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This work reports on the validation of a liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of more than 700 mycotoxins and other secondary fungal metabolites and plant toxins in pasta, biscuits, crackers and musli. The "dilute and shoot" approach was found to be fully applicable to these complex matrices, as only 7-14% of the analytes exhibited significant matrix effects while recoveries of the extraction were outside the target range of 70-120% for only 26 compounds. Data on repeatability (based on 7 brands per matrix) and on intermediate precision was compliant to the related < 20% criterion for 95-98% and 99% of all analytes, respectively. The limits of quantification were much lower than the related regulatory limits set for mycotoxins in cereal products. Application of the method to 157 samples from the European market revealed the presence of enniatins and deoxynivalenol in the majority of the samples. No regulatory limits were exceeded except the sum of ergot alkaloids being higher in a few samples than the 50-150 µg/kg to be implemented as of July 2024.
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Ochratoxin A (OTA) is a nephrotoxin that contaminates grains in storage. Moisture and temperature sensors give delayed responses due to their slow kinetic movement within the silo. This study examines if CO2 production could predict OTA contamination and identify storage conditions exceeding the maximum limit (5 µg/kg). The impact of water activity levels (0.70-0.90 aw), temperatures (15 and 20 °C), and storage duration on (a)Penicillium verrucosum population, (b)CO2 respiration rates (RR), and (c)ochratoxins concentrations in stored wheat was investigated. 96 samples were analysed for ochratoxins with LCMS-MS. RR was >7 times higher at wetter conditions than at drier aw levels. A positive correlation between CO2, OTA, OTB, and OTα was observed at the wettest conditions. OTA exceeded the limit at >0.80 aw (16% moisture content) with RR > 0.01 mg CO2 kg-1 h-1. The knowledge of the RR of stored grain would alert grain farmers/managers to improve grain storage management.
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Bread is an important staple food that is susceptible to spoilage, making it one of the most wasted foods. To determine the safety of partially moldy bread, five types of bread were inoculated with common mold species. After incubation, the metabolite profile was determined in and under the inoculation spot, as well as at a lateral distance of 3 cm from the moldy spot. The result showed that the metabolites were exclusively concentrated in the inoculation area and directly below the inoculation area. The only exception was citrinin, a mycotoxin produced by Penicillia such as Penicillium citrinum, which was detected in almost all tested bread areas when inoculated with the corresponding strains. The results of our study suggest that the removal of moldy parts may be a solution to reduce food waste if the remaining bread is to be used, for example for insect farming to produce animal feed.
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The presence of mycotoxins and other toxic metabolites in hops (Humulus lupulus L.) was assessed for the first time. In total, 62 hop samples were sampled in craft breweries, and analyzed by a multi-toxin LS-MS/MS method. The study collected samples from craft breweries in all of the Croatian counties and statistically compared the results. Based on previous reports on Alternaria spp. and Fusarium spp. contamination of hops, the study confirmed the contamination of hops with these toxins. Alternaria toxins, particularly tenuazonic acid, were found in all tested samples, while Fusarium toxins, including deoxynivalenol, were present in 98% of samples. However, no Aspergillus or Penicillium metabolites were detected, indicating proper storage conditions. In addition to the Alternaria and Fusarium toxins, abscisic acid, a drought stress indicator in hops, was also detected, as well as several unspecific metabolites. The findings suggest the need for monitoring, risk assessment, and potential regulation of Alternaria and Fusarium toxins in hops to ensure the safety of hop usage in the brewing and pharmaceutical industries. Also, four local wild varieties were tested, with similar results to the commercial varieties for toxin contamination, but the statistically significant regional differences in toxin occurrence highlight the importance and need for targeted monitoring.
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Alternaria , Contaminación de Alimentos , Fusarium , Humulus , Micotoxinas , Humulus/química , Humulus/microbiología , Micotoxinas/análisis , Contaminación de Alimentos/análisis , Alternaria/metabolismo , Fusarium/metabolismo , Espectrometría de Masas en Tándem , Croacia , Ácido Abscísico/análisis , Ácido Abscísico/metabolismoRESUMEN
Environmental factors influence fungal growth and mycotoxin production in stored grains. However, the concentrations of free mycotoxins and their conjugates and how they are impacted by different interacting environment conditions have not been previously examined. The objectives of this study were to examine the impact of storage conditions (0.93-0.98 aw) and temperature (20-25 °C) on (a) the concentrations of deoxynivalenol and zearalenone and their respective glucosides/conjugates and (b) the concentrations of emerging mycotoxins in both naturally contaminated and irradiated wheat grains inoculated with Fusarium graminearum. Contaminated samples were analysed for multiple mycotoxins using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Method validation was performed according to the acceptable performance criteria set and updated by the European Commission regulations No. 2021/808/EC. As an important conjugate of deoxynivalenol, the concentrations of deoxynivalenol-3-glucoside were significantly different from its precursor deoxynivalenol at 0.93 aw (22% moisture content- MC) at 25 °C in the naturally contaminated wheat with a ratio proportion of 56:44% respectively. The high concentrations of deoxynivalenol-3-glucoside could be influenced by the wheat's variety and/or harvested season/fungal strain type/location. Zeralenone-14-sulfate concentrations were surprisingly three times higher than Zearalenone in the naturally contaminated wheat at 0.98 aw (26% MC) at both temperatures. Emerging mycotoxins such as moniliformin increased with temperature rise with the highest concentrations at 0.95 aw and 25 °C. These findings highlight the influence and importance of storage aw x temperature conditions on the relative presence of free vs conjugated mycotoxins which can have implications for food safety.
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Maize grain samples collected from 129 small-scale farmers' stores in southern and southwestern Ethiopia were analysed by LC-MS/MS for a total of 218 mycotoxins and other fungal metabolites of which 15% were regulated mycotoxins. Mycotoxins produced by Penicillium, Aspergillus, and Fusarium accounted for 31%, 17%, and 12% of the metabolites, respectively. Most of the current samples were contaminated by masked and/or emerging mycotoxins with moniliformin being the most prevalent one, contaminating 93% of the samples. Each sample was co-contaminated by 3 to 114 mycotoxins/fungal metabolites. Zearalenone, fumonisin B1, and deoxynivalenol were the dominant mycotoxins, occurring in 78%, 61%, and 55% of the samples with mean concentrations of 243, 429, and 530 µg/kg, respectively. The widespread co-occurrence of several mycotoxins in the samples may pose serious health risks due to synergistic/additional effects.
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Contaminación de Alimentos , Fumonisinas , Micotoxinas , Espectrometría de Masas en Tándem , Zea mays , Zea mays/química , Zea mays/microbiología , Etiopía , Micotoxinas/análisis , Contaminación de Alimentos/análisis , Fumonisinas/análisis , Humanos , Zearalenona/análisis , Fusarium/química , Fusarium/metabolismo , Tricotecenos/análisis , Penicillium , Aspergillus , Almacenamiento de Alimentos , Cromatografía Liquida/métodos , CiclobutanosRESUMEN
This case report investigated the outbreak of aflatoxicosis in a dairy herd in Pakistan, which resulted in 30 abortions of 40 confirmed (75%) pregnant cows in a period of 35 days and in 18.8% depression of farm average milk production for the entire herd. The analysis of the concentrate feed of the total mixed ration (TMR), using enzyme-linked immunosorbent assay (ELISA) procedures from two different local laboratories, indicated concentrations of 60 µg/kg dry matter (DM) of aflatoxin B1 (AFB1) and 100 µg/kg DM of total aflatoxins (AFs: sum of B1, B2, G1 and G2). Subsequently, a confirmatory analysis with a more sensitive and validated multi-metabolite liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was performed. This analysis detected a concentration of total AFs in the TMR of 166 µg/kg DM ± 3.5 (AFB1:134, AFB2:17.4 and AFM1:14.9 µg/kg DM). The concentrate feed (55% of the TMR DM) was confirmed as a source of contamination, presenting a concentration >29 times higher than the EU-maximum limit value (5.68 µg/kg DM). Additionally, the multi-mycotoxin analysis evidenced the co-occurrence of 81 other toxic and potentially toxic fungal metabolites in the fed TMR. After replacing the contaminated concentrate feed with feedstuffs of the same formulation but from a new charge of ingredients, the abortion episodes ceased, and milk production increased significantly. In conclusion, the data of this case report suggest that AFs may be associated with pregnancy losses in dairy cattle and milk production depression. From the public health perspective, the data also indicate the need for a more careful examination of dairy animal feed in Pakistan. Since the high concentration of AFB1 detected in feed and considering the literature-reported transfer rates (1-6%) of this toxin to AFM1 (carcinogen for humans) in milk, the milk produced during the outbreak period is expected to be contaminated with AFM1, which raises public health concerns.
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Envenenamiento por Aflatoxinas , Brotes de Enfermedades , Leche , Animales , Bovinos , Femenino , Embarazo , Aborto Veterinario/epidemiología , Envenenamiento por Aflatoxinas/veterinaria , Alimentación Animal/análisis , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/inducido químicamente , Industria Lechera , Brotes de Enfermedades/veterinaria , Contaminación de Alimentos/análisis , Lactancia , Leche/química , Pakistán/epidemiología , Espectrometría de Masas en TándemRESUMEN
Fifty-four maize grain samples freshly harvested from subsistence farmers' fields in southwestern Ethiopia were analyzed for multiple mycotoxins using liquid chromatography-tandem mass spectrometric (LC-MS/MS) method following extraction by acetonitrile/water/acetic acid on a rotary shaker. The grain samples were contaminated with a total of 164 metabolites, of which Fusarium and Penicillium metabolites were the most prevalent accounting for 27 and 30%, respectively. All the major mycotoxins and derivatives except one (citrinin) were of Fusarium origin. Zearalenone was the most frequent major mycotoxin occurring in 74% of the samples at concentrations of 0.32-1310 µg/kg. It was followed by nivalenol (63%), zearalenone-sulfate (44%), and fumonisin B1 (41%). Nivalenol, nivalenol glucoside, and fusarenon-X were detected at unusually high levels of 8-1700 µg/kg, 21-184 µg/kg, and 33-149 µg/kg, respectively. Deoxynivalenol and DON-3 glucoside contaminated 32% of the samples, each at levels of 15.9-5140 µg/kg and 10-583 µg/kg, respectively. Moniliformin and W493B occurred in 96 and 22% samples at levels of 3.27-4410 µg/kg and 3-652 µg/kg, respectively. Fumonisins were also detected in the samples at levels of 9-6770 µg/kg (B1), 16-1830 µg/kg (B2), 9.5-808 µg/kg (B3), and 1.3-128 µg/kg (A1). This study confirmed the presence of an array of mycotoxins contaminating maize grains right from the field. The effect of the co-occurring mycotoxins on consumers' health should be investigated along with that of the newly emerging ones. Results of the current study call for application of pre-harvest mycotoxin mitigation strategies to safeguard maize-based food and feed.
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Contaminación de Alimentos , Micotoxinas , Espectrometría de Masas en Tándem , Zea mays , Zea mays/microbiología , Zea mays/química , Micotoxinas/análisis , Etiopía , Cromatografía Liquida , Contaminación de Alimentos/análisis , Fusarium/metabolismo , Fusarium/aislamiento & purificación , Agricultores , Grano Comestible/microbiología , Grano Comestible/química , Penicillium/aislamiento & purificación , Penicillium/metabolismoRESUMEN
BACKGROUND: Mycotoxin surveys play an essential role in our food safety system. The obtained occurrence data form the basis for the assessment of the exposure of humans and animals to these toxic fungal secondary metabolites. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become the gold standard for mycotoxin determination because it enables selective and sensitive multi-toxin analysis. Simultaneous determination of several hundreds of secondary fungal metabolites is feasible using this technique. In this study, we combined a targeted dilute-and-shoot LC-MS/MS-based multi-analyte approach with multivariate statistics for the analysis of Austrian wheat from two different years and different geographical origins. RESULTS: We quantified 47 secondary fungal metabolites, including regulated emerging and masked mycotoxins. The resulting multi-mycotoxin occurrence data were further analyzed using both multivariate and univariate statistics. Principal component analysis (PCA) and analysis of variance (ANOVA) simultaneous component analysis (ASCA) were employed to identify regional and yearly trends within the dataset and to quantify the variance in metabolite occurrence attributed to the different effects. In addition, secondary fungal metabolites significantly impacted by these factors were selected via ANOVA. Of the 47 secondary metabolites identified, 39 were affected by the year, region or a combined effect. Moreover, our findings show that 43 of the secondary fungal metabolites were significantly influenced by the weather conditions. CONCLUSION: The results presented in this study underline the added value of combining targeted LC-MS/MS with multivariate statistics for monitoring a broad spectrum of secondary fungal metabolites in food crops. Through multivariate statistics, trends associated with the year or region can be readily studied. The approach presented could pave the way for a better understanding of the impact of climate change on plant pathogenic fungi and its implications for food safety. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.
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Fusarium , Enfermedades de las Plantas , Tricotecenos , Triticum , Triticum/microbiología , Triticum/metabolismo , Fusarium/patogenicidad , Fusarium/genética , Fusarium/metabolismo , Tricotecenos/metabolismo , Virulencia , Enfermedades de las Plantas/microbiología , Micotoxinas/metabolismo , DepsipéptidosRESUMEN
An HPLC-MS/MS multi-class method for quantitation of 15 different classes of veterinary drug residues (>140 analytes) in milk and poultry feed was developed and validated. Accuracy criteria for routine laboratories were met for the majority of analytes, > 83 % in milk and between 50 and 60 % in chicken feed, with an apparent recovery of 60-140 %. Extraction efficiency criteria were met for >95 % of the analytes for milk and > 80 % for chicken feed. Intermediate precision meets the SANTE criterion of RSD < 20 % for 80-90 % of the analytes in both matrices. For all analytes with an existing MRL in milk, the LOQ was below the related MRL. Twenty-nine samples of commercial milk and chicken feed were analyzed within the interlaboratory comparison. No residues of veterinary drugs were found in the milk samples. However, the feed samples exhibited high levels of nicarbazin, salinomycin, and decoquinate.
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Alimentación Animal , Residuos de Medicamentos , Contaminación de Alimentos , Leche , Drogas Veterinarias , Animales , Bovinos , Alimentación Animal/análisis , Pollos , Residuos de Medicamentos/análisis , Contaminación de Alimentos/análisis , Cromatografía Líquida con Espectrometría de Masas , Leche/química , Aves de Corral , Espectrometría de Masas en Tándem , Drogas Veterinarias/análisisRESUMEN
The climate crisis further exacerbates the challenges for food production. For instance, the increasingly unpredictable growth of fungal species in the field can lead to an unprecedented high prevalence of several mycotoxins, including the most important toxic secondary metabolite produced by Fusarium spp., i.e., deoxynivalenol (DON). The presence of DON in crops may cause health problems in the population and livestock. Hence, there is a demand for advanced strategies facilitating the detection of DON contamination in cereal-based products. To address this need, we introduce infrared attenuated total reflection (IR-ATR) spectroscopy combined with advanced data modeling routines and optimized sample preparation protocols. In this study, we address the limited exploration of wheat commodities to date via IR-ATR spectroscopy. The focus of this study was optimizing the extraction protocol for wheat by testing various solvents aligned with a greener and more sustainable analytical approach. The employed chemometric method, i.e., sparse partial least-squares discriminant analysis, not only facilitated establishing robust classification models capable of discriminating between high vs low DON-contaminated samples adhering to the EU regulatory limit of 1250 µg/kg but also provided valuable insights into the relevant parameters shaping these models.
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BACKGROUND: Many developing countries, Namibia included, have a high prevalence of malnutrition among children, especially in rural subsistence farming areas where inadequate food supply is common. Poor diets in children under 5 years may result in negative health impacts. This study determined the association of food consumption patterns and nutritional status of children under 5 years from rural households in Oshana and Oshikoto regions in Namibia. METHOD: Employing a cross-sectional descriptive design, 377 children under 5 years participated in this study using purposive sampling. Validated dietary diversity and food frequency questionnaires were used to obtain information on demographic characteristics, commonly consumed food per week, and meal frequencies for the recruited children. Anthropometric measurements were obtained to assess nutritional status of children using Emergency Nutrition Assessment (ENA) software. Descriptive and inferential statistics were computed using the IBM® SPSS® Statistics (Statistical Package for Social Sciences) version 27. RESULTS: Staple foods, mostly grains, roots and tubers, along with flesh foods, legumes and nuts were commonly consumed. Vitamin A-rich fruits and vegetables were solely consumed in Oshana region (10.7%) and not in Oshikoto. Oshana exhibited a lower dietary diversity score (4±1 SD) compared to Oshikoto (5±1 SD). The prevalence of adequate feeding practices varied, with Oshana having 38.8% meeting minimum milk feeding frequency (MMFF), 55.6% minimum dietary diversity (MDD), 69.8% minimum meal frequency (MMF), and 27% minimum acceptable diet (MAD). In Oshikoto, these figures were lower at 2%, 7%, 32%, and 0.5%, respectively. Stunting, underweight, wasting, and overweight were also documented, with slight differences between the two regions. The study did not find association between nutritional status and MMFF, MDD and MAD. However, significant associations were found between specific food types, amount of food, breastfeeding length, MMF and malnutrition indicators in both regions (p<0.05). CONCLUSION: Most study participants consumed locally available staple foods. Stunting, underweight, and wasting were prevalent among children in the two regions which were significantly associated to the amount of food consumed, MMF and/ some food types. Improving food environments and eliminating access barriers to diversified diets can mitigate high prevalence of malnutrition among rural children.
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Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusarium sporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3-O-alpha-glucoside and HT-2-4-O-alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomyces cerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium.
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Fusarium , Micotoxinas , Oryza , Toxina T-2/análogos & derivados , Humanos , Glucósidos/metabolismo , Fusarium/metabolismo , Oryza/metabolismo , Micotoxinas/metabolismoRESUMEN
In the original publication [...].
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The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.
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Genes Bacterianos , Ríos , Animales , Humanos , Antibacterianos/farmacología , Antibacterianos/análisis , Aguas Residuales , Farmacorresistencia Microbiana/genética , Agua/análisisRESUMEN
In this study, secondary metabolites produced by Alternaria were investigated for their presence in milling oats. For this purpose, pre-cleaned milling oat samples (n = 193), intended for human consumption, out of harvest years 2017 to 2021 originating from different northern European countries were analysed by LC-MS/MS. Alternariol and alternariol methyl ether were positively identified in 38% of the samples with mean values of 2.1 µg/kg and 1.2 µg/kg, respectively. The highest concentrations of 50.5 µg/kg alternariol and 24.2 µg/kg of alternariol methyl ether were detected in a Latvian sample. Tenuazonic acid was found in 45% of all samples, with a mean concentration of 28.9 µg/kg and a maximum concentration of 1430 µg/kg, also in a Latvian sample. Tentoxin was detected in 49% of all samples with a mean value of 1.7 µg/kg. The Alternaria metabolite most frequently detected in 96% of all samples was infectopyrone with a mean concentration of 593 µg/kg and a maximum value reaching up to 3990 µg/kg in a German sample. In addition, eight oat samples were selected to investigate to what extent the Alternaria metabolites are distributed between the oat hulls and the oat kernels. After de-hulling, approximately 23% of Alternaria metabolites were found in the remaining oat kernels. According to the results, alternariol, infectopyrone and altersetin were present in the kernels with the lowest proportion of 10%-20% on average, respectively. The values for tentoxin showed that about 60% of tentoxin was contained in the hulls, while almost 40% remained in the oat kernel. This suggests that potential health risks posed by Alternaria secondary metabolites and metabolites of other fungal genera in milling oats can be reduced by de-hulling.
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Lactonas , Éteres Metílicos , Micotoxinas , Humanos , Micotoxinas/análisis , Avena , Alternaria/metabolismo , Cromatografía Liquida , Contaminación de Alimentos/análisis , Espectrometría de Masas en Tándem , Grano Comestible/química , Éteres Metílicos/análisis , Éteres Metílicos/metabolismoRESUMEN
Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.
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Fusarium , Fusarium/genética , Histonas/genética , Histonas/metabolismo , Silenciador del GenRESUMEN
BACKGROUND: The increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. RESULTS: We describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg-1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. SIGNIFICANCE: Our findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with material costs of 2.5, relies on biodegradable 3D printing filament and can be produced with consumer-grade printers, making the prototype readily available. As a future perspective, the modular character of our developed tool kit will allow for adaptation to other hard food commodities beyond the determination of DON in wheat.
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Micotoxinas , Micotoxinas/análisis , Cromatografía Liquida/métodos , Triticum/química , Espectrometría de Masas en Tándem/métodos , Contaminación de Alimentos/análisisRESUMEN
Pastures are used for grazing and the production of conserved roughage in horses. Yet, the nutritional profile of the forage varies from spring to late summer, affecting equine nutrient supply and health. In addition, environmental factors may also favor plant contaminants such as mycotoxins. This study aimed to determine the nutritional profile and contaminant load of selected horse pastures from early spring till late summer. The nutrient composition (main macronutrients, macro elements and trace elements), as well as mycotoxins, metabolites, pesticides, and plant-derived compounds of seven horse pastures were analyzed. Each pasture was sampled three times and the samples were categorized according to the status of the pasture plants: ear emergence, early- till full bloom, and drought-damaged vegetation. Drought-damaged pastures demonstrated a rise in the acid to neutral detergent fiber ratio, calcium, iron, and magnesium but lower potassium contents. Mycotoxins and other contaminants were found in the pastures including 64 fungal compounds (ergot alkaloids (13) and metabolites from Fusarium (21), Aspergillus (2), Penicillium (8), Alternaria (8) and other fungal species (12), one bacterial metabolite (cereulide), twelve plant metabolites (including eight phytoestrogens and three cyanogenic glycosides (linamarin, lotaustralin and prunasin)), 11 nonspecific metabolites and six pesticides. Fusarium metabolites showed the highest concentrations among the fungal metabolites and drought-induced stress increased the contamination levels (range: 123-3873 µg/kg DM). In conclusion, there was a dominant effect of the developmental stages of the plants, botanical composition of the pastures and weather conditions on the nutritional composition and presence of contaminants on pastures.