Produção de pigmentos pelo fungo antártico Pseudogymnoascus sp. 6DC415-I: otimização, purificação e identificação
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Data
2022-07-29
Autores
Silva, Isabela Fernanda da
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Universidade Estadual Paulista (Unesp)
Resumo
A demanda por pigmentos naturais vem crescendo devido a efeitos nocivos de alguns corantes sintéticos e a busca por formas mais sustentáveis de consumo. Nesse contexto, pigmentos fúngicos fornecem uma boa alternativa aos sintéticos. As comunidades microbianas antárticas são de grande importância para a produção de metabólitos secundários, tais como os pigmentos, pois são compostos importantes para a adaptação desses organismos ao ambiente extremo em que vivem. Além disso, devido ao seu isolamento, essas comunidades podem ser uma fonte rica de novos produtos naturais com estruturas químicas únicas. Este trabalho visou analisar a produção dos pigmentos vermelhos pelo fungo Pseudogymnoascus sp. 6DC415-I, isolado de sedimentos marinhos da Antártica, bem como otimizá-la, seguida da purificação e identificação dos pigmentos produzidos. Para tal, avaliou-se a produção dos pigmentos vermelhos nas temperaturas de 15 e 25 ºC e nos pHs 3, 5, 7 e 9 em meio líquido Malte 2% por 20 dias de incubação a 150 rpm. Nas condições de 15 ºC e pH 5 se obteve a maior produção dos pigmentos, avaliada na leitura de absorbância em 500 nm, sendo que a 25 ºC não se detectou produção de pigmentos vermelhos. O fungo foi cultivado em meio Malte Ágar 1% e os pigmentos produzidos no meio de cultura foram extraídos com acetona. O extrato bruto foi submetido a cromatografia de camada reversa C18 para sua purificação, na qual obteve-se três frações vermelhas eluídas em Metanol 90 %. As frações foram submetidas às análises de RMN de 1H e FT-IR e obteve-se a confirmação da purificação de duas delas como compostos muito similares. Os espectros dessas frações indicaram tratarem-se de compostos da classe das melaninas, da categoria das feomelaninas. A atividade antibacteriana do extrato bruto e das frações obtidas foi testada contra as bactérias Xanthomonas citri, X. passiflorae, Escherichia coli, Streptococcus aureus e Bacillus subtilis, através do método de microdiluição em poços e leitura da D.O600, não sendo observada nenhuma inibição de crescimento superior a 50%. Também avaliou-se a atividade antioxidante das mesmas amostras, por meio do método de captura de DPPH, com resultado de IC50 a 1000 µg/mL para a fração 1 e 1143 µg/mL para a fração 2. Apesar do gênero Pseudogymnoascus sp. ser bastante comum ao ambiente antártico, até o momento, não há muitos estudos referentes à sua produção de pigmentos, principalmente de feomelanina. O pigmento vermelho extracelular e solúvel em água produzido pelo Pseudogymnoascus sp. 6DC415-I pode então representar um potencial para aplicações biotecnológicas.
The demand for natural pigments has been growing due to the harmful effects of some synthetic dyes and the search for more sustainable forms of consumption. In this context, fungal pigments provide a good alternative to synthetics ones. Antarctic microbial communities are of great importance for the production of secondary metabolites, such as pigments, due to their role in helping these organisms adapt to the extreme environment in which they live. Furthermore, because of their isolation, these communities can be a rich source of new products with unique structures. This study aimed to analyze the production of red pigments by the fungus Pseudogymnoascus sp. 6DC415-I, isolated from antarctic marine sediments, as well as optimizing it, followed by purification and identification of the pigments produced. In order to do that, the production of red pigments was evaluated at temperatures of 15 and 25 ºC and at pHs 3, 5, 7 and 9 in 2% Malt broth for 20 days of incubation at 150 rpm. The biggest production of pigments obtained, quantified in the absorbance reading at 500 nm, was at 15 ºC and pH 5. No red pigments production was detected at 25 ºC. The fungus was cultivated in 1% Malte Agar medium and pigments produced were extracted from the culture medium with acetone. The crude extract was subjected to C18 reversed-phase chromatography for its purification, in which three red fractions were eluted in 90 % methanol. The fractions were submitted to 1H NMR and FT-IR analysis. Two of them were purified and were similar compounds. According to their spectra, the fractions are melanins, belonging to the pheomelanin category. The antibacterial activity of the crude extract and fractions was tested against the bacteria Xanthomonas citri, X. passiflorae, Escherichia coli, Streptococcus aureus e Bacillus subtilis, using the microdilution in wells method and reading of their O.D600. No growth inhibition greater than 50% was observed. The antioxidant activity of the samples was also evaluated, using the DPPH scavenging activity method, with an IC50 result of 1000 µg/mL for fraction 1 and 1143 µg/mL for fraction 2. Although the genus Pseudogymnoascus sp. is quite common in the Antarctic environment, there are not many studies regarding its production of pigments so far, specially pheomelanin. The extracellular, water-soluble red pigment produced by Pseudogymnoascus sp. 6DC415-I could then represent a potential for biotechnological applications.
The demand for natural pigments has been growing due to the harmful effects of some synthetic dyes and the search for more sustainable forms of consumption. In this context, fungal pigments provide a good alternative to synthetics ones. Antarctic microbial communities are of great importance for the production of secondary metabolites, such as pigments, due to their role in helping these organisms adapt to the extreme environment in which they live. Furthermore, because of their isolation, these communities can be a rich source of new products with unique structures. This study aimed to analyze the production of red pigments by the fungus Pseudogymnoascus sp. 6DC415-I, isolated from antarctic marine sediments, as well as optimizing it, followed by purification and identification of the pigments produced. In order to do that, the production of red pigments was evaluated at temperatures of 15 and 25 ºC and at pHs 3, 5, 7 and 9 in 2% Malt broth for 20 days of incubation at 150 rpm. The biggest production of pigments obtained, quantified in the absorbance reading at 500 nm, was at 15 ºC and pH 5. No red pigments production was detected at 25 ºC. The fungus was cultivated in 1% Malte Agar medium and pigments produced were extracted from the culture medium with acetone. The crude extract was subjected to C18 reversed-phase chromatography for its purification, in which three red fractions were eluted in 90 % methanol. The fractions were submitted to 1H NMR and FT-IR analysis. Two of them were purified and were similar compounds. According to their spectra, the fractions are melanins, belonging to the pheomelanin category. The antibacterial activity of the crude extract and fractions was tested against the bacteria Xanthomonas citri, X. passiflorae, Escherichia coli, Streptococcus aureus e Bacillus subtilis, using the microdilution in wells method and reading of their O.D600. No growth inhibition greater than 50% was observed. The antioxidant activity of the samples was also evaluated, using the DPPH scavenging activity method, with an IC50 result of 1000 µg/mL for fraction 1 and 1143 µg/mL for fraction 2. Although the genus Pseudogymnoascus sp. is quite common in the Antarctic environment, there are not many studies regarding its production of pigments so far, specially pheomelanin. The extracellular, water-soluble red pigment produced by Pseudogymnoascus sp. 6DC415-I could then represent a potential for biotechnological applications.
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Palavras-chave
Fungos antárticos, Pigmentos naturais, Purificação, Biotecnologia, Antarctic fungi, Natural pigments, Purification, Biotechnology, Feomelanina, Pheomelanin