycotoxins studied were 69.0% (40/58), 69.8% (37/53), 85.9% (67/78), 78.9% (75/95), and 30.0% (18/60) for rice, wheat, corn, wheat flour, and corn flour, respectively. BEA was the predominant toxin in rice (average = 37.2 μg/kg) and wheat (average = 58.4 μg/kg), followed by ENN B and ENN B1, while less ENN A and ENN A1 were detected. ENN A was most common in corns with an average level of 28.1 μg/kg, while BEA had a higher average of 62.8 μg/kg. The levels of ENNs and BEA significantly decreased in wheat flours and corn flours, presumably due to the production process. The co-occurrences of ENNs and BEA in cereal samples were common in the combination of two and three mycotoxins and the significant positive correlations in concentrations were also obtained among them. Besides, agroclimate was considered as an important factor for Fusarium production and the mycotoxin contamination was found more serious in Jizhou district, which had more rainfall and less sunshine, than the other agriculture regions. These results suggested the necessity of carrying out in-depth and large-scale monitoring of mycotoxins in cereals and their products nationwide.

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.