Enniatin Research Articles

Emerging Alternaria and Fusarium mycotoxins in tomatoes and derived tomato products from the China market: Occurrence, methods of determination, and risk evaluation

Emerging Alternaria and Fusarium mycotoxins are increasingly becoming a public health concern due to the frequent contamination of various food commodities with these mycotoxins, posing potential health risks to humans. This study developed QuEChERS extraction method coupled with LC–MS/MS for simultaneous quantification of alternariol monomethyl ether (AME), alternariol (AOH), tentoxin (TEN), tenuazonic acid (TeA), altenuene (ALT), enniatins (ENNs) A, A1, B, and B1, and beauvericin (BEA) in tomatoes and derived tomato products. The validated method was successfully applied for a survey of 194 tomatoes and derived tomato products. Overall, one or more mycotoxins were detected in 58.8% of the samples, 100% of tomato powders, 100% of canned tomatoes, 89.6% of tomato sauces, 82.1% of ketchups, 46.7% of tomato juices, and 20% of dried tomatoes, with no mycotoxins detected in fresh tomatoes. Various mycotoxins were detected at relatively higher contamination levels in tomato powders than in other tomato products. TeA, the most predominant mycotoxin, was detected in 56.7% of the samples with an occurrence level of up to 7,985 μg/kg. Approximately 39.2% of positive samples presented the co-occurrence of more than one mycotoxins, and AME + AOH + TeA was the most frequently detected combination. This study is the first to perform the deterministic risk assessment for children and adults on Alternaria and Fusarium mycotoxins that are present in tomatoes and derived tomato products in China. The results showed that the average and 95th percentile dietary exposures to these mycotoxins, especially to AME and AOH, in children might be higher than those in adults by a factor of approximately 2. The estimation of exposure to either Alternaria mycotoxins or Fusarium mycotoxins indicated that the presence of Alternaria and Fusarium mycotoxins in derived tomato products might be a health concern, especially among children and the populations consuming a high amount of these products. However, no firm conclusion could be drawn due to the lack of relevant in vivo toxicity data. This study serves as a useful reference for individuals and food safety authorities concerning mycotoxin contamination in food commodities. • Methods for emerging toxins determination in various tomato products were developed. • About 58.8% (114/194) of tomato-based products were contaminated with emerging toxins. • Co-occurrence of various emerging toxins was evident in 39.2% (76/194) of samples. • Dietary exposures to Alternaria and Fusarium toxins varied among children and adults.

In Vitro Effects of Enniatin A on Steroidogenesis and Proliferation of Bovine Granulosa Cells.

The emerging Fusarium mycotoxins enniatins (ENNs) have been the focus of new research because of their well-documented existence in various cereal and grain products. Research findings indicate that reproductive disorders may be caused by exposure to Fusarium mycotoxins, but little work has evaluated ENNs on reproductive function. Therefore, to determine the effects of ENNA on the proliferation and steroidogenesis of granulosa cells (GC), experiments were conducted using bovine GC cultures. In vitro, ENNA (1–5 μM) inhibited (p < 0.05) hormone-induced GC progesterone and estradiol production. The inhibitory effect of ENNA on estradiol production was more pronounced in small- than large-follicle GC. In large-follicle GC, 0.3 μM ENNA had no effect (p > 0.10) whereas 1 and 3 μM ENNA inhibited GC proliferation. In small-follicle GC, ENNA (1–5 μM) dramatically decreased (p < 0.05) GC proliferation. Using cell number data, the IC50 of ENNA was estimated at 2 μM for both follicle sizes. We conclude that ENNA can directly inhibit ovarian function in cattle, decreasing the proliferation and steroid production of GC.

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.

Distribution, pathogenicity and disease control of Fusarium tricinctum

Plant pathogenic fungi such as Fusarium tricinctum cause various plant diseases worldwide, especially in temperate regions. In cereals, F. tricinctum is one of the most common species causing Fusarium head blight (FHB) and root rot. Infection with F. tricinctum results in high yield losses and reduction in quality, mainly due to mycotoxin contamination of grain. Mycotoxins produced by F. tricinctum, such as enniatins (ENs) and moniliformin (MON), which are the most studied mycotoxins, have been reported to have multiple toxic effects on humans and animals. Although chemical control of Fusarium infection has been applied to grains, it is not always effective in controlling disease or reducing the level of mycotoxins in wheat grains. To the contrary, chemical control may significantly increase infection of F. tricinctum in fungicide-treated plots after treatment. Our studies show that the bacterium Bacillus amyloliquefaciens, has good control effects against F. tricinctum. Therefore, its use as a biological control agent against various plant pathogens may be an effective strategy to control the spread of Fusarium pathogens. Here, we conduct a review of the literature involving this plant pathogen, its diversity, virulence, and methods to control.

Phylogenetic Diversity and Mycotoxin Potential of Emergent Phytopathogens Within the Fusarium tricinctum Species Complex.

Recent studies on multiple continents indicate members of the Fusarium tricinctum species complex (FTSC) are emerging as prevalent pathogens of small-grain cereals, pulses, and other economically important crops. These understudied fusaria produce structurally diverse mycotoxins, among which enniatins (ENNs) and moniliformin (MON) are the most frequent and of greatest concern to food and feed safety. Herein a large survey of fusaria in the Fusarium Research Center and Agricultural Research Service culture collections was undertaken to assess species diversity and mycotoxin potential within the FTSC. A 151-strain collection originating from diverse hosts and substrates from different agroclimatic regions throughout the world was selected from 460 FTSC strains to represent the breadth of FTSC phylogenetic diversity. Evolutionary relationships inferred from a five-locus dataset, using maximum likelihood and parsimony, resolved the 151 strains as 24 phylogenetically distinct species, including nine that are new to science. Of the five genes analyzed, nearly full-length phosphate permease sequences contained the most phylogenetically informative characters, establishing its suitability for species-level phylogenetics within the FTSC. Fifteen of the species produced ENNs, MON, the sphingosine analog 2-amino-14,16-dimethyloctadecan-3-ol (AOD), and the toxic pigment aurofusarin (AUR) on a cracked corn kernel substrate. Interestingly, the five earliest diverging species in the FTSC phylogeny (i.e., F. iranicum, F. flocciferum, F. torulosum, and Fusarium spp. FTSC 8 and 24) failed to produce AOD and MON, but synthesized ENNs and/or AUR. Moreover, our reassessment of nine published phylogenetic studies on the FTSC identified 11 additional novel taxa, suggesting this complex comprises at least 36 species.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

Inhibition of the Growth of Fusarium tricinctum and Reduction of Its Enniatin Production by Erwinia gerundensis Isolated from Barley Kernels

Fusarium tricinctum is among the pathogenic fungi that contaminate cereal crops, including barley. It produces toxic metabolites such as enniatins (ENNs) that affect the quality and safety of barley-based products such as malt and beer, thus representing a significant concern for the cereal industries. Therefore, it is critical to reduce Fusarium spp. development and enniatin contamination in cereal-based products. Fungicides are commonly used during agricultural practices, but they are facing growing fungal resistance and environmental and health concerns. Hence, there is need to define environmentally friendly control strategies. This study highlights the ability of a bacterial strain isolated from barley kernels, namely Erwinia gerundensis 6E4, to inhibit the growth of Fusarium tricinctum and to reduce its toxigenic potential. First, E. gerundensis 6E4 exhibited a significant fungistatic activity against pathogenic fungal strains during co-cultures, with fungal growth reduced by up to 100% on Petri dishes and by about 69% in liquid medium. The bacterial strain also led to a drastic reduction (74 to 100%) of ENNs production by F. tricinctum strains over 72 h co-cultures. Based on these results, our study supports the use of E. gerundensis 6E4 as a biocontrol agent in strategies aiming at reducing the presence of Fusarium tricinctum and enniatins in cereal-based products, or as a food and feed supplement for the bio-detoxification of enniatins.

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。

Mycotoxins occurrence in medicinal herbs dietary supplements and exposure assessment

The multimycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), ochratoxin A (OTA), enniatins (ENNs) and beauvericin (BEA) was performed in 85 samples of medicinal herbs dietary supplements. The samples were classified in 64 samples of one herbal ingredient and 21 mixed samples. The extraction was performed by QuEChERS method and the determination by liquid chromatography coupled to ion-trap tandem mass spectrometry (LC–MS/MS-IT). Then, the risk characterization to mycotoxins through the consumption of medicinal herbs dietary supplements was assessed. The results showed that ZEA, OTA, ENNs and BEA showed in the samples with incidences between 1 and 34%, being ENNB the most detected mycotoxin. Mycotoxins contents ranged from LOQ to 3850.5 µg/kg while the mean of positives samples were 65.5 µg/kg (ENNA), 82.7 µg/kg (ENNA1), 88.7 µg/kg (ENNB), 324.9 µg/kg (ENNB1), 137.9 µg/kg (BEA) and 1340.11 µg/kg (ZEA), respectively. OTA was detected in one herbal mix tablet for insomnia at concentration of 799 μg/kg. In herbal drugs the European Pharmacopoeia Commission has implemented limits of 2 µg/kg for AFB1 and 4 µg/kg for total AFs. In the present study AFs have not been detected in the analyzed medicinal herbs dietary supplements. The Estimated Daily Intakes (EDIs) values were calculated using a deterministic method, considering two exposure scenarios (lower bound (LB) and upper bound (UB)). The values obtained were in general far below the Tolerable Daily Intakes (TDIs) established.Graphical abstract

Enniatin B and Deoxynivalenol Activity on Bread Wheat and on Fusarium Species Development.

Fusarium head blight (FHB) is a devastating wheat disease, mainly caused by Fusarium graminearum (FG)—a deoxynivalenol (DON)-producing species. However, Fusarium avenaceum (FA), able to biosynthesize enniatins (ENNs), has recently increased its relevance worldwide, often in co-occurrence with FG. While DON is a well-known mycotoxin, ENN activity, also in association with DON, is poorly understood. This study aims to explore enniatin B (ENB) activity, alone or combined with DON, on bread wheat and on Fusarium development. Pure ENB, DON, and ENB+DON (10 mg kg−1) were used to assess the impacts on seed germination, seedling growth, cell death induction (trypan blue staining), chlorophyll content, and oxidative stress induction (malondialdehyde quantification). The effect on FG and FA growth was tested using ENB, DON, and ENB+DON (10, 50, and 100 mg kg−1). Synergistic activity in the reduction of seed germination, growth, and chlorophyll degradation was observed. Conversely, antagonistic interaction in cell death and oxidative stress induction was found, with DON counteracting cellular stress produced by ENB. Fusarium species responded to mycotoxins in opposite directions. ENB inhibited FG development, while DON promoted FA growth. These results highlight the potential role of ENB in cell death control, as well as in fungal competition.

Pilot Study: Does Contamination with Enniatin B and Beauvericin Affect the Antioxidant Capacity of Cereals Commonly Used in Animal Feeding?

Increasing consumption of cereals has been associated with reduced risk of several chronic diseases, as they contain phytochemicals that combat oxidative stress. Cereal contamination by the “emerging mycotoxins” beauvericin (BEA) and enniatins (ENs) is a worldwide health problem that has not yet received adequate scientific attention. Their presence in feeds represents a risk for animals and a potential risk for humans because of their carry-over to animal-derived products. This preliminary study aimed to investigate if the total antioxidant capacity (TAC) of corn, barley, and wheat flours could be influenced by contamination with increasing levels of BEA and ENN B. The highest TAC value was observed in barley compared with wheat and corn (p < 0.001) before and after contamination. No effect of mycotoxin or mycotoxin level was found, whereas cereal x mycotoxin exhibited a significant effect (p < 0.001), showing a lower TAC value in wheat contaminated by ENN B and in barley contaminated by BEA. In conclusion, barley is confirmed as a source of natural antioxidants with antiradical potentials. Additional studies with a larger sample size are necessary to confirm the obtained results, and investigations of the toxic effects of these emergent mycotoxins on animals and humans should be deepened.

Phylogeny and mycotoxin profile of Fusarium species isolated from sugarcane in Southern Iran

Sugarcane is an important crop in Southern Iran for agri-food, energy, and pharmaceutical industries. Among the pathogens that colonize sugarcane, mycotoxigenic Fusarium species are reason of serious concern for both their pathogenicity on plants and ability to produce harmful mycotoxins to humans and animals. We studied 104 Fusarium strains, selected within a wider Fusarium set isolated from sugarcane in Southern Iran, for molecular identification, phylogeny and mycotoxin analyses. Most of Fusarium strains belonged to Fusarium fujikuroi Species Complex (FFSC) and identified mainly as F. proliferatum, at minor extent as F. sacchari, and rarely as F. thapsinum, and F. verticillioides. Moreover, 14 strains identified as FFSC could not be assigned to any known species, although they were phylogenetically closely related to F. andiyazi, likely representing a new phylogenetic species. A subset of FFSC strains were analyzed for in vitro production of fumonisins (FBs), beauvericin (BEA), and enniatins (ENNs). Fusarium proliferatum strains produced FBs at high amount, and, at a lesser extent, BEA, and ENNs; F.sacchari produced only BEA and B ENNs at very low level; Fusarium sp. strains produced only B ENNs. The paper provides new insights on the genetic diversity of Fusarium species and their mycotoxin profile occurring on sugarcane in Iran.

Transcriptional Changes after Enniatins A, A1, B and B1 Ingestion in Rat Stomach, Liver, Kidney and Lower Intestine

Enniatins (ENs) are depsipeptide mycotoxins produced by Fusarium fungi. They are known for their capacity to modulate cell membrane permeability and disruption of ionic gradients, affecting cell homeostasis and initiating oxidative stress mechanisms. The effect of the acute toxicity of ENs A, A1, B and B1 at two different concentrations after 8 h of exposure was analysed in Wistar rats by a transcriptional approach. The following key mitochondrial and nuclear codified genes related to the electron transport chain were considered for gene expression analysis in stomach, liver, kidney and lower intestine by quantitative Real-Time PCR: mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded cytochrome c oxidase 1 (MT-COX1), succinate dehydrogenase flavoprotein subunit A and ATP synthase F1 subunit alpha, respectively. Moreover, the expression of markers involved in oxidative stresssuperoxide dismutase 1 (SOD1), glutathione peroxidase 1 (Gpx1), heme oxygenase 1, apoptosis B-cell lymphoma 2, Bcl2 Associated protein X (Bax), tumor suppressor protein (p53), inhibition of apoptosis nuclear factor kappa of activated B cells, immune system interleukin 1β and intestinal tight junction Occludin merely in lower intestine tissues have been investigated. For mitochondrial genes, the main differences were observed for MT-ND1 and MT-COX1, showing its deficiency in all selected organs. With regard to the intestinal barrier’s cellular response to oxidative stress, the activity of the antioxidant gene SOD1 was decreased in a dose-dependent manner. Similarly, the catalytic enzyme GPx1 was also downregulated though merely at medium dose employed. On the contrary, the pro-apoptotic Bax and p53 regulators were activated after ENs exposure, reporting a significant increase in their expression. Furthermore, the alteration of intestinal permeability was assessed by the abnormal activity of the tight junction protein occludin. In summary, ENs may generate mitochondrial disorders and induce oxidative stress in intestinal barrier function.

Phylogeny and Mycotoxin Profile of Pathogenic Fusarium Species Isolated from Sudden Decline Syndrome and Leaf Wilt Symptoms on Date Palms (Phoenix dactylifera) in Tunisia.

In 2017–2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, Fusarium was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative Fusarium strains at species level and investigate their phylogenetic relationships. The main species detected was Fusarium proliferatum, and at a much lesser extent, Fusarium brachygibbosum, Fusarium caatingaense, Fusarium clavum, Fusarium incarnatum, and Fusarium solani. Pathogenicity on the Deglet Nour variety plantlets and the capability to produce mycotoxins were also assessed. All Fusarium species were pathogenic complying Koch’s postulates. Fusarium proliferatum strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All F. brachygibbosum strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. Fusarium caatingaense, F. clavum, F. incarnatum produced only BEA. Fusarium solani strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of Fusarium species on date palms, concerning both phytopathological and food safety issues.

Species diversity and mycotoxin production by members of the Fusarium tricinctum species complex associated with Fusarium head blight of wheat and barley in Italy

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. In Europe, the main species responsible for FHB are F. graminearum, F. culmorum and F. poae. However, members of the F. tricinctum species complex (FTSC) have become increasingly important. FTSC fusaria can synthesize mycotoxins such as moniliformin (MON), enniatins (ENNs) and several other biologically active secondary metabolites that could compromise food quality. In this study, FTSC isolates primarily from Italian durum wheat and barley, together with individual strains from four non-graminaceous hosts, were collected to assess their genetic diversity and determine their potential to produce mycotoxins in vitro on rice cultures. A multilocus DNA sequence dataset (TEF1, RPB1 and RPB2) was constructed for 117 isolates from Italy and 6 from Iran to evaluate FTSC species diversity and their evolutionary relationships. Phylogenetic analyses revealed wide genetic diversity among Italian FTSC isolates. Among previously described FTSC species, F. avenaceum (FTSC 4) was the most common species in Italy (56/117 = 47.9%) while F. tricinctum (FTSC 3), and F. acuminatum (FTSC 2) accounted for 11.1% (13/117) and the 8.5% (10/117), respectively. The second most detected species was a new and unnamed Fusarium sp. (FTSC 12; 32/117 = 19%) resolved as the sister group of F. tricinctum. Collectively, these four phylospecies accounted for 111/117 = 94.9% of the Italian FTSC collection. However, we identified five other FTSC species at low frequencies, including F. iranicum (FTSC 6) and three newly discovered species (Fusarium spp. FTSC 13, 14, 15). Of the 59 FTSC isolates tested for mycotoxin production on rice cultures, 54 and 55 strains, respectively, were able to produce detectable levels of ENNs and MON. In addition, we confirmed that the ability to produce bioactive secondary metabolites such as chlamydosporol, acuminatopyrone, longiborneol, fungerin and butanolide is widespread across the FTSC.

Tea: Transfer of Mycotoxins from the Spiked Matrix into an Infusion.

Recent surveys report the occurrence of Aspergillus and Penicillium metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STC), citrinin), Fusarium (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and Alternaria (alternariol (AOH), its methyl ether (AME), tentoxin (TE), and tenuazonic acid (TNZ)) toxins in dry Camellia sinensis and herbal tea samples. Since tea is consumed in the form of infusion, correct risk assessment needs evaluation of mycotoxins’ transfer rates. We have studied the transfer of AFLs, OTA, STC, deoxynivalenol (DON), ZEA, FBs, T-2, and HT-2 toxins, AOH, AME, TE, ENN A and B, beauvericin (BEA), and MPA from the spiked green tea matrix into an infusion under variation of preparation time and water characteristics (total dissolved solids (TDS) and pH). Analytes were detected by HPLC-MS/MS. The main factors affecting transfer rate proved to be mycotoxins’ polarity, pH of the resulting infusion (for OTA, FB2, and MPA) and matrix-infusion contact period. The concentration of mycotoxins increased by 20–50% within the first ten minutes of infusing, after that kinetic curve changed slowly. The concentration of DON and FB2 increased by about 10%, for ZEA, MPA, and STC it stayed constant, while for T-2, TE, AOH, and AFLs G1 and G2 it went down. Maximum transfer correlated well with analytes polarity. Maximum transfer of ENNs, BEA, STC, ZEA, and AOH into infusion was below 25%; AFLs—25–45%; DON, TE, and T-2 toxins 60–90%, FB1—80–100%. The concentration of OTA, MPA, and FB2 in the infusion depended on its pH. At pH about four, 20%, 40%, and 60% of these toxins transferred into an infusion, at pH about seven, their concentrations doubled. Water TDS did not affect transfer significantly.

Herbs and herbal infusions: Determination of natural contaminants (mycotoxins and trace elements) and evaluation of their exposure

Herbal infusions are amongst the world's most popular and widely enjoyed beverages, due to both large variety and convenience. However, natural contaminants, such as mycotoxins and trace elements can accumulate in aromatic herbs, which may have serious food safety and public health implications. In this study, the presence of mycotoxins, as well as the content of trace elements was evaluated in herbs and herbal infusions commercialized in Brazil. For the determination of fourteen mycotoxins, including the emerging mycotoxins enniatins (EN), beauvericin (BEA), and sterigmatocystin (STE), a liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was validated. Overall, 42 out of 58 herb samples (72%) were contaminated, being BEA the most usual mycotoxin, present in 43% of the samples, followed by STE and HT-2 toxin, present in 37% and 24% of the samples, respectively. In herbal infusions, the occurrence of mycotoxins was 88% lesser than those verified in raw products. Despite these low levels, the hazard quotient (HQ) calculated revealed a potential health concern for HT-2 in infusions. The margin of exposure values for aflatoxins (AF), and ochratoxin A (OTA) from six herbal infusions were below 10,000, indicating also potential health risks. The twenty-one trace elements comprising toxic elements such as arsenic (As), cadmium (Cd), and lead (Pb) were determined in herb raw materials by inductively coupled plasma-mass spectrometry (ICP-MS). The levels of trace elements in herbs were very varied, with aluminum (Al) presenting the highest amount. The levels of legislated elements (As, Cd, Pb) analyzed in herbs were lower than 3.03 µg g−1 (Pb), thus not exceeding the legal limits defined for herbal medicinal by both European and Brazilian pharmacopeia.

Proteomics evaluation of enniatins acute toxicity in rat liver

Enniatins (ENs) are emerging mycotoxins produced by Fusarium fungi which are cytotoxic also at low concentrations due to its ionophoric properties. The aim of this study was to evaluate the hepatic toxicity of ENs exposure at different concentrations in Wistar rats through a proteomic approach. Animals were intoxicated by oral gavage with medium (EN A 256, ENA1 353, ENB 540, ENB1 296 μg/mL) and high concentrations (ENA 513, ENA1 706, ENB 1021, ENB1 593 μg/mL) of an ENs mixture and sacrificed after 8 h. Protein extraction was performed using powdered liver. Peptides were analyzed using a liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Proteins were filtered by abundance using Mass Professional Profiler software (Agilent Technologies) and 57 were differentially expressed when compared to the control. In terms of abundance, the liver biomarker Carboamoyl-phosphate synthase showed the highest levels in all conditions employed while actin-1 had the lowest. Bioinformatic analysis using DAVID platform reported acetylation, nucleotide phosphate-binding region:NAD and catalytic activity as the most represented terms. Furthermore, metabolism was the most significant and enriched pathway in Reactome overrepresentation. In conclusion, ENs acute exposure caused protein expression changes related to major cellular processes in rats, hinting its involvement in liver disturbance.

Cellulose-ferrite nanocomposite for monitoring enniatins and beauvericins in paprika by liquid chromatography and high-resolution mass spectrometry

Paprika is considered a high-quality product being one of the most consumed spices in the world. Contamination with mycotoxins may appear due to inappropriate practices during processing or resulting from invading mould in the final manufactured products. A sample treatment based on dispersive magnetic solid-phase extraction (DMSPE) has been proposed for emerging mycotoxin determination, enniatins (ENNs) and beauvericins (BEAs), in paprika. Different magnetic nanoparticles were tested, and cellulose-ferrite nanocomposite was selected for the extraction and preconcentration of the mycotoxins. Nanocomposite was characterised using field emission scanning electron microscopy and energy dispersive X-ray spectroscopy in terms of morphology and elemental composition. High-resolution mass spectrometry allowed the quantification of the five main emerging mycotoxins and the monitoring of unexpected members of this class of toxic fungal secondary metabolites. The method has been validated, obtaining limits of quantification between 9.5 and 9.9 μg kg−1 and testing its trueness through recovery studies, with satisfactory values of between 89.5 and 97.7%. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 8% were obtained in all cases. The analysis of 26 samples, including conventional and organic, demonstrated the presence of ENNB1 at 12.0 ± 0.6 μg kg−1 in one of the samples studied. Other analogues ENNs and BEAs were not detected.