Hidrólise enzimática de hemicelulose do pseudocaule de bananeira com endoxilanase I de Aspergillus versicolor para produção de xilo-oligossacarídeos e avaliação do seu efeito prebiótico
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Data
2019-02-22
Autores
Freitas, Caroline de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Hemiceluloses são polissacarídeos com ligações do tipo β-1,4 sendo o principal tipo encontrado em gramíneas as xilanas, estruturas com cadeia principal de xilose. O pseudocaule de bananeira é um resíduo agrícola da colheita de banana que apresenta em média 19,1 % de xilana em sua composição. Xilanas apresentam grande potencial econômico e uma importante aplicação dessa macromolécula é a produção de xilo-oligossacarídeos (XOS). XOS são oligômeros de xilose, reconhecidos por seu efeito prebiótico, além de possuírem diversas propriedades biológicas que promovem a saúde. A produção de XOS pode ser realizada por hidrólise enzimática através da ação de endoxilanase na cadeia da xilana. No entanto, é importante que a hemicelulose seja extraída da biomassa lignocelulósica e que o extrato enzimático seja livre de β-xilosidase, enzima que hidrolisa XOS a xilose. Tendo em vista o aumento da demanda de compostos que previnem doenças e promovem a saúde, o objetivo desse trabalho foi a produção de xilo-oligossacarídeos, a partir da hidrólise enzimática de hemiceluloses extraídas de pseudocaule de bananeira, usando a endoxilanase I de Aspergillus versicolor e avaliar a ação prebiótica dos compostos produzidos. A xilanase I de Aspergillus versicolor foi produzida pelo crescimento do microrganismo em meio contendo xilana proveniente de farelo de trigo. A enzima foi purificada por cromatografia de troca iônica (DEAE-Sephadex A-50) e cromatografia de exclusão molecular (Sephadex G-75). Previamente a extração de hemicelulose do pseudocaule de bananeira, foram avaliados diferentes pré-tratamentos/lavagens para remoção de extrativos. A extração da hemicelulose foi realizada com peróxido de hidrogênio a 6 % (m/v) em meio alcalino (pH 11,6). Após a extração da hemicelulose, as amostras foram quimicamente caracterizadas e o rendimento foi maior após o pré-tratamento alcalino com NaOH. A reação de hidrólise foi realizada variando-se os parâmetros de carga enzimática (10 UI/g, 30 UI/g, 60 UI/g, 100 UI/g e 200 UI/g), concentração de substrato (2%, 4%, 6%, 8% e 10% m/v) e tempo de reação (2h, 4h, 6h, 8h, 16h, 24h e 48h), a fim de se obter as melhores condições para produção de XOS. A maior quantidade de XOS foi obtida pela hidrólise realizada com carga enzimática de 30 UI/g, 8% de substrato e tempo de reação de 24 horas, produzindo 20,51 g/L de XOS, a maioria deles xilopentoses/xilohexoses e não houve produção de xilose. A ação prebiótica dos XOS produzidos foi avaliada através do crescimento de duas bactérias probióticas e uma bactéria patogênica em meio contendo XOS como fonte de carbono. Ambas as cepas probióticas utilizadas demonstraram habilidade em utilizar XOS de cadeia longa para seu crescimento, assim como também a cepa patogênica. Dessa maneira, pode-se afirmar que foi possível produzir compostos prebióticos (XOS) através da hidrólise enzimática de hemicelulose de pseudocaule de bananeira, utilizando a endoxilanase I de Aspergillus versicolor.
Hemicelluloses are polysaccharides with β-1,4 bonds and the main type found in grasses is xylan, xylose main chain structures. The banana pseudostem is an agricultural residue of the banana crop that presents on average 19.1% of xylan in its composition. Xylans present great economic potential and an important application of this macromolecule is the production of xylooligosaccharides (XOS). XOS are sugar oligomers composed of xylose units recognized for their prebiotic effect, besides having diverse biological properties that promote the health. The production of XOS can be carried out by enzymatic hydrolysis through the action of endoxylanase on the xylan chain. However, it is important that the hemicellulose is extracted from the lignocellulosic biomass and that the enzyme extract is free from β-xylosidase, an enzyme that hydrolyzes XOS to xylose. Because of the increase demand for compounds that prevent disease and promote health, the objective of this study was xylo-oligosaccharides production from the enzymatic hydrolysis of hemicelluloses extracted from banana pseudostem using Aspergillus versicolor endoxylanase I and evaluate the prebiotic action of the compounds produced. A xylanase from Aspergillus versicolor was produced by the growth of the microorganism in medium containing xylan (wheat bran). The enzyme was purified through ion exchange chromatography (DEAE-Sephadex A-50) and molecular exclusion chromatography (Sephadex G-75). Pior to the hemicellulose extraction from banana pseudostem, different pre-treatments/washes were evaluated for the removal of extractives. The hemicellulose extraction was performed with 6% (m/v) hydrogen peroxide in alkaline medium (pH 11,6). After extraction, the samples were chemically characterized and the higher yield of hemicellulose was obtained after alkaline pre-treatment with NaOH. The hydrolysis reaction was performed by varying the parameters of enzymatic concentration (10 IU/g, 30 IU/g, 60 IU/g, 100 IU/g and 200 IU/g), substrate concentration (2%, 4%, 6%, 8% and 10%) and reaction time (2h, 4h, 6h, 8h, 16h, 24h and 48h), in order to obtain the best conditions for XOS production. The final enzymatic hydrolysis was performed with enzymatic concentration of 30 IU / g, 8% of substrate and 24 hours reaction time, producing 20.51 g / L of XOS, most of them xylopentaose and xylohexaose and there was no xylose production. The prebiotic action of the XOS produced was evaluated by the growth of two probiotic and a pathogenic bacterias in medium containing XOS as carbon source. Both probiotic strains demonstrated the ability to use long chain XOS for their growth, as well as the pathogenic strain. It can be affirmed that it was possible to produce prebiotic compounds (XOS) through the enzymatic hydrolysis of banana pseudostem hemicellulose.
Hemicelluloses are polysaccharides with β-1,4 bonds and the main type found in grasses is xylan, xylose main chain structures. The banana pseudostem is an agricultural residue of the banana crop that presents on average 19.1% of xylan in its composition. Xylans present great economic potential and an important application of this macromolecule is the production of xylooligosaccharides (XOS). XOS are sugar oligomers composed of xylose units recognized for their prebiotic effect, besides having diverse biological properties that promote the health. The production of XOS can be carried out by enzymatic hydrolysis through the action of endoxylanase on the xylan chain. However, it is important that the hemicellulose is extracted from the lignocellulosic biomass and that the enzyme extract is free from β-xylosidase, an enzyme that hydrolyzes XOS to xylose. Because of the increase demand for compounds that prevent disease and promote health, the objective of this study was xylo-oligosaccharides production from the enzymatic hydrolysis of hemicelluloses extracted from banana pseudostem using Aspergillus versicolor endoxylanase I and evaluate the prebiotic action of the compounds produced. A xylanase from Aspergillus versicolor was produced by the growth of the microorganism in medium containing xylan (wheat bran). The enzyme was purified through ion exchange chromatography (DEAE-Sephadex A-50) and molecular exclusion chromatography (Sephadex G-75). Pior to the hemicellulose extraction from banana pseudostem, different pre-treatments/washes were evaluated for the removal of extractives. The hemicellulose extraction was performed with 6% (m/v) hydrogen peroxide in alkaline medium (pH 11,6). After extraction, the samples were chemically characterized and the higher yield of hemicellulose was obtained after alkaline pre-treatment with NaOH. The hydrolysis reaction was performed by varying the parameters of enzymatic concentration (10 IU/g, 30 IU/g, 60 IU/g, 100 IU/g and 200 IU/g), substrate concentration (2%, 4%, 6%, 8% and 10%) and reaction time (2h, 4h, 6h, 8h, 16h, 24h and 48h), in order to obtain the best conditions for XOS production. The final enzymatic hydrolysis was performed with enzymatic concentration of 30 IU / g, 8% of substrate and 24 hours reaction time, producing 20.51 g / L of XOS, most of them xylopentaose and xylohexaose and there was no xylose production. The prebiotic action of the XOS produced was evaluated by the growth of two probiotic and a pathogenic bacterias in medium containing XOS as carbon source. Both probiotic strains demonstrated the ability to use long chain XOS for their growth, as well as the pathogenic strain. It can be affirmed that it was possible to produce prebiotic compounds (XOS) through the enzymatic hydrolysis of banana pseudostem hemicellulose.
Descrição
Palavras-chave
Xilana, Pseudocaule de bananeira, Xilanase, Xilo-oligossacarídeos, Prebióticos, Xylan, Banana pseudostem, Xylanase, Xylooligosaccharides, Prebiotics