Evaluación del peligro potencial y real de la presencia de ocratoxina A, tricotecenos B y patulina en trigo y manzana mediante técnicas microbiológicas y cromatográficas

Las micotoxinas son metabolitos secundarios producidos por hongos filamentosos capaces de crecer en gran variedad de sustratos, contaminando con frecuencia alimentos, en especial, los de origen vegetal, pudiendo producir una respuesta tóxica en animales o seres humanos después de la ingestión del alimento contaminado. Los principales hongos productores de micotoxinas pertenecen a los géneros Penicillium, Aspergillus, Fusarium y Alternaria que pueden producir entre otras, las micotoxinas: alternarioles, aflatoxinas, zearalenona, patulina, ocratoxinas y tricotecenos. Dada la peligrosidad de la ingesta de micotoxinas, la Unión Europea, preocupada por la calidad y salubridad de los alimentos, ha aprobado el Reglamento (CE) 1881/2006 de la Comisión en el que controla la presencia de micotoxinas en productos alimentarios que llegan al consumidor. De entre las diferentes micotoxinas las que están adquiriendo mayor relevancia debido a que están siendo encontradas en gran variedad de sustratos son: la ocratoxina A (OTA) y los tricotecenos tipo B, así como la patulina (PAT). La OTA es nefrotóxica, nefrocarcinogénicas, inmunosupresora, teratogénica y posiblemente genotóxica. Los niveles más altos de incidencia de OTA se han encontrado en cereales y productos derivados, así como en uva y derivados. Los tricotecenos pueden producir diferentes efectos tóxicos en animales y humanos como rechazo del alimento, vomito, hemorragia, anemia, immunosupresión, reducción de ganancia de peso y diarrea. También son unos potentes inhibidores de la síntesis de la proteína. El desoxinivalenol es el más frecuente tricoteceno detectado en muestras de cereal, aunque también se puede encontrar nivalenol, 3- y 15-acetildesoxinivalenol. Todas estas micotoxinas están clasificadas como tricotecenos tipo B. La patulina, produce hiperemia, congestión y lesiones hemorrágicas, especialmente en el tracto digestivo; así como náuseas y vómitos. La PAT puede detectarse en frutas, hortalizas, cereales y piensos. Para la determinación de las micotoxinas se utilizan técnicas cromatográficas que ofrecen los mejores resultados, sin embargo es imprescindible su optimización para adaptarlas a cada matriz, así como de un método previo de preparación, extracción y purificación adecuado para cada tipo de sustrato. La tesis doctoral está enfocada a: i) La realización de estudios de la micobiota presente en trigo y manzana. ii) La evaluación de la incidencia de especies fúngicas potencialmente productoras de OTA y tricotecenos tipo B en trigo y de patulina en manzana. iii) La mejora de los métodos de detección de la OTA, tricotecenos tipo B y patulina en los productos a estudiar. Para ello se realiza un estudio de la metodología existente y se procede al análisis comparativo y a una posterior optimización con procedimientos desarrollados en nuestro laboratorio. iv) La optimización de técnicas de análisis sensibles, fiables y reproducibles para la determinación de las toxinas en los cereales y manzanas y en sus derivados considerados mediante GC, LC, GC-MS, LC-MS. v) El análisis de los tricotecenos tipo B y ocratoxina A en trigo y sus derivados como pan, y comprobar su incidencia y cantidad de toxina encontrada para establecer el grado de peligrosidad para el consumo humano, y comparar los resultados con los de otros países. De igual modo se abordó el estudio de la patulina para la manzana y sus derivados. vi) Los estudios de la influencia de la temperatura de incubación, actividad de agua y tipo de aislado en la producción de tricotecenos tipo B, OTA y patulina. vii) El desarrollo de métodos predictivos para poder predecir la existencia de micotoxinas y poder eliminar riesgos controlando los diversos factores de la manipulación del trigo. viii) Los estudios sobre el uso de fitosanitarios con respecto al control de la presencia de estas especies fúngicas contaminantes en las materia primas y el efecto que estos pueden ejercer sobre la producción de diferentes micotoxinas.The mycotoxins are secondary metabolites produced by filamentous fungi able to grow in great variety of substrates, contaminating frequently foods such as wheat and apple. These foods can produce a toxic effects in humans. The main producing fungi of mycotoxins belong to the genera Penicillium, Aspergillus and Fusarium that can produce among others, patulin, ochratoxin and trichothecene mycotoxins. Research carried out in the development of the Doctoral Thesis has permitted: i) Mycobiota study of wheat coming from the Spanish market with special attention to the potentially producing species of ochratoxin A (OTA), nivalenol, deoxynivalenol, and 3–acetyldeoxynivalenol and 15–acetyldeoxynivalenol and mycobiota study of apple especially by Penicillium expansum, the main patulin producing species. ii) Critical study and optimization of chromatographic methods for ochratoxin, type–B trichothecene and patulin determination in vitro culture media of the main producing fungal species. iii) Chemotyping of isolates belonging to species that are potential producers of ochratoxins, type–B trichothecenes and patulin in wheat and apple. iv) Study of the influence of physicochemical factors on mycelial growth of the fungi and mycotoxin production. v) Design and application of response surface models to predict deoxynivalenol accumulation by Fusarium graminearum and ochratoxin A accumulation by Aspergillus ochraceus in wheat in different conditions. vi) Optimization of the methodology for determining of ochratoxin A and type–B trichothecenes in wheat and wheat products, and of patulin in apple and apple products. Analysis of foodstuffs in Spain. vii) Stability study of ochratoxin A and type– B trichothecenes for baking process. viii) Critical study of mycotoxin multi–detection methods by LC–MS in wheat flour. Preparation and optimization of an analytical method to determine the maximum number of mycotoxins present in wheat flour

On-line database of voltammetric data of immobilized particles for identifying pigments and minerals in archaeometry, conservation and restoration (ELCHER database)

[EN] A web-based database of voltammograms is presented for characterizing artists' pigments and corrosion products of ceramic, stone and metal objects by means of the voltammetry of immobilized particles methodology. Description of the website and the database is provided. Voltammograms are, in most cases, accompanied by scanning electron microphotographs, X-ray spectra, infrared spectra acquired in attenuated total reflectance Fourier transform infrared spectroscopy mode (ATR-FTIR) and diffuse reflectance spectra in the UV-Vis-region. For illustrating the usefulness of the database two case studies involving identification of pigments and a case study describing deterioration of an archaeological metallic object are presented. (C) 2016 Elsevier B.V. All rights reserved.Research was conducted within the "Grupo de analisis cientifico de bienes culturales y patrimoniales y estudios de ciencia de la conservacion" Microcluster of the University of Valencia Excellence Campus. Financial support is gratefully acknowledged from the MINECO Projects CTQ2014-53736-C3-1-P and CTQ2014-53736-C3-2-P which are also supported with ERDF funds. The authors would like to thank to Gonzalo Girones Sarrio manager of GongDisseny Co. by the technical support for building the site structure and the structure of the database, Archbishop of Valencia, Dr. Ignacio Bosch Reig and Dr. Pilar Roig Picazo directors of the intervention project in the Basilica de la Virgen de los Desamparados de Valencia, the conservator Estrella Arcos Von Haartman (Quibla Restaura Company) and City Council Town of Malaga, the Museum of Archaeology of Xativa, its director Angel Velasco and the conservators Isabel Martinez Lazaro and Betlem Martinez for facilitating access to samples as well as Manuel Planes Insausti and Dr Jose Luis Moya Lopez technical supervisors of the Electron Microscopy Service of the Universitat Politecnica de Valencia where were carried out SEM-EDX analyses.Domenech-Carbo, A.; Domenech Carbo, MT.; Valle-Algarra, FM.; Gimeno-Adelantado, J.; Osete Cortina, L.; Bosch-Reig, F. (2016). On-line database of voltammetric data of immobilized particles for identifying pigments and minerals in archaeometry, conservation and restoration (ELCHER database). Analytica Chimica Acta. 927:1-12. https://doi.org/10.1016/j.aca.2016.04.052S11292

Study of Spanish Grape Mycobiota and Ochratoxin A Production by Isolates of Aspergillus tubingensis and Other Members of Aspergillus Section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time

Rapid characterization of potential ochratoxin-producing fungi isolated from grapes and study of natamycin efficacy for its control

Fungal flora on ripe grape and fungi characterization are very critical for assessing the risk of OTA presence in wine, a mycotoxin classified as possible carcinogen to humans. In the present work, the PCR-RFLP technique has been applied to the ITS1- 5.SS-ITS2 region of the rDNA to carry out the characterization of potential OTA producing species of Aspergillus section Nigri from different grape varieties grown in Spain. The toxin production levels of the different isolates have also been analysed and the ability of natamycin, a fungicide, to control fungal development and OTA production in vitro has been studied, as well as the interaction with environmental factors {water activity, temperature). The results obtained from in vitro cultures of 205 isolates of species in Aspergillus section Nigri showed that 74.2 o of A. carbonarius isolates and 14.3 o of A. tubingensis isolates were capable of producing OTA at different levels. No isolate of A. niger showed OTA producing capacity under the experimental conditions. A. carbonarius was the most frequently isolated ochratoxigenic species. The effect of natamycin against isolates of A. carbonarius has been studied according to different level, water activity and temperature. It can be seen that the efficacy of very low concentrations of this fungicide on fungal growth depends on water activity and temperature

Composition and Color of Maya Blue: Reexamination of Literature Data Based On the Dehydroindigo Model

[EN] An analysis of literature data studying the composition and color of Maya blue (MB) type materials prepared from indigo, dehydroindigo, and different aluminosilicates, accompanied by new spectral data, is presented. After thermal treatment at above 100 degrees C, indigo-based specimens displayed Raman and UV-vis spectroscopic features common to those of equivalent dehydroindigo-based replicants, thus supporting the socalled dehydroindigo model (J. Phys. Chem. B 2006, 110, 6027-6039) in which the dehydroindigo/indigo ratio, increasing with temperature, is crucial to determine the color of MB and its variability. The current analysis supports the view of MB as a polyfunctional hybrid material just characterized by the presence of different organic components, each one distributed in topological isomers differently attached to the clay support. On the basis of the comparison of spectral data for different channeled silicates (among others, palygorskite, sepiolite, and montmorillonite clays, and different zeolites), we proposed a view on the color characteristics of Maya blue-type specimens as defined by the compromise between trapping ability for indigo molecules and facility to promote the oxidation of indigo to dehydroindigo.Project CTQ2017-85317-C2-1-P, supported with Ministerio de Economia, Industria y Competitividad (MINECO), Fondo Europeo de Desarrollo Regional (ERDF), and Agencia Estatal de Investigacion (AEI) is gratefully acknowledged.Doménech-Carbó, A.; Holmwood, S.; Di Turo, F.; Montoya, N.; Valle-Algarra, FM.; Edwards, HG.; Domenech Carbo, MT. (2019). Composition and Color of Maya Blue: Reexamination of Literature Data Based On the Dehydroindigo Model. The Journal of Physical Chemistry C. 123(1):770-782. https://doi.org/10.1021/acs.jpcc.8b08448770782123

Redox Tuning and Species Distribution in Maya Blue-Type Materials: A Reassessment

Maya Blue-type specimens prepared from indigo (1 wt %) plus kaolinite, montmorillonite, palygorskite, sepiolite, and silicalite are studied. Liquid chromatography with diode array detection, ultra-performance liquid chromatography coupled with mass spectrometry, and pyrolysis-silylation gas chromatography–mass spectrometry analyses of the extracts from these specimens combined with spectral and solid-state voltammetry, electrochemical impedance spectroscopy, and scanning electrochemical microscopy techniques provide evidence for the presence of a significant amount of dehydroindigo and isatin accompanying indigo and other minority organic compounds in all samples. Solid-state electrochemistry data permits the estimatation of indigo loading in archeological Maya Blue, which is in the range of 0.2 to 1.5 wt %. These results support a view of ‘genuine’ Maya Blue-type materials as complex polyfunctional organic–inorganic hybrids