Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-04T11:32:19Z No. of bitstreams: 1 2016 - Felipe Machado Trombete.pdf: 1613295 bytes, checksum: 957f0b025def400e446f7c3176c76b4c (MD5) / Made available in DSpace on 2017-01-04T11:32:19Z (GMT). No. of bitstreams: 1 2016 - Felipe Machado Trombete.pdf: 1613295 bytes, checksum: 957f0b025def400e446f7c3176c76b4c (MD5) Previous issue date: 2016-03-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Mycotoxins are natural contaminants produced by filamentous fungi and they are widely present in food. In wheat grains, the presence of trichothecenes produced by Fusarium spp. and also aflatoxins, produced by Aspergillus spp. are a serious public health problem because they are toxic metabolites to man and animals that remain stable during the industrial processes to which wheat is subjected when manufacturing derived products. One way to reduce such contaminants is to use ozone (O3) in food processing. Due to its high potential as an oxidant, O3 may react with mycotoxins and reduce their toxicity due to molecular degradation. This research aimed to evaluate the effects of different conditions of ozonation on the i) reduction in mycotoxins levels (deoxynivalenol and total aflatoxins); ii): reduction in total fungal count; iii) the effects on chemical, mineral and technological profiles and; iv) the possible differences sensorial profile of the flour obtained from the ozonized grains. In a second phase of this project, a methodology for determination of D3G (deoxynivalenol-3-glucoside), a masked form of DON, using High Performance Liquid Chromatography with photodiode array detector (HPLC-PDA) was optimized and in-house validated. Results obtained from ozonation study showed that O3 reduced total fungal count in approximately 3.0 cycles log CFU/g of wheat grain and deoxynivalenol and total aflatoxins contamination up to 64.3% and 48.0%, respectively. The gaseous ozonation can be applied without negatively changing the chemical, technological and sensory characteristics of the grains and can be considered an excellent method for remediation of fungal and mycotoxin contaminations. Also, the method optimized and in-house validated for determination of D3G by HPLC-PDA showed adequate results and, it could be considered an alternative to mass spectrometry determination of D3G in wheat grains. / Micotoxinas s?o contaminantes naturais, produzidos por fungos filamentosos e, podem ocorrer em altos n?veis nos alimentos. Nos gr?os de trigo, a presen?a de tricotecenos, um grupo de micotoxinas produzidas por Fusarium spp. e, aflatoxinas, produzidas por Aspergillus spp., representam um importante problema de sa?de p?blica por serem t?xicas ao homem e animais e muito est?veis aos processos no qual o trigo ? submetido para obten??o de produtos industrializados. Uma forma de reduzir a contamina??o dos alimentos por micotoxinas ? atrav?s do uso do oz?nio (O3) no processamento do alimento. Devido ao alto potencial oxidante do O3, esse pode degradar as mol?culas das micotoxinas, tendo como consequ?ncia a elimina??o ou redu??o de seus efeitos t?xicos. Essa pesquisa teve como objetivos principais avaliar os efeitos de diferentes condi??es de ozoniza??o na i) redu??o de micotoxinas (desoxinivalenol e aflatoxinas) em gr?os de trigo; ii) redu??o nos n?veis de fungos filamentosos; iii) influ?ncia nos par?metros qu?micos, perfil de minerais e par?metros tecnol?gico dos gr?os e da farinha obtida ap?s o processamento e; iv) influ?ncia nas caracter?sticas sensoriais da farinha elaborada a partir dos gr?os ozonizados. Em uma segunda etapa do projeto, um m?todo para determina??o de uma forma modificada do desoxinivalenol (DON), o desoxinivalenol-3-glicos?deo (D3G) foi otimizada e validada intralaboratorialmente, utilizando cromatografia l?quida de alta efici?ncia com detector de arranjo de diodos (CLAE-DAD). Os resultados obtidos dos ensaios de ozoniza??o demonstraram que o O3, nas condi??es experimentais utilizadas, reduziu a contagem de fungos totais em cerca 3,0 logs UFC/g de gr?os e a contamina??o por desoxinivalenol e aflatoxinas totais em at? 64,3 % e 48,0 %, respectivamente. O processo de ozoniza??o n?o influenciou de modo negativo a qualidade qu?mica, tecnol?gica e sensorial dos gr?os de trigo, podendo ser utilizado como um excelente m?todo para remedia??o da contamina??o dos gr?os por fungos e micotoxinas. Resultados adequados tamb?m foram obtidos na valida??o do m?todo de determina??o de D3G por CLAE-DAD, demonstrando que o m?todo ? confi?vel para a determina??o dessa forma mascarada do DON em gr?os e trigo e, pode ser utilizado como um m?todo alternativo a espectrometria de massas para tal an?lise.

No description available.

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses. / The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination.