Síntese de lipídeo estruturado a partir do óleo de abóbora (cucurbita pepo. L) extraído por via enzimática empregando celulases e pectinases
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Data
2021-02-26
Autores
Atsakou, Abra Eli
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Universidade Estadual Paulista (Unesp)
Resumo
Resumo: Os oleaginosos são matérias-primas ricas em óleos vegetais que têm interesse principalmente para os industriais de alimentos. Os óleos vegetais são potenciais fontes de vários componentes cuja as utilizações em diferentes áreas são demonstradas. A grande demanda pelo esse grupo de alimento é um desafio para as indústrias de alimentos procurando novos processos adequados para sua produção. Os processos convencionais mais usados (métodos físicos e químicos) têm limitações no rendimento de extração e na qualidade de óleo extraído. Objetivo: Esse trabalho visou fazer a extração aquosa enzimática do óleo de sementes de abóbora (Cucurbita pepo. L) a partir de celulase comercial e pectinase produzida pelo fungo Aspergillus sp. 391 em fermentação submersa. Além disso, produzir os lipídeos estruturados especificamente triacilglicerol dietético do tipo MLM pela reação de acidólise usando óleo de abóbora extraído e ácido cáprico (C10:0) em meio livre de solvente catalisada pela lipase imobilizada comercial Lipozyme RM IM ®. Métodos: A primeira etapa desse trabalho foi a otimização da produção de pectinase em função do crescimento celular do fungo Aspergillus sp.391 em fermentação submersa. Os efeitos da temperatura e pH durante a produção de pectinase foram avaliados. Em seguida, foram realizados experimentos para caracterização parcial de pectinase e celulase em função de pH, temperatura, energia de ativação e o tempo da meia vida. A segunda etapa foi a extração aquosa enzimática do óleo de sementes de abóbora (Cucurbita pepo. L). As condições reacionais foram estabelecidas a partir dos resultados obtidos das propriedades cinéticas das duas enzimas. A mistura de sementes de abóbora moída e enzima foi incubado a 50°C, pH 4 por 24h com agitação constante (120 rpm) onde as enzimas foram usadas separadamente e em combinação. A última etapa foi a produção de triacilglicerol dietético do tipo MLM pela reação de acidólise a partir do óleo de abóbora extraído com o ácido cáprico (C10:0) catalisada pela lipase imobilizada comercial Lipozyme RM IM® em meio livre de solvente. Um planejamento experimental foi estabelecido avaliando a influência da temperatura e razão molar (RM) óleo: ácido durante a maximização da incorporação do ácido cáprico. Resultados: Na primeira etapa, os resultados mostraram que as melhores condições para produção de pectinase foram pH 5,0 e 30ºC. A caracterização parcial das enzimas apresentou máximas atividades em pH 5 e 50°C para pectinase e em pH 5 e 60° C para celulase. Os resultados de estabilidade térmica mostraram que as enzimas foram mais estáveis a 50°C e apresentaram maior estabilidade em pH 4,0. A pectinase e a celulase apresentaram tempo de meia vida de (t1/2=27h) e (t1/2=26h) respectivamente a 50°C e demonstraram uma energia de ativação de 11,04 Kj/mol e 81,20 Kj/mol respectivamente. Na segunda etapa, os resultados mostraram que a combinação das duas enzimas foi considerada mais eficaz do que o uso separadamente, com 58,84% de rendimento do óleo. As propriedades físico-químicas do óleo extraído, como índice de acidez e composição de ácidos graxos, não foram significativamente afetadas (p>0,05) pelos diferentes métodos. Foi observada uma diferença significativa (p<0,05) de índice de peróxido entre o óleo extraído com enzima e o óleo extraído com solvente. O óleo de abóbora é rico em ácido linoleico (C18:2). Na última etapa, o óleo extraído pela combinação das enzimas foi escolhido para a síntese do triacilglicerol dietético do tipo MLM. Os resultados mostraram que o máximo grau de incorporação de ácido cáprico no óleo de abóbora após 24h pela reação de acidólise foi observado a 60°C com razão molar ácido cáprico: óleo de 9:1 com 47,99±0,5% no valor do grau de incorporação. Conclusão: O fungo Aspergillus sp.391 mostrou ser um microrganismo capaz de produzir a pectinase. O óleo de abóbora extraído pelo processo aquoso enzimático foi uma adequada matéria-prima usada para obtenção de triacilglicerol dietético do tipo MLM com propriedades nutricionais.
Oilseeds are raw materials rich in vegetable oils that are of interest mainly to food industry. Vegetable oils are potential sources of several components whose uses in different areas have been demonstrated. The great demand for this food group is a challenge for the food industries looking for new processes suitable for their production.The most used conventional processes (physical and chemical methods) have limitations in the extraction recovery and in the quality of extracted oil.Objective: This work aimed to carry out an aqueous enzymatic extraction of pumpkin seed oil (Cucurbita pepo L) with cellulase commercial and pectinase produced by the fungus Aspergillus sp. 391 in submerged fermentation. In addition, to produce the structured lipids specifically dietary triacylglycerol of the MLM-type by acidolysis using pumpkin oil extracted with capric acid (C10: 0) in solvent-free medium catalyzed by the commercial immobilized lipase Lipozyme RM IM ®. Methods: The first step of this work was the optimization of pectinase production depending of cell growth of the fungus Aspergillus sp.391 in submerged fermentation. The effects of temperature and pH during pectinase production were evaluated. Then, experiments were carried out for partial characterization of pectinase and cellulase depending of pH, temperature, activation energy and half-life time. The second step was the aqueous enzymatic oil extraction of pumpkin seed (Cucurbita pepo. L). The reaction conditions were established from the results obtained from the kinetic properties of the two enzymes. The mixture of pumpkin seeds crushed and enzyme was incubated at 50°C, pH 4 for 24h with constant agitation (120 rpm) where the enzymes were used either separately and in combination. The last step was the production of dietary triacylglycerol of the MLM-type by acidolysis from pumpkin oil extracted with capric acid (C10:0) catalyzed by the commercial immobilized lipase Lipozyme RM IM® in solvent-free medium. An experimental methodology was established evaluating the influence of temperature and molar ratio (RM) oil: acid during the maximization of the incorporation of capric acid. Results: In the first step, the results showed that the best conditions for pectinase production were pH 5.0 and 30ºC. The partial characterization of the enzymes showed maximum activities at pH 5 and 50°C for pectinase and at pH 5 and 60°C for cellulase. The results of thermal stability showed that the enzymes were more stable at 50°C and showed higher stability at pH 4.0. Pectinase and cellulase presented half-life time (t1/2=27h) and (t1/2=26h) respectively at 50°C and demonstrated an activation energy of 11.04 Kj /mol and 81.20 Kj /mol respectively. In the second step, the results showed that the combination of the two enzymes was considered more effective than the use separately, with 58.84% of oil recovery. The physiochemical properties of the extracted oil such as acidez value and fatty acid composition were not significantly affected (p>0.05) by the different methods. A significant difference (p <0.05) in the peroxide index was observed between the oil extracted with enzyme and the oil extracted with solvent. Pumpkin oil is rich in linoleic acid (C18:2). In the last step, the oil extracted by combination of the enzymes was chosen for the synthesis of dietary triacylglycerol of the MLM-type. The results showed that the maximum incorporation degree of capric acid in pumpkin oil after 24h by acidolysis reaction was observed at 60°C with acid capric: oil molar ratio 9:1 with 47.99 ± 0.5% in incorporation degree value. Conclusion: The fungus Aspergillus sp.391 proved to be a microorganism capable to produce pectinase. Pumpkin oil extracted by the aqueous enzymatic process was an adequate raw material used to obtain MLM-type dietary triacylglycerol presenting nutritional properties.
Oilseeds are raw materials rich in vegetable oils that are of interest mainly to food industry. Vegetable oils are potential sources of several components whose uses in different areas have been demonstrated. The great demand for this food group is a challenge for the food industries looking for new processes suitable for their production.The most used conventional processes (physical and chemical methods) have limitations in the extraction recovery and in the quality of extracted oil.Objective: This work aimed to carry out an aqueous enzymatic extraction of pumpkin seed oil (Cucurbita pepo L) with cellulase commercial and pectinase produced by the fungus Aspergillus sp. 391 in submerged fermentation. In addition, to produce the structured lipids specifically dietary triacylglycerol of the MLM-type by acidolysis using pumpkin oil extracted with capric acid (C10: 0) in solvent-free medium catalyzed by the commercial immobilized lipase Lipozyme RM IM ®. Methods: The first step of this work was the optimization of pectinase production depending of cell growth of the fungus Aspergillus sp.391 in submerged fermentation. The effects of temperature and pH during pectinase production were evaluated. Then, experiments were carried out for partial characterization of pectinase and cellulase depending of pH, temperature, activation energy and half-life time. The second step was the aqueous enzymatic oil extraction of pumpkin seed (Cucurbita pepo. L). The reaction conditions were established from the results obtained from the kinetic properties of the two enzymes. The mixture of pumpkin seeds crushed and enzyme was incubated at 50°C, pH 4 for 24h with constant agitation (120 rpm) where the enzymes were used either separately and in combination. The last step was the production of dietary triacylglycerol of the MLM-type by acidolysis from pumpkin oil extracted with capric acid (C10:0) catalyzed by the commercial immobilized lipase Lipozyme RM IM® in solvent-free medium. An experimental methodology was established evaluating the influence of temperature and molar ratio (RM) oil: acid during the maximization of the incorporation of capric acid. Results: In the first step, the results showed that the best conditions for pectinase production were pH 5.0 and 30ºC. The partial characterization of the enzymes showed maximum activities at pH 5 and 50°C for pectinase and at pH 5 and 60°C for cellulase. The results of thermal stability showed that the enzymes were more stable at 50°C and showed higher stability at pH 4.0. Pectinase and cellulase presented half-life time (t1/2=27h) and (t1/2=26h) respectively at 50°C and demonstrated an activation energy of 11.04 Kj /mol and 81.20 Kj /mol respectively. In the second step, the results showed that the combination of the two enzymes was considered more effective than the use separately, with 58.84% of oil recovery. The physiochemical properties of the extracted oil such as acidez value and fatty acid composition were not significantly affected (p>0.05) by the different methods. A significant difference (p <0.05) in the peroxide index was observed between the oil extracted with enzyme and the oil extracted with solvent. Pumpkin oil is rich in linoleic acid (C18:2). In the last step, the oil extracted by combination of the enzymes was chosen for the synthesis of dietary triacylglycerol of the MLM-type. The results showed that the maximum incorporation degree of capric acid in pumpkin oil after 24h by acidolysis reaction was observed at 60°C with acid capric: oil molar ratio 9:1 with 47.99 ± 0.5% in incorporation degree value. Conclusion: The fungus Aspergillus sp.391 proved to be a microorganism capable to produce pectinase. Pumpkin oil extracted by the aqueous enzymatic process was an adequate raw material used to obtain MLM-type dietary triacylglycerol presenting nutritional properties.
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Aspergillus sp, Óleo de sementes de abóbora (Cucurbita pepo L)., Lipídios estruturados, Lipase, Enzymatic aqueous extraction, Pumpkin seed oil (Cucurbita pepo. L), Structured lipids, Extração aquosa enzimática