Metabolic pathways of degradation of wine coloured monomeric compounds by yeasts
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2018-11-07
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As antocianinas são substâncias hidrossolúveis, pertencentes ao grupo dos compostos fenólicos, também conhecidos como polifenóis. São responsáveis pela cor dos vinhos tintos, e são sintetizados durante o metabolismo secundário das plantas. Estão bem descritas na literatura algumas características importantes destes compostos, nomeadamente a atividade antioxidante, o seu efeito sinérgico com outros compostos fenólicos contra a peroxidação lipídica, a atuação como potente anti-inflamatório e também a atividade antimicrobiana. A biodegradação é um processo de transformação biológica de substâncias utilizando microrganismos que as convertem em produtos finais não degradáveis, idealmente CO2 e H2O. Os processos de biodegradação são realizados principalmente por bactérias e fungos devido à sua capacidade de metabolizar uma grande variedade de compostos. Alguns géneros de leveduras podem degradar compostos fenólicos usando-os como fonte de carbono, nomeadamente o género Candida.
Este trabalho visou propor uma via ou metabolismo de degradação de compostos monoméricos corados encontrados no vinho, especificamente a malvidina-3-O-monoglucósido (Mv3G), e a identificação dos produtos resultantes da sua degradação.
Um isolado selvagem da espécie Candida oleophila foi utilizado como modelo biológico, para verificar se os compostos corados encontrados no vinho seguem a mesma via metabólica de degradação que os derivados do benzeno. Pretendeu-se ainda verificar se os produtos resultantes apresentam toxicidade. Foram testadas três concentrações diferentes de Mv3G como fonte única de carbono e energia para o crescimento do isolado, durante um ensaio de biodegradação, com amostragem nos dias 0, após 7 dias e após 14 dias.
Os resultados mostraram que a levedura foi responsável pela remoção de cerca de 30% da concentração inicial de Mv3G para as concentrações superiores testadas (1,0 g/L, 0,5 g/L) e cerca de 14% na menor concentração testada (0,25 g/L). As amostras resultantes do ensaio de biodegradação foram analisadas por HPLC-DAD-MS e o principal metabolito identificado foi o ácido siríngico, juntamente com alguns glucósidos de chalcona. Simultaneamente com esta análise, foi confirmada a atividade da catecol-2,3-dioxigenase, indicando uma meta-clivagem do anel dos compostos fenólicos resultantes do processo de biodegradação.
Neste trabalho foi ainda pesquisada a atividade biológica da Mv3G, assim como dos metabolitos resultantes da sua biodegradação. Os resultados mostraram atividadeantimicrobiana contra Salmonella enteritidis, com valores de MIC a variar entre 0,25 e 0,5 mg/mL. O composto (Mv3G) à concentração de 0,6 g/L apresentou elevada toxicidade, causando redução da viabilidade celular em cerca de 95% nas linhas celulares Caco-2 e HaCaT. O efeito genotóxico ao nível do dano ao DNA também foi elevado, com identificação de danos severos no DNA em várias células.
Anthocyanins are water-soluble compounds, pertaining to phenolic compounds, also known as polyphenols, responsible for the colour of red wines, and synthetized during the secondary metabolism of plants. It is quite well described in literature some important features of these compounds namely the antioxidant activity, their synergic effect with other phenolic compounds against lipid peroxidation, the action as potent anti-inflammatory compound, and also antimicrobial activity. Biodegradation is a process of biological transformation of substances using microorganisms that convert them into non-degradable final products, ideally CO2 and H2O. Biodegradation processes mostly use bacteria and fungi due to their ability to degrade a wide variety of compounds. Some yeast genera may degrade phenolic compounds using them as carbon source, namely the genus Candida. This work aimed the proposal of the degradation metabolism of coloured monomeric compounds found in wine, specifically malvidin-3-O-monoglucoside (Mv3G), and the identification of the resulting degradation products. A wild isolate of the species Candida oleophila was used as biological model, to ascertain if coloured compounds found in wine follow the same metabolic pathway of degradation as benzene derivatives. It was also intended to check whether the resulting products present toxicity. Three different concentrations of Mv3G were tested as single carbon and energy source for the growth of the isolate, during a biodegradation assay, with sampling at days 0, after 7 days and after 14 days. The results showed that the yeast was responsible for the removal of about 30% of the initial concentration of Mv3G for the highest concentrations tested (1.0 g/L, 0.5 g/L), and about 14% in the lowest concentration tested (0.25 g/L). The samples resulting from the biodegradation assay were analyzed by HPLC-DAD-MS, and the main metabolite identified was syringic acid, along with some chalcone glucosides. Simultaneously with this analysis the activity of catechol-2,3-dioxygenase was confirmed, indicating a meta-ring cleavage of the phenolic compounds resultant from the biodegradation process. This work also searched for biological activities of Mv3G and biodegradation resulting metabolites. The results showed antimicrobial activity against Salmonella enteritidis, with a measured MIC value varying from 0.25 and 0.5 mg/mL. The compound (Mv3G) atconcentrations of 0.6 g/L showed high toxicity causing inhibition in cell viability of about 95% in Caco-2 and HaCaT cell lines. The genotoxic effect at DNA damage level was also high, with identification of severe damages in DNA in several cells.
Anthocyanins are water-soluble compounds, pertaining to phenolic compounds, also known as polyphenols, responsible for the colour of red wines, and synthetized during the secondary metabolism of plants. It is quite well described in literature some important features of these compounds namely the antioxidant activity, their synergic effect with other phenolic compounds against lipid peroxidation, the action as potent anti-inflammatory compound, and also antimicrobial activity. Biodegradation is a process of biological transformation of substances using microorganisms that convert them into non-degradable final products, ideally CO2 and H2O. Biodegradation processes mostly use bacteria and fungi due to their ability to degrade a wide variety of compounds. Some yeast genera may degrade phenolic compounds using them as carbon source, namely the genus Candida. This work aimed the proposal of the degradation metabolism of coloured monomeric compounds found in wine, specifically malvidin-3-O-monoglucoside (Mv3G), and the identification of the resulting degradation products. A wild isolate of the species Candida oleophila was used as biological model, to ascertain if coloured compounds found in wine follow the same metabolic pathway of degradation as benzene derivatives. It was also intended to check whether the resulting products present toxicity. Three different concentrations of Mv3G were tested as single carbon and energy source for the growth of the isolate, during a biodegradation assay, with sampling at days 0, after 7 days and after 14 days. The results showed that the yeast was responsible for the removal of about 30% of the initial concentration of Mv3G for the highest concentrations tested (1.0 g/L, 0.5 g/L), and about 14% in the lowest concentration tested (0.25 g/L). The samples resulting from the biodegradation assay were analyzed by HPLC-DAD-MS, and the main metabolite identified was syringic acid, along with some chalcone glucosides. Simultaneously with this analysis the activity of catechol-2,3-dioxygenase was confirmed, indicating a meta-ring cleavage of the phenolic compounds resultant from the biodegradation process. This work also searched for biological activities of Mv3G and biodegradation resulting metabolites. The results showed antimicrobial activity against Salmonella enteritidis, with a measured MIC value varying from 0.25 and 0.5 mg/mL. The compound (Mv3G) atconcentrations of 0.6 g/L showed high toxicity causing inhibition in cell viability of about 95% in Caco-2 and HaCaT cell lines. The genotoxic effect at DNA damage level was also high, with identification of severe damages in DNA in several cells.
Descrição
Dissertation of Master degree in Biochemistry
Palavras-chave
malvidina-3-O-monoglucósido , Candida oleophila , vias metabólicas , HPLC-DAD-MS