RESUMO
Naringin and limonin are the two main bitter compounds of citrus products such as grapefruit juice. The aim of this investigation was to evaluate the reduction in both bitter components simultaneously using a combined biochemical and physical approach. The proposed strategy was based on the use of heterofunctional supports with glyoxyl groups that allow for the covalent immobilization of naringinase, which hydrolyses naringin and alkyl groups that allow for the adsorption of limonin. The supports were butyl-glyoxyl agarose (BGA) and octyl-glyoxyl agarose (OGA), which were characterized in terms of aldehyde group quantification and FTIR analysis. The optimal pH and temperature of free and immobilized enzymes were assessed. The maximum enzyme loading capacity of supports was analyzed. Debittering of grapefruit juice was evaluated using soluble enzyme, enzyme-free supports, and immobilized catalysts. Enzyme immobilized in BGA reduced naringin and limonin concentrations by 54 and 100%, respectively, while the use of catalyst immobilized in OGA allowed a reduction of 74 and 76%, respectively, obtaining a final concentration of both bitter components under their detection threshold. The use of OGA biocatalyst presented better results than when soluble enzyme or enzyme-free support was utilized. Biocatalyst was successfully applied in juice debittering in five repeated batches.
Assuntos
Citrus paradisi , Limoninas , Adsorção , Estabilidade Enzimática , Enzimas Imobilizadas/química , Flavanonas , Hidrólise , Complexos Multienzimáticos , Sefarose , beta-GlucosidaseRESUMO
The enzymatic deglycosylation of the plant flavonoid rutin (quercetin-3-O-(6-O-α-l-rhamnopyranosyl-ß-d-glucopyranoside) is usually assessed by means of high performance liquid chromatography (HPLC). We have developed a spectrophotometric method for the quantification of the released quercetin. After the enzymatic reaction, quercetin is extracted with ethyl acetate, and subsequently oxidized under basic conditions. The absorbance of quercetin autooxidation products at 320nm was correlated with the quercetin concentration by linear regression (molar extinction coefficient 23.2 (±0.3)×103M-1cm-1). With this method, rutin-deglycosylation activity in buckwheat flour and a commercial naringinase was measured, and showed no significant differences with the results obtained by HPLC. The convenience of this method resides on the enzymatic activity quantification using the natural substrate by UV-visible spectrometry. Moreover, the simplicity and speed of analysis allows its application for a large number of samples.
Assuntos
Flavonoides/química , Complexos Multienzimáticos/química , Rutina/química , Espectrofotometria/métodos , beta-Glucosidase/química , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Rutina/análise , Raios UltravioletaRESUMO
Naringinase is a complex enzyme composed of α-L-rhamnosidase and ß-D-glucosidase, which has a vast potential application in the field of industrial biotechnology. The novel aspect in the present study is employing a three-phase partitioning (TPP) technique for the purification of naringinase by solid-state fermentation using Aspergillus brasiliensis MTCC 1344. At optimum conditions of 28±2 °C and 30% (w/v) ammonium sulfate along with a 1:1 ratio of t-butanol to crude extract, the purification is enhanced by 4.2-fold .Temperature and pH profile of TPP purified naringinase was found to be active with an optimal activity of 719.6 units at an elevated temperature of 60 °C. The kinetic constants K(m) and V(max) using naringin as substrate were 3.21 mM and 321 U/ml. The purified enzyme was not inhibited by any metal ions except Hg(2+) but completely inhibited by adding chelating agents such as EDTA and SDS at a concentration of 10 mM. These results can be inevitable to establish the TPP method to be an inexpensive, economical and attractive technology for better recovery and to find its application in the industrial sector.
Assuntos
Proteínas Fúngicas/química , Complexos Multienzimáticos/química , beta-Glucosidase/química , Aspergillus/enzimologia , Precipitação Química , Estabilidade Enzimática , Proteínas Fúngicas/isolamento & purificação , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Extração Líquido-Líquido , Metais Pesados/química , Complexos Multienzimáticos/isolamento & purificação , beta-Glucosidase/isolamento & purificaçãoRESUMO
Statistics based optimization, Plackett-Burman design (PBD) and response surface methodology (RSM) were employed to screen and optimize the media components for the production of naringinase from Aspergillus brasiliensis MTCC 1344, using solid state fermentation. Cassava waste (CW) was used as both the solid support and carbon source for the growth of A. brasiliensis. Based on the positive influence of the Pareto chart obtained from PBD on naringinase activity, three media components--maltose, peptone and calcium chloride were screened. Box-Behnken design (BBD) was employed using these three factors at three levels, for further optimization, and the second order polynomial equation was derived, based on the experimental data. Derringer's desired function methodology showed that the concentrations of maltose (7.74 g/L), peptone (4.19 g/L) and calcium chloride (7.63 mM) were the optimal levels for maximal naringinase activity (889.91 U/mg) which were validated through experiments.
Assuntos
Aspergillus/metabolismo , Meios de Cultura , Fermentação , Complexos Multienzimáticos/biossíntese , beta-Glucosidase/biossíntese , Algoritmos , Meios de Cultura/química , Ativação Enzimática , Modelos Estatísticos , Reprodutibilidade dos TestesRESUMO
O Brasil é o maior produtor mundial de laranja e de suco concentrado destinado à exportação. Sucos cítricos concentrados para exportação com níveis elevados de naringina são excessivamente amargos, o que reduz a qualidade e o valor comercial do produto. A redução do amargor pode ser obtida pela naringinase, um complexo enzimático que degrada a naringina. Neste trabalho Aspergillus niger 426 foi usado como produtor de naringinase utilizando matérias-prima da agroindústria, melaço como fonte de carbono e extrato de levedura como fonte de nitrogênio. Naringina foi usada como indutor. Dentre as cinco fontes de nitrogênio pesquisadas, extrato de levedura apresentou o melhor resultado de 225,6 mU/mL de naringinase em 120 horas de cultivo. Para otimização da produção de naringinase, foi aplicada a metodologia de superfície de resposta, com planejamento fatorial incompleto 22, obtendo 354,26 mU/mL de atividade de naringinase, tendo como variáveis independentes o extrato de levedura (14,0g/L) e naringina (0,2g/L). Quando aplicadas ondas de ultrassom com intensidade de 20 kHz por 2 minutos na cultura, a atividade de naringinase atingiu o valor máximo de 473,6 mU/mL. Assim, a naringinase, enzima de grande potencial de aplicação biotecnológica, teve a produção aumentada pelo uso do planejamento estatístico e ultrassom.
Brazil is the world's largest producer of orange and concentrated juice for export. Concentrated juice with high levels of naringin has excessive bitterness, which reduces the quality and value on the market. The debittering can be obtained by using naringinase, an enzymatic complex that degrades naringin. This study reports the production of naringinase by Aspergillus niger 426 utilizing both sugar cane molasses as carbon source and yeast extract as nitrogen source. Naringin was used as inducer. Five nitrogen sources were studied and yeast extract was found as the best one as 225.6 mU/mL of naringinase activity at 120 hours of fermentation was achivied. For optimization of naringinase production it was applied response-surface methodology, with 22 incomplete factorial design. An activity value of 354.26 mU/mL of naringinase was achivied when as independent variables yeast extract (14.0g/L) and naringin (0.2g/L) were used. When applied ultrasound waves at 20 kHz of intensity for 2 minutes in the fermented broth, the activity reached the highest value of 473.6 mU/mL. Thus, naringinase, this enzyme of great potential for biotechnological applications, has its production increased by using statistical design and ultrasound.
Assuntos
Aspergillus niger , Ultrassom , Melaço , Sucos de Frutas e VegetaisRESUMO
Brazil is the world"s largest producer of orange and concentrated juice for export. Concentrated juice with high levels of naringin has excessive bitterness, which reduces the quality and value on the market. The debittering can be obtained by using naringinase, an enzymatic complex that degrades naringin. This study reports the production of naringinase by Aspergillus niger 426 utilizing both sugar cane molasses as carbon source and yeast extract as nitrogen source. Naringin was used as inducer. Five nitrogen sources were studied and yeast extract was found as the best one as 225.6 mU/mL of naringinase activity at 120 hours of fermentation was achivied. For optimization of naringinase production it was applied response-surface methodology, with 22 incomplete factorial design. An activity value of 354.26 mU/mL of naringinase was achivied when as independent variables yeast extract (14.0g/L) and naringin (0.2g/L) were used. When applied ultrasound waves at 20 kHz of intensity for 2 minutes in the fermented broth, the activity reached the highest value of 473.6 mU/mL. Thus, naringinase, this enzyme of great potential for biotechnological applications, has its production increased by using statistical design and ultrasound.
O Brasil é o maior produtor mundial de laranja e de suco concentrado destinado à exportação. Sucos cítricos concentrados para exportação com níveis elevados de naringina são excessivamente amargos, o que reduz a qualidade e o valor comercial do produto. A redução do amargor pode ser obtida pela naringinase, um complexo enzimático que degrada a naringina. Neste trabalho Aspergillus niger 426 foi usado como produtor de naringinase utilizando matérias-prima da agroindústria, melaço como fonte de carbono e extrato de levedura como fonte de nitrogênio. Naringina foi usada como indutor. Dentre as cinco fontes de nitrogênio pesquisadas, extrato de levedura apresentou o melhor resultado de 225,6 mU/mL de naringinase em 120 horas de cultivo. Para otimização da produção de naringinase, foi aplicada a metodologia de superfície de resposta, com planejamento fatorial incompleto 22, obtendo 354,26 mU/mL de atividade de naringinase, tendo como variáveis independentes o extrato de levedura (14,0g/L) e naringina (0,2g/L). Quando aplicadas ondas de ultrassom com intensidade de 20 kHz por 2 minutos na cultura, a atividade de naringinase atingiu o valor máximo de 473,6 mU/mL. Assim, a naringinase, enzima de grande potencial de aplicação biotecnológica, teve a produção aumentada pelo uso do planejamento estatístico e ultrassom.