Analytical study and exposure to Fusarium mycotoxins = Estudio analítico y de exposición a micotoxinas de Fusarium

Las micotoxinas son metabolitos secundarios de determinadas especies fúngicas que se encuentran habitualmente en los alimentos. El Reglamento No. 1881/2006 de la Comisión Europea, parcialmente modificado por posteriores Reglamentos, establece los contenidos máximos de micotoxinas para diferentes productos alimenticios. La introducción de estas normas pone de manifiesto el interés por el conocimiento de valores relativos a la concentración de estos tóxicos para poder evaluar riesgos y adoptar las medidas necesarias para proteger la salud de los consumidores. Los productos agrícolas contaminados, especialmente cereales y sus derivados, son la principal fuente de micotoxinas en la dieta de la población mundial. Es por ello que en la presente Tesis Doctoral se han desarrollado procedimientos analíticos rápidos, precisos y selectivos basados en la cromatografía de gases acoplada a espectrometría de masas en tándem (GC-MS/MS) con analizador de triple cuadrupolo para detectar y cuantificar micotoxinas de Fusarium en distintas matrices alimentarias, principalmente en las de elevado consumo. Las metodologías propuestas se validaron de acuerdo con las recomendaciones recogidas en las guías europeas obteniendo resultados satisfactorios. Se detectó la presencia de micotoxinas en un porcentaje importante de las muestras analizadas siendo los productos a base de trigo los que presentaron una mayor incidencia (67,6%); no obstante, las concentraciones halladas fueron inferiores a los contenidos máximos permitidos. También se detectó la presencia simultánea de micotoxinas en el 10,8% de las matrices alimentarias analizadas (n = 418) siendo la combinación más frecuente deoxinivalenol y la toxina HT-2. Con los resultados obtenidos se llevó a cabo una evaluación de la exposición combinando los datos de contaminación y los datos de consumo. En general se obtuvieron ingestas diarias de micotoxinas inferiores a las ingestas tolerables, y por tanto la exposición a estos contaminantes alimentarios no supondría un riesgo importante para el consumidor medio. Especial atención hay que prestar a grupos de población de menor edad ya que para los niños se obtuvieron datos de exposición a micotoxinas significativamente mayores, como consecuencia de un mayor consumo de cereales y menor peso corporal. Por otra parte, se desarrolló y validó un método de análisis para la determinación de micotoxinas y metabolitos en orina humana. Para ello, se aplicó una hidrólisis enzimática con β-glucuronidasa para separar los conjugados de azúcares y micotoxinas, productos formados como consecuencia de la fase II del metabolismo (principal vía de detoxificación de micotoxinas como el deoxinivalenol) y así identificar y confirmar la presencia de estos contaminantes alimentarios en orina. Los resultados mostraron presencia de micotoxinas en el 69% de las muestras analizadas (n = 55) siendo el deoxinivalenol la toxina más frecuente. La tasa de conjugación de esta micotoxina obtenida experimentalmente fue superior al 70%. Además se evaluó la exposición a las micotoxinas a partir de las concentraciones obtenidas de las muestras de orina de donde se dedujo una ingesta de deoxinivalenol superior al 50% de la ingesta diaria tolerable provisional e incluso casi una décima parte de los sujetos expuestos sobrepasó la ingesta tolerable. Se obtuvieron así valores de exposición mayores a los calculados a partir del análisis individual de las matrices alimentarias. Finalmente, teniendo en cuenta la correlación entre los niveles de ingesta y excreción de micotoxinas y a fin de reducir la incertidumbre asociada a la evaluación a través del análisis de alimentos, se propone llevar a cabo estudios de exposición a micotoxinas utilizando biomarcadores de exposición en orina.Mycotoxins are secondary metabolites produced by fungal species which can usually be found in foodstuffs. Regulation (EC) 1881/2006, partially amended by other Regulations, sets maximum contents of some mycotoxins in different foodstuffs allowing to evaluate risks and take actions to protect public health. Contaminated crops such as cereals and cereal-based products are the main source of mycotoxins and they represent one of the most consumed commodity. Therefore, in this PhD Thesis rapid and selective analytical procedures based on gas chromatography tandem mass spectrometry (GC-MS/MS), with triple quadrupole mass analyzer (QqQ), were developed in order to determine Fusarium mycotoxins in several food matrices, mainly in those widely consumed. Proposed methodologies were validated according to the European guidelines recommendations and satisfactory results were obtained. Occurrence of mycotoxins was found in an important percentage of samples. Wheat-based products showed the highest incidence of mycotoxins (67.6%); nonetheless, the mycotoxin levels found were lower than the maximum permitted contents. Co-occurrence of mycotoxins was also found in 10.8% of analyzed food matrices (n = 418), being deoxynivalenol and HT-2 toxin the most frequent combination found. An exposure assessment was carried out by combining food consumption data and the results of mycotoxin contamination here obtained. In general terms, mycotoxin daily intakes were lower than tolerable daily intakes and thus, the exposure to these food contaminants would not imply a risk for the average consumer. However, special attention should be paid to susceptible population groups such as children since they show a higher exposure due to their wide cereal consumption and their lower body weight. On the other hand, an analytical method for determining mycotoxins and metabolites in human urine was developed and validated. Moreover, an enzymatic hydrolysis by β-glucuronidase was applied to liberate mycotoxins from Phase II metabolism products. The cleavage of DON glucuronide by β-glucuronidase hydrolysis allowed the total DON quantitation. Results showed occurrence of mycotoxins in 69% of analyzed samples (n = 55) being deoxynivalenol the most frequent toxin. Conjugation rate here experimentally obtained was greater than 70%. The exposure to mycotoxins was also assessed throughout the obtained concentration from the analyzed urine samples. It was obtained a deoxynivalenol intake above 50% of the provisional tolerable daily intake in over 50% of the individuals. One tenth of exposed subjects exceeded the safety levels. So, exposure data obtained by urine analysis were higher than those calculated by food analysis. Finally, taken into account the correlation between intake and excretion of mycotoxins, and in order to reduce the uncertainty associated to the assessment through the analysis of food, mycotoxin biomarkers in urine are suggested to carry out exposure studies

Mycotoxins: An Under-evaluated Risk for Human Health

Mycotoxins are secondary toxic metabolites produced mainly by fungi belonging to the Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps genera. These moulds can colonize agricultural crops and produce mycotoxins during pre- and post-harvest practices, processing, and storage. Animals fed with feed contaminated with mycotoxins may be a natural and unwanted bioenhancer way to transfer mycotoxins, eventually metabolized, to animal-derived food addressed to humans. The natural occurrence of mycotoxins, also a low concentration, in food may cause adverse health effects in humans, rarely showing acute symptoms but the chronic exposure causes problems ranging from gastrointestinal and kidney disorders to immune deficiency and to develop some types of cancers. Human exposure to mycotoxins can happen by eating directly contaminated foods or through contaminated animal products. This alternative entry of mycotoxin into the human food chain is a signal of animals fed with contaminated feed. The exposure danger to mycotoxins can be monitored by following the biotransformation product occurrence in tissues and biological fluids, and these data are needed to evaluate their potential risk for humans, in particular for weak subpopulations like babies, children, old, or pressed by food security troubles. In this regard, the main aim of this volume is to evaluate the occurrence of mycotoxins and other contaminants in food, nutraceuticals, and biological fluids in order to ensure human safety. To guarantee effective consumer safety, reliable methods have been validated for the analysis of contaminants in various matrices. In addition, the risk associated with the assumption of contaminated food was assessed. Risk characterization is an indispensable aspect to safeguard public health, which helps to identify risks threatening consumers

Preliminary Estimation of Deoxynivalenol Excretion through a 24 h Pilot Study

A duplicate diet study was designed to explore the occurrence of 15 Fusarium mycotoxins in the 24 h-diet consumed by one volunteer as well as the levels of mycotoxins in his 24 h-collected urine. The employed methodology involved solvent extraction at high ionic strength followed by dispersive solid phase extraction and gas chromatography determination coupled to mass spectrometry in tandem. Satisfactory results in method performance were achieved. The method"s accuracy was in a range of 68%108%, with intra-day relative standard deviation and inter-day relative standard deviation lower than 12% and 15%, respectively. The limits of quantitation ranged from 0.1 to 8 μg/Kg. The matrix effect was evaluated and matrix-matched calibrations were used for quantitation. Only deoxynivalenol (DON) was quantified in both food and urine samples. A total DON daily intake amounted to 49.2 ± 5.6 μg whereas DON daily excretion of 35.2 ± 4.3 μg was determined. DON daily intake represented 68.3% of the established DON provisional maximum tolerable daily intake (PMTDI). Valuable preliminary information was obtained as regards DON excretion and needs to be confirmed in large-scale monitoring studie

Determination of deoxynivalenol in wheat-based snacks by gas chromatography-triple quadrupole tandem mass spectrometry

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal and cereal-based products, and a continuous monitoring of this toxin in foodstuffs is highly desirable. In this sense,a QuEChERS based extraction and gas chromatography-tandem mass spectrometry detection is proposed to determine DON in an appetizer largely consumed, the wheat-based snacks. In this study, a total of 40 samples were analyzed. The samples were divided into two groups based on the composition. Extraction was carried out with acetonitrile followed by a dispersive solid phase extraction and analyzed for DON content by gas chromatography-tandem mass spectrometry (GC-QqQ-MS/MS) method. The overall occurrence of samples with DON was 67.5%, with maximum content of 61μg/kg. In spite of its high incidence, DON concentrations found in samples were much lower than the maximum limit established in the current European legislation (500 μg/kg) for the foodstuff evaluated. Data obtained indicated a low exposure to DON through the consumption of this food commodity. Resumen: Determinación de deoxinivalenol en rosquilletas mediante cromatografía de gases acoplada a espectrometría de masas en tándem. El deoxinivalenol (DON) es la micotoxina producida por hongos del género Fusarium que con más frecuencia se detecta en cereales y productos a base de cereales. Por ello, es recomendable realizar una continua monitorización de su incidencia en los alimentos. Este trabajo propone un procedimiento analítico basado en una extracción tipo QuEChERS seguido de una cromatografía de gases acoplada a un detector de triple cuadrupolo para la determinación de DON en rosquilletas. Se analizaron un total de 40 muestras las cuales se dividieron según su composición en dos grupos. El DON fue identificado en el 67,5% de las muestras analizadas con un contenido máximo de 61 μg/kg. A pesar de su incidencia elevada, los niveles de DON hallados fueron muy inferiores a los límites máximos legislados en la actual legislación europea (500 μg/kg). Los resultados obtenidos muestran una baja exposición a DON a través del consumo de esta matriz alimentaria

Target analysis and retrospective screening of mycotoxins and pharmacologically active substances in milk using an ultra-high-performance liquid chromatography/high-resolution mass spectrometry approach

Milk is a nutritious food suitable for infants and adults, and it plays an important role in the human diet. However, it may also be a vehicle for food contaminants. In this report, we developed a method using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS; Thermo Fisher Scientific, Waltham, MA) for simultaneous identification of target pharmacologically active substances and mycotoxins in milk. We also used the Q-Orbitrap operating in full scan mode to identify other possible drugs and microbial metabolites that occurred in samples. Fifty-six commercially available milk samples from the Italian market were analyzed. Investigated analytes were extracted using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach. Method detection and quantification limits and performance criteria set by European regulations were fulfilled. Pharmacologically active substances were detected in 49% of samples (range 0.007–4.53 ng/mL), including nontarget mycotoxins. Retrospective analysis allowed us to identify other antibiotics and pharmacologically active substances, as well as nonregulated fungal/bacterial metabolites at a relatively high incidence. From the obtained values, the need for continuous monitoring of contaminants in the milk production chain is clear. This is the first study to assess the presence of pharmacologically active substances, mycotoxins, and other microbial metabolites in Italian milk samples using the UHPLC-Q-Orbitrap HRMS system

Effect of Phenolic Extract from Red Beans (Phaseolus vulgaris L.) on T-2 Toxin-Induced Cytotoxicity in HepG2 Cells

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells

Interiorización del método científico en alimentación sostenible mediante la experimentación en el aula

[ES] La alimentación sostenible hace referencia a aquella alimentación saludable que se adapta al entorno y la cultura, que disminuye el impacto ambiental, respeta los recursos naturales y la biodiversidad y es económicamente accesible. El alumnado que cursa alguno de los grados de Ciencias de la Alimentación debe ser consciente del concepto de sostenibilidad, integrarlo en su vida diaria, así como saber transmitirlo a la sociedad. Este alumnado busca adquirir conocimientos a través de metodologías diferentes y complementarias. La combinación de prácticas tradicionales y modernas son vitales para promover diferentes habilidades cognitivas en el estudiantado. Es importante aumentar su conocimiento y sus competencias en la resolución de problemas pero también lo es incrementar sus habilidades de razonamiento. Teniendo en cuenta que los y las estudiantes en Ciencias de la Alimentación deben trabajar desde la aplicación del método científico, una de las maneras más adecuadas de interiorizar este es llevándolo a la práctica. Por ello, en este proyecto se pretende que el alumnado sea capaz de plantear una hipótesis de trabajo en alimentación sostenible en consonancia con los ODS relacionados y llevar a cabo un taller práctico que potencie sus competencias comunicativas en el ámbito universitario.[EN] Sustainable food refers to healthy food that is adapted to the environment and culture, that reduces environmental impact, respects natural resources and biodiversity and is economically accessible. Students taking a degree in Food Science must be aware of the concept of sustainability, integrate it into their daily lives and know how to transmit it to society. These students want to acquire knowledge through different and complementary methodologies. The combination of traditional and modern practices are vital to promote different cognitive skills in students. It is important to increase their knowledge and problem solving competences but it is also important to increase their reasoning skills. Taking into account that students in Food Science must work from the application of the scientific method, one of the most appropriate ways to learn this method is by putting it into practice. Therefore, the aim of this project is to ensure that students are able to propose a working hypothesis on sustainable food in line with the related SDGs and to carry out a practical workshop that enhances their communicative skills in the university environment.Gandía Gómez, M.; Rodríguez-Carrasco, Y.; Cabrera-Pastor, A.; Pardo, E.; Gamero, A. (2023). Interiorización del método científico en alimentación sostenible mediante la experimentación en el aula. Editorial Universitat Politècnica de València. 581-589. https://doi.org/10.4995/INRED2023.2023.1653758158

Quantitative determination of trichothecenes in breadsticks by gas chromatography-triple quadrupole tandem mass spectrometry

Breadsticks are pencil-sized sticks of dry bread widely consumed as a pre-meal appetiser. They are basically wheat-based snacks, which makes them a good matrix to evaluate mycotoxin contamination, since wheat is very susceptible to fungal attack. In this sense, the fast, selective and sensitive gas chromatography-triple quadrupole tandem mass spectrometry (GCQqQ- MS/MS) method proposed here allows simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, nivalenol, neosolaniol, HT-2 and T-2 toxin in breadsticks after QuEChERS extraction and clean-up. The performance of the method was assessed with respect to European Commission Regulations by studying the selectivity and specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, matrix effect, accuracy, precision and trueness. Satisfactory results in terms of validation parameters were obtained for all selected mycotoxins (recovery range of 70110%, RSD 1). The trueness of the method was supported by using certified reference material (DON 1062 ± 110 μg kg1). The method was successfully used to evaluate the occurrence of the studied Fusarium toxins in 61 breadstick samples. A total of 64% of the samples showed mycotoxin contamination, DON being the most frequently detected toxin. Nonetheless, mean levels obtained were far below the maximum levels permitted by European Union legislation. An additional goal was to carry out a risk-characterisation approach to DON by comparing probable daily intake and provisional maximum tolerable daily intake (PMTDI)

Biomonitoring of Enniatin B1 and Its Phase I Metabolites in Human Urine: First Large-Scale Study

Enniatins (Enns) are mycotoxins produced by Fusarium spp. which are a fungus widely spread throughout cereals and cereal-based products. Among all the identified enniatins, Enn B1 stands as one of the most prevalent analogues in cereals in Europe. Hence, the aim of this study was to evaluate for the first time the presence of Enn B1 and its phase I metabolites in 300 human urine samples using an ultrahigh-performance liquid chromatography high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Enn B1 was detected in 94.3% of samples ranging from 0.007 to 0.429 ng/mL (mean value: 0.065 ng/mL). In accordance with previous in vitro and in vivo analysis, hydroxylated metabolites (78.0% samples) and carbonylated metabolites (66.0% samples) were tentatively identified as the major products. Results from this biomonitoring study point to a frequent intake of Enn B1 in the studied population, suggesting that in-depth toxicological studies are needed in order to understand the potential effects in humans

Development and Validation of a LC-ESI-MS/MS Method for the Determination of Alternaria Toxins Alternariol, Alternariol Methyl-Ether and Tentoxin in Tomato and Tomato-Based Products

Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are some of the main Alternaria mycotoxins that can be found as contaminants in food. In this work, an analytical method based on liquid chromatography (LC) tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of AOH, AME, and TEN in tomato and tomato-based products was developed. Mycotoxin analysis was performed by dispersive liquid-liquid microextraction (DLLME) combined with LC-ESI-MS/MS. Careful optimization of the MS/MS parameters was performed with an LC/MS system with the ESI interface in the positive ion mode. Mycotoxins were efficiently extracted from sample extract into a droplet of chloroform (100 µL) by DLLME technique using acetonitrile as a disperser solvent. Method validation following the Commission Decision No. 2002/657/EC was carried out by using tomato juice as a blank matrix. Limits of detection and quantitation were, respectively, in the range 0.7 and 3.5 ng/g. Recovery rates were above 80%. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤ 9% and ≤ 15%, respectively, at levels of 25 and 50 ng/g. Five out of 30 analyzed samples resulted positive to at least one Alternaria toxin investigated. AOH was the most common Alternaria toxin found, but at levels close to LOQ (average content: 3.75 ng/g)