Imobilização de lipase em criogel supermacroporoso para a síntese de lipídios estruturados
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2013-12-04
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Universidade Federal de Viçosa
Resumo
A exigência atual por alimentos com elevada qualidade nutricional e sensorialmente atraentes têm tornado a tecnologia enzimática uma alternativa cada vez mais atrativa. Neste contexto, a modificação da composição dos triacilgliceróis por meio de reações enzimáticas de interesterificação é uma alternativa para a obtenção de lipídios com características nutricionais, sensoriais e tecnológicas superiores. Para esse propósito é interessante a aplicação de lipases (E.C.3.1.1.3) na sua forma imobilizada devido a vantagens como a reutilização do biocatalisador, a facilidade de separação do produto final e o potencial para aplicações em processos contínuos. Recentemente, os criogéis supermacroporosos têm despontado como potenciais suportes para a imobilização de enzimas por apresentar características de interesse tecnológico como excelente biocompatibilidade, baixo custo de aquisição e alta porosidade de estrutura, o que facilita o processo de escoamento e de transferência de massa. Assim, neste trabalho foi desenvolvido um novo biorreator enzimático constituído de lipases de Aspergillus niger (ANL) confinadas por meio do processo de polimerização in situ em um criogel de poliacrilamida supermacroporoso, visando a síntese de lipídios estruturados. Inicialmente, as lipases de Aspergillus niger em sua forma livre foram caracterizadas em relação a sua atividade hidrolítica e de esterificação na síntese do butirato de butila, um aroma de interesse industrial. Para isso, empregou-se um delineamento composto central de face centrada (DCCF) a fim de avaliar o efeito dos parâmetros reacionais temperatura (°C), razão molar de substrato (ácido butírico:n-butanol) e conteúdo de água (% ( v/v)) adicionada sobre o rendimento de esterificação (Y). Verificou-se que o uso de uma maior concentração molar de n-butanol em relação à concentração de ácido butírico proporcionou um efeito positivo sobre Y, enquanto que o aumento da temperatura foi prejudicial para a síntese do aroma. A adição de água também favoreceu o rendimento de esterificação (%). Por meio da metodologia de superfície de resposta (MSR) verificou-se que um rendimento máximo de esterificação (98,8%) foi obtida na condição de temperatura de 40,0 °C, razão molar de substrato de 1:2,41 (ácido:álcool) e conteúdo de água adicionada de 1,05% (v/v). Em seguida, as lipases foram imobilizadas por meio do confinamento em um criogel monolítico supermacroporoso. O sistema imobilizado (biorreator) foi caracterizado em termos das suas propriedades morfológicas e parâmetros de imobilização. Também foi avaliado o efeito da adição de aditivos estabilizantes (polietilenoglicol de massa molar média de 1500 g/mol, Triton X-100 e Tween 80), durante a etapa de imobilização, nos parâmetros de imobilização e propriedades morfológicas. O uso de aditivos estabilizantes não alterou as propriedades morfológicas dos criogéis (p>0,05), enquanto que o emprego do aditivo triton X-100 resultou em um aumento significativo (p<0,05) na atividade hidrolítica aparente (U/gcriogel), no rendimento de imobilização (%) e no conteúdo de proteína imobilizada (mgproteína/gcriogel). Avaliou-se também o desempenho do biorreator na síntese do butirato de butila e em reações de interesterificação. Por meio MSR verificou-se que o rendimento máximo de esterificação (46,1%) na síntese do butirato de butila foi obtido na condição reacional de 40,0 °C, razão molar de substrato de 1:1,43 (ácido:álcool) e conteúdo de água adicionada de 65% (v/v). O maior rendimento de interesterificação (17,35%) foi obtido no meio reacional contendo 15 mmol/L de tripalmitina e 15 mmol/L trioleína, conteudo de água adicionada de 35% (v/v) e a temperatura de 40 °C. Estes resultados indicam que o biorreator desenvolvido é um promissor biotecnológicas. biocatalisador para aplicações.
The current requirements for foods with high nutritional quality and sensory attractive has become the enzyme technology an attractive alternative compared to the conventional chemical processes. In this context, the modification of the triglycerides composition through enzymatic interesterification reactions is an alternative for obtaining lipids with high nutritional, sensory and technological properties. For this purpose is interesting the use of lipases (E.C.3.1.1.3) in its immobilized form due to advantages such as the reuse of the biocatalyst, ease separation of the final product and the potential for applications in continuous processes. Recently, the supermacroporous cryogels have emerged as potential supports for enzyme immobilization for presenting characteristics of technological interest as excellent biocompatibility, low cost and highly porous structure which facilitates the flow process and mass transfer. In this work we developed a new enzymatic bioreactor consisting of Aspergillus niger (ANL) lipase entrapped into a polyacrylamide based supermacroporous cryogels by "in situ" polymerization for the synthesis of structured lipids. Initially, free lipases from Aspergillus niger were characterized in terms to their hydrolytic and esterification activity in the synthesis of butyl butyrate, an flavor of industrial interest. For this purpose, it was used a central composite face-centered design (CCFD) in order to evaluate the effect of reaction parameters temperature (°C), substrate molar ratio (butyric acid:n-butanol) and added water (% (v/v)) on the esterification yield (Y). It has found that the use of a higher molar concentration of n-butanol in relation to the molar concentration of butyric acid promoted a positive effect on Y while increasing the temperature was detrimental for the flavor synthesis. The addition of water also favored the esterification yield (%). Through the Response Surface Methodology (RSM) it was found that a maximum yield of esterification (98.8%) was obtained in the condition of temperature at 40.0 °C, substrate molar ratio of 1:2.41 (acid:alcohol) and added water of 1.05 % (v/v). Then, lipases were entrapped in a supermacroporoso monolith cryogel. The immobilized system (bioreactor) was characterized in terms of their morphological properties and immobilization parameters. The effect of addition of stabilizing additives (polyethylene glycol of molar mass 1500 g/mol, Triton X-100 and Tween 80) in the parameters of immobilization and morphological properties was also evaluated. The use of stabilizing additives did not alter the morphological properties of cryogels (p>0.05), while the use of the additive Triton X-100 resulted in a significant increase (p<0.05) in the apparent hydrolytic activity (U/gcryogel), immobilization yield (%) and the immobilized protein content (mgprotein/gcryogel). It was also evaluated the performance of the bioreactor in the synthesis of butyl butyrate and interesterification reactions. Through the RSM it was found that the maximum yield of esterification (46.1%) in the synthesis of butyl butyrate was obtained in the reaction condition at 40.0 °C, substrate molar ratio of 1:1.43 (acid:alcohol) and added water of 65 % (v/v). A higher interesterification yield (17.35 %) was obtained in the reaction medium containing 15 mmol/L of tripalmitin and 15 mmol/L of triolein, added water content of 35 % (v/v) and 40 °C. These results indicated that the bioreactor developed is a promising biocatalyst for biotechnological applications.
The current requirements for foods with high nutritional quality and sensory attractive has become the enzyme technology an attractive alternative compared to the conventional chemical processes. In this context, the modification of the triglycerides composition through enzymatic interesterification reactions is an alternative for obtaining lipids with high nutritional, sensory and technological properties. For this purpose is interesting the use of lipases (E.C.3.1.1.3) in its immobilized form due to advantages such as the reuse of the biocatalyst, ease separation of the final product and the potential for applications in continuous processes. Recently, the supermacroporous cryogels have emerged as potential supports for enzyme immobilization for presenting characteristics of technological interest as excellent biocompatibility, low cost and highly porous structure which facilitates the flow process and mass transfer. In this work we developed a new enzymatic bioreactor consisting of Aspergillus niger (ANL) lipase entrapped into a polyacrylamide based supermacroporous cryogels by "in situ" polymerization for the synthesis of structured lipids. Initially, free lipases from Aspergillus niger were characterized in terms to their hydrolytic and esterification activity in the synthesis of butyl butyrate, an flavor of industrial interest. For this purpose, it was used a central composite face-centered design (CCFD) in order to evaluate the effect of reaction parameters temperature (°C), substrate molar ratio (butyric acid:n-butanol) and added water (% (v/v)) on the esterification yield (Y). It has found that the use of a higher molar concentration of n-butanol in relation to the molar concentration of butyric acid promoted a positive effect on Y while increasing the temperature was detrimental for the flavor synthesis. The addition of water also favored the esterification yield (%). Through the Response Surface Methodology (RSM) it was found that a maximum yield of esterification (98.8%) was obtained in the condition of temperature at 40.0 °C, substrate molar ratio of 1:2.41 (acid:alcohol) and added water of 1.05 % (v/v). Then, lipases were entrapped in a supermacroporoso monolith cryogel. The immobilized system (bioreactor) was characterized in terms of their morphological properties and immobilization parameters. The effect of addition of stabilizing additives (polyethylene glycol of molar mass 1500 g/mol, Triton X-100 and Tween 80) in the parameters of immobilization and morphological properties was also evaluated. The use of stabilizing additives did not alter the morphological properties of cryogels (p>0.05), while the use of the additive Triton X-100 resulted in a significant increase (p<0.05) in the apparent hydrolytic activity (U/gcryogel), immobilization yield (%) and the immobilized protein content (mgprotein/gcryogel). It was also evaluated the performance of the bioreactor in the synthesis of butyl butyrate and interesterification reactions. Through the RSM it was found that the maximum yield of esterification (46.1%) in the synthesis of butyl butyrate was obtained in the reaction condition at 40.0 °C, substrate molar ratio of 1:1.43 (acid:alcohol) and added water of 65 % (v/v). A higher interesterification yield (17.35 %) was obtained in the reaction medium containing 15 mmol/L of tripalmitin and 15 mmol/L of triolein, added water content of 35 % (v/v) and 40 °C. These results indicated that the bioreactor developed is a promising biocatalyst for biotechnological applications.
Descrição
Palavras-chave
Criogel, Imobilização, Lipase, Lipídeos estruturados, Cryogel, Immobilization, Lipase, Structured lipids
Citação
ALCÂNTARA, Lizzy Ayra Pereira. Lipase immobilization in supermacroporous cryogel for the synthesis of structured lipids. 2013. 104 f. Tese (Doutorado em Ciência de Alimentos; Tecnologia de Alimentos; Engenharia de Alimentos) - Universidade Federal de Viçosa, Viçosa, 2013.