Imobilização de lipases em suportes poliméricos

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2015O presente trabalho apresenta como objetivo principal a síntese de partículas poliméricas via miniemulsão para a imobilização das lipases de Candida antarctica B (CalB) e Thermomyces lanuginosus (TLL) e aplicação dos derivados enzimáticos na produção de ésteres etílicos de ácidos graxos. Na primeira etapa do trabalho, CalB foi imobilizada em nanopartículas de poliuretano via miniemulsão utilizando diisocianato de isoforona (IPDI) e poli(e-caprolactona) (PCL530) e crodamol. A fase aquosa foi composta por água deionizada, SDS, PEG400 e enzima. Diferentes intensidades da sonda de ultrassom foram testadas (70 e 90%) por 1, 2 ou 3 min. A atividade enzimática foi determinada por esterificação do ácido láurico e n-propanol, e o maior valor alcançado foi de 21 U/mg (70% por 2 min), com Dp=158 nm. CalB também foi imobilizada em nanopartículas de polimetilmetacrilato (PMMA) via miniemulsão. AIBN e KPS foram testados como iniciadores. A atividade enzimática foi avaliada por hidrólise do pNPP. CalB imobilizada em PMMA manteve a atividade por 20 ciclos de hidrólise, com atividade relativa acima de 40%. Na segunda etapa do trabalho, o suporte de poliuretano (PU) foi sintetizado como citado anteriormente, com exceção do crodamol, e antes de utilizado, as partículas foram liofilizadas. TLL foi imobilizada em PU sintetizado com diferentes tamanhos de cadeia de PEG. A atividade enzimática foi determinada por hidrólise do pNPB. TLL-PU-PEG6000 apresentou os melhores resultados de Km (0,183 mM) e Vmax (45,79 µmol/min/mg). O derivado se destacou também da produção de ésteres etílicos (EE) de ácidos graxos (260 mM.U-1). Os derivados foram recobertos com polietilenoimina (PEI) a 10 e 20% e trealose (10%). Os derivados recobertos com PEI 20% apresentaram os melhores resultados em termos de estabilidade à temperatura. O uso de agentes de ligação possibilitou um aumento de no mínimo 4 vezes na produção de EE de ácidos graxos. CalB também foi imobilizada em PU PEGuilado com diferentes cadeias de PEG. CalB-PU-PEG6000 apresentou os melhores valores de Km (0,815 mM) e Vmax (41,15 µmol/min/mg). O recobrimento com trealose possibilitou maior estabilidade térmica. CalB-PU-PEG400 apresentou os melhores valores de produção de EE, 43,72 e 16,83 mM.U-1 EE-EPA e DHA, respectivamente. C imobilizada em PU-PEG400, 4000 e 6000 também foi aplicada na hidrólise do etil éster de (R,S)ácido mandélico, apresentando razões enantioméricas satisfatórias (E>20). Foi possível imobilizar as lipases CalB e TLL nas partículas poliméricas via miniemulsão com elevadas porcentagens de imobilização e atividade recuperada. Elevadas concentrações de EE de EPA e DHA foram obtidas com os derivados propostos. Abstract: This current work shows as the main objective, the synthesis of polymeric particles via miniemulsion for the immobilization of Candida antarctica lipase B (CalB) and Thermomyces lanuginosus lipase (TLL) and application of the enzymatic derivatives for production of ethyl esters of fatty acids. In the first phase of the work, CalB was immobilized on polyurethane nanoparticles via miniemulsion using isophorone diisocyanate (IPDI) and poly(e-caprolactone) (PCL530) and crodamol. The aqueous phase was comprised of deionized water, SDS, PEG400 and enzyme. Differents ultrasound power intensities were tested (70 and 90%) by 1, 2 or 3 min. The enzyme activity was determined by esterification of lauric acid and n-propanol, and the highest value achieved was 21 U/mg (70% for 2 min), with Dp=158 nm. CalB was also immobilized on polymethylmethacrylate (PMMA) nanoparticles via miniemulsion. AIBN and KPS were tested as initiators. The enzymatic activity was evaluated by hydrolysis of pNPP. CalB immobilized on PMMA retained its activity over 20 cycles of hydrolysis, with relative activity above 40%. In the second phase of the work, the polyurethane support (PU) was synthesized as previously mentioned, with the exception of crodamol, and before using it, the particles were lyophilized. TLL was immobilized on synthesized PU with different sizes of PEG chain. The enzymatic activity was determined by hydrolysis of pNPB. TLL-PU-PEG6000 showed the best results of Km (0.183 mM) and Vmax (45.79 mmol/min/mg). The derivatives also highlighted the production of ethyl esters (EE) of fatty acids (260 mM.U-1). The derivatives were coated with polyethyleneimine (PEI) at 10 and 20%, and trehalose (10%). The derivatives coated with PEI 20% showed the best results in terms of temperature stability. The use of linking agents has enabled an increase of at least 4 times the production of EE of fatty acids. CalB was also immobilized on PU PEGylated with different PEG chains. CalB-PU-PEG6000 showed the best values of Km (0.815 mM) and Vmax (41.15 mmol/min/mg). The coating with trehalose allowed greater thermal stability. CalB-PU-PEG400 showed the best values of EE production, 43.72 and 16.83 mM.U-1 of EE-EPA and DHA, respectively. Calb immobilized on PU-PEG400, 4000 and 6000 was also applied for the hydrolysis of the (R,S) mandelic acid ethyl ester, presenting satisfactory enantiomeric ratios (E> 20). It was possible to immobilize the CalB and TLL lipases in polymeric particles via miniemulsion with high percentages of immobilization and recovered activity. High concentrations of EE of EPA and DHA were obtained with by proposed enzymatic derivatives

Stabilization of lipase from Thermomyces lanuginosus by crosslinking in PEGylated polyurethane particles by polymerization: Application on fish oil ethanolysis

The adsorption of Thermomyces lanuginosus lipase (TLL) on PEGylated polyurethane particles as support permitted the development of several strategies to improve the properties of this commercial low-cost enzyme. The supports were synthesized by miniemulsion technique using isophoronediisocyanate (IPDI) and poly(ε-caprolactone) diol (PCL530) as monomers. The aqueous phase was composed of distilled water, surfactant sodium dodecyl sulfate (SDS), and poly(ethylene glycol) with different molar mass (PEG 400, 4000 or 6000). Polyethyleneimine (PEI) and trehalose were used to coat the PU-PEG polyurethane particles in order to increase the stability. In general, the coating with PEI (20%) allowed a greater stability of the derivatives. (100% of relative activity at 50 °C during 8 h). TLL immobilized on PEGylated polyurethane particles was efficiently used in the production of ethyl esters from fish oil compared to the free TLL (data not shown). The values of ethyl esters production of EPA and DHA were dependent on the support used for immobilization, which proved to be a determining factor in the activity. The highest selectivity obtained value was 45.8 for the PU-PEG4000-PEI20 derivative.The authors thank the financial support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the Programa de Doutorado Sanduíche no Exterior, Project BEX 2569/14-0 and the direction of the Instituto de Investigación en Ciencias de la Alimentación for helping in the development of this work.Peer Reviewe

Phenolic compounds and antioxidant activity in fermented rice (Oryza sativa) bran

This study investigated the content of total phenolic compounds and antioxidant activity in fermented rice bran in order to evaluate the effect of solid state fermentation on these properties. The process was performed with the fungus Rhizopus oryzae CTT 1217 in tray reactors at 30 °C for 120 hours. Samples of fermented rice bran were collected every 24 hours. Antioxidant property was evaluated by the diphenyl-1-picrylhydrazyl radical scavenging method and through the inhibition of enzymatic oxidation and lipid peroxidation of olive oil. The methanol extract of the biomass obtained at 96 hours of fermentation inactivated 50% of free radical in 15 minutes. The same extract reduced the peroxide value in the olive oil by 57% after 30 days of storage. The aqueous extract of the biomass obtained at 120 hours was the most efficient inhibitor of the darkening reaction catalyzed by peroxidase

Phenolic compounds and antioxidant activity in fermented rice (Oryza sativa) bran Compostos fenólicos e atividade antioxidante em farelo de arroz (Oryza sativa) fermentado

This study investigated the content of total phenolic compounds and antioxidant activity in fermented rice bran in order to evaluate the effect of solid state fermentation on these properties. The process was performed with the fungus Rhizopus oryzae CTT 1217 in tray reactors at 30 °C for 120 hours. Samples of fermented rice bran were collected every 24 hours. Antioxidant property was evaluated by the diphenyl-1-picrylhydrazyl radical scavenging method and through the inhibition of enzymatic oxidation and lipid peroxidation of olive oil. The methanol extract of the biomass obtained at 96 hours of fermentation inactivated 50% of free radical in 15 minutes. The same extract reduced the peroxide value in the olive oil by 57% after 30 days of storage. The aqueous extract of the biomass obtained at 120 hours was the most efficient inhibitor of the darkening reaction catalyzed by peroxidase.Este trabalho investigou o conteúdo de compostos fenólicos totais e a atividade antioxidante em farelo de arroz fermentado, com o objetivo de avaliar o efeito da fermentação em estado sólido nessas propriedades. O processo foi realizado com o fungo Rhizopus oryzae CTT 1217 em reatores de bandeja a 30 °C por 120 horas. Amostras de farelo de arroz fermentado foram coletadas a cada 24 horas. A capacidade antioxidante foi avaliada pelo método do sequestro do radical livre difenil-1-picrilhidrazil e também por meio da inibição da oxidação enzimática e da peroxidação lipídica do óleo de oliva. O extrato metanólico produzido com a biomassa de 96 horas sequestrou 50% do radical livre em 15 minutos. O mesmo extrato reduziu o índice de peróxido no óleo em 57% após 30 dias de armazenamento. O extrato aquoso obtido da biomassa de 120 horas foi o inibidor mais eficiente da reação de escurecimento enzimático catalisada pela peroxidase

Study on the extracting conditions of phenolic compounds from onion (Allium cepa L.)

Submitted by Paula Gautério ([email protected]) on 2014-05-14T20:12:08Z No. of bitstreams: 1 Estudo das condições de extração de compostos fenólicos de cebola (Allium cepa L.).pdf: 1561741 bytes, checksum: 1b8faafe3a00801f93fd1d25593ffe30 (MD5)Approved for entry into archive by cristiane soares ([email protected]) on 2014-07-22T21:36:49Z (GMT) No. of bitstreams: 1 Estudo das condições de extração de compostos fenólicos de cebola (Allium cepa L.).pdf: 1561741 bytes, checksum: 1b8faafe3a00801f93fd1d25593ffe30 (MD5)Made available in DSpace on 2014-07-22T21:36:49Z (GMT). No. of bitstreams: 1 Estudo das condições de extração de compostos fenólicos de cebola (Allium cepa L.).pdf: 1561741 bytes, checksum: 1b8faafe3a00801f93fd1d25593ffe30 (MD5) Previous issue date: 2009O objetivo deste trabalho foi estabelecer as melhores condições para efetuar extração de compostos fenólicos totais de diferentes classes comerciais de cebola (Allium cepa L.). Por meio de Planejamento Experimental Fatorial foram determinadas as variáveis que infl uenciam signifi cativamente nas etapas de extração. As variáveis estudadas foram: natureza do solvente, procedimento de agitação, tempo de extração e tempo de agitação com e sem interrupções. A melhor combinação resultou em um modelo preditivo, empregandose metanol como solvente, agitação de 120 minutos a 200 rpm. O maior conteúdo fenólico em diferentes classes de cebola foi 2275 μg/g, 88% de recuperação e o limite de quantifi cação foi de 31 μg fenóis/g.The present study was carried out to establish the best conditions for performing the total phenolic compounds extraction from diverse kinds of commercialized onion (Allium cepa L.). By means of the factorial experimental design the variables that signifi cantly infl uence the extraction stages were determined. The following variables were analyzed: type of solvent, shaking process, time of extraction, and time with and without shaking interruptions. The best combining proceeding resulted in a predictive model, by using methanol as solvent, shaking for 120 minutes, and at 200 rpm. The highest phenolic content in different kinds of onion was 2275 μg/g, the recovery was 88%, and the quantifi cation limit of 31 μg/g

Estudo das condições de extração de compostos fenólicos de cebola (Allium cepa L.)

O objetivo deste trabalho foi estabelecer as melhores condições para efetuar extração de compostos fenólicos totais de diferentes classes comerciais de cebola (Allium cepa L.). Por meio de Planejamento Experimental Fatorial foram determinadas as variáveis que infl uenciam signifi cativamente nas etapas de extração. As variáveis estudadas foram: natureza do solvente, procedimento de agitação, tempo de extração e tempo de agitação com e sem interrupções. A melhor combinação resultou em um modelo preditivo, empregandose metanol como solvente, agitação de 120 minutos a 200 rpm. O maior conteúdo fenólico em diferentes classes de cebola foi 2275 μg/g, 88% de recuperação e o limite de quantifi cação foi de 31 μg fenóis/g.The present study was carried out to establish the best conditions for performing the total phenolic compounds extraction from diverse kinds of commercialized onion (Allium cepa L.). By means of the factorial experimental design the variables that signifi cantly infl uence the extraction stages were determined. The following variables were analyzed: type of solvent, shaking process, time of extraction, and time with and without shaking interruptions. The best combining proceeding resulted in a predictive model, by using methanol as solvent, shaking for 120 minutes, and at 200 rpm. The highest phenolic content in different kinds of onion was 2275 μg/g, the recovery was 88%, and the quantifi cation limit of 31 μg/g

Application of protein-phenolic based coating on tomatoes (Lycopersicum esculentum) Aplicação de coberturas proteicas e fenólicas em tomates (Lycopersicum esculentum)

The aim of this study was to investigate the use of protein-phenolic based coating made from fermented rice bran on cherry tomatoes (Lycopersicum esculentum). Tests were performed with glycerol 3% (v/v), glycerol with protein-phenolic rice bran extract (5%), glycerol with protein-phenolic extract after 96 hours of fermentation (5%), and a control (without coating). The coated cherry tomatoes were kept at room temperature for 28 days. Mass loss, pH and acidity, total soluble solids, and carotenoids were determined every 96 hours. The coating made from the biomass extract reduced the carotenoid and acidity levels in the fruits studied by 17 and 21.1%, respectively, compared to the control. The coating proved an efficient barrier to water vapor with mass loss of 57% less than the control suggesting that it can be used as an alternative for vegetable tissue conservation.<br>Este trabalho teve como objetivo estudar a utilização de películas, à base de compostos proteicos e fenólicos provenientes de farelo de arroz fermentado, em tomates (Lycopersicum esculentum). Foram realizados testes com: glicerol 3% (v/v); glicerol com extrato fenólico e proteico do farelo de arroz (5%); glicerol com extrato fenólico e proteico da biomassa gerada em 96 hours (5%), e um controle (sem a película). Os tomates revestidos foram mantidos à temperatura ambiente durante 28 dias, sendo determinados, a cada 96 horas, os seguintes aspectos: a perda de massa, o pH e a acidez, os sólidos solúveis totais e os carotenoides. A película elaborada com os extratos da biomassa reduziu os níveis de carotenoides e acidez dos frutos estudados em 17 e 21,1%, respectivamente, em relação ao controle. A película também foi eficiente como barreira ao vapor de água; assim, com perda de massa 57% inferior à do controle, sugere-se que esta poderá ser utilizada como alternativa para conservação desse tecido vegetal

Solid-State Fermentation for the Enrichment and Extraction of Proteins and Antioxidant Compounds in Rice Bran by Rhizopus oryzae

The objective of this work was to evaluate the solid-state fermentation with Rhizopus oryzae CCT 7560 of rice bran for the enrichment of proteins and the antioxidant compounds in the fermented biomass. Fermentation was performed in tray bioreactors at 30ºC for 120 h. Protein extraction was done at alkaline pH, followed by precipitation with acetone. Phenolic compounds were extracted with cold methanol. The maximum protein was recovered from after 120 h (26.6%). The content of total phenolic compounds increased during the fermentation and was maximum after 96 h, which inhibited the DPPH radical by 87%. The promising characteristics of the protein and phenolic extracts of the biomass suggested the application in the coating composition for vegetal tissues preservation