Orientadores: Telma Teixeira Franco, Gustavo Paim Valença / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T21:59:10Z (GMT). No. of bitstreams: 1 Ayres_BiancaMairaTeixeira_D.pdf: 23160216 bytes, checksum: 967289613483c430d41cd88562085d44 (MD5) Previous issue date: 2014 / Resumo: O objetivo deste trabalho foi a biocatálise de acrilatos de carboidratos, desde glicose até maltodextrina. Maltodextrinas (MD) são produtos da hidrólise do amido, caracterizadas por cadeias de 5 a 20 unidades de ?-D-glicose unidas por ligações ?-1,4 (principalmente). Análises por cromatografia de permeação em gel (HPSEC) caracterizou ampla distribuição de massa molar de amostras padrão ou industriais de MD, apresentando de 1 a 100 unidades de glicose (G1). O fracionamento por etanol para seleção de menores cadeias de glicose não foi efetivo pois variadas frações volumétricas de etanol forneceu contaminação de cadeias curtas no precipitado. Os principais fatores limitantes para a esterificação enzimática da MD com ácido acrílico são baixa solubilidade do substrato sacarídico, inibição por ácido acrílico, polaridade do solvente orgânico que permita atividade enzimática, a qual depende das características da lipase imobilizada como tipo de suporte e origem da lipase. Lipases disponíveis comercialmente na forma imobilizada de Thermomyces lanuginosa (TL IM) ou Candida antarctica (Novozyme 435) e, imobilização por adsorção das lipases de T.lanuginosa, C.antarctica, Candida rugosa e Rizomucor miehei em Accurel EP-100 foram investigadas em triagem associada de solventes orgânicos. Dioxano e Novozyme 435 foram a melhor associação que alcançou maior conversão de G1 e G2. Acrilatos de maltodextrina foram também observados sob incubação com TL IM em TBA. A solubilidade da MD é completa em água, piridina e DMSO, cerca de 1 kg L-1, mas as lipases não são ativas para esterificação nestes solventes. A água é um subproduto da esterificação e sua presença pode deslocar a reação em favor da hidrólise. A adição de DMSO como co-solvente para o sistema reacional contendo 2M2B ou TBA foi comprovado ser menos eficiente que sistemas com 100% dos solventes apolares. A maior área de taxa de aumento da produção direta de acrilatos de maltodextrina foi alcançada com 2-metil-2- butanol como meio reacional. Analogicamente, a acrilação enzimática de n-butanol foi catalisada pela Novozyme 435. Diferentemente dos acrilatos de carboidratos, o produto acrilato de butila pôde ser quantificado por cromatografia em fase gasosa (CG). Sistemas de solventes e co-solventes (tolueno, ciclohexano, 2-metil-2-butanol (2M2B), álcool terc-butílico (TBA) e associações com parcial volume de dimetilsulfoxido (DMSO)) foram estudados para determinar o efeito destes na atividade enzimática específica. O uso de ciclohexano e tolueno resultaram em atividade da enzima três vezes maior que em TBA e 2M2B. n-Butanol e MD são substratos diferenciados pela solubilidade nos solventes orgânicos, os quais devem ser apolares ou pouco polares para permitir atividade catalítica. Uma reação secundária de acrilação com o solvente orgânico, TBA ou 2M2B, foi verificada por cromatografia gasosa acoplada à espectrometria de massas. A esterificação enzimática de maltose, de maltotriose e maltodextrina com ácido acrílico catalisada pela lipase Novozyme 435 em 2M2B foram analisadas por espectrometria de massas com ionização por eletrospray, ou acoplada à cromatografia líquida de alta eficiência. Essas análises confirmaram a presença de uma até quatro hidroxilas acriladas da maltose (G2) ou da maltotriose (G3). Um processo em duas etapas de biocatálise para produção de acrilatos de carboidratos foi investigado. Inicialmente, G1 ou G2 foram esterificadas enzimaticamente com propionato de vinila ou acrilato de etila por incubação com Novozyme 435 em dioxano. Em subsequente etapa, a cadeia glicosídica destes ésteres foram alongadas a partir da atividade da cicloglicosil transferase de Bacillus macerans com ?-ciclodextrina como doador de grupo glicosil. Cromatografia líquida de alta eficiência com detecção amperométrica e aerossol carregado e cromatografia em camada delgada foram utilizados para identificar os ésteres de oligossacarídeos. Cerca de 75% ou 55% de ?-ciclodextrina foi convertida com consumo de 40.5% de propionato de glicose (G1P) ou 86.3% de propionato de maltose (G2P) pela atividade da CGTase. A composição da solução final foi, desde 2 a 14 unidades de glicose com uma unidade acrilada ou propilada / Abstract: The aim of this work was the biocatalysis of the acrylates of carbohydrates, from glucose up to maltodextrins. Maltodetrins (MD) are starch hydrolysates consisting of ?-D-glucose units bounded by ?-1,4 glycosidic linkages (primarily). Analysis of standard or industrial samples of MD in gel permeation chromatography presented a huge molecular mass distribution, from 1 to 100 units of glucose (G1). An ethanol fractionation step for selection of narrow range of the chains of glucose was unsuccesfull because the precipitate and supernatant were contamined with small molecules. The main limitant factors for enzymatic esterification of MD with acrylic acid are the catering saccharidic substrate (increase its solubility) to the lipase, to avoid (or overcome) inhibition from acrylic acid, and to allow enzymatic activity, which depends on the characteristics of the immobilized lipases (type of support, source of the lipase) and solvent of the system. Commercial immobilized lipases from Thermomyces lanuginosa (TL IM) or Candida antarctica (Novozyme 435) and, lipases from T.lanuginosa, C.antarctica, Candida rugosa and Rizomucor miehei immobilized by absorption in Accurel EP-100 were investigated in a screening of organic solvents. Dioxane and Novozyme 435 were the best association for higher conversion of G1 and G2. TL IM in tert-butanol (TBA) was the only system, which produced acrylates of maltodextrina on TLC plates. The solubility of the MD is complete in water, pyridine and dimethylsulfoxide (DMSO), about 1 kg L-1, but lipases are not active for esterification in these solvents. The water is a byproduct of the esterification and shifts the reversible reaction toward the hydrolysis. The partial addition of the co- solvent DMSO to the reactional system containing 2-methyl-2-butanol (2M2B) or TBA was tested for partial solubilization of the maltodextrin. The systems with only one organic solvent were more efficient than the presence of DMSO. The highest area in high performance liquid chromatography (HPLC) for production of the MD acrylates was achieved with 2M2B as adjuvant. Analogically, the acrylation of n-butanol by Novozyme 435 was studied to better understand the enzymatic acrylation, since the quantification of the product butyl acrylate is possible by gas chromatography (GC). Solvent systems (toluene, cyclohexane, 2M2B, TBA and partial volume of DMSO) were studied to determine the effect of the solvents on the specific enzymatic activity. It was found that cyclohexane and toluene achieved 3-fold the enzyme activity that in TBA and 2M2B. However, n-butanol and MD as substrate are mainly differentiated regards to the solubility in non polar organic solvents which are more suitable for lipase activity. A side reaction of acrylation of the organic solvent, TBA or 2M2B, was verified by GC-Mass Spectrometry. Meanwhile, the enzymatic esterification of maltose, maltotriose and maltodextrin with acrylic acid by Novozyme 435 in 2M2B were analyzed in electrospray ionization (ESI) mass spectrometry (MS) associated to HPLC, which confirmed the presence of mono- until tetra- acrylated hydroxyls for maltose (G2) and maltotriose (G3). A two-step process of biocatalysis for producton of sugar acrylates was investigated. G1 or G2 was acylated with either vinyl propionate or ethyl acrylate by Novozyme 435 in dioxane. Then, the elongation of the chain by cyclodextrin glucanotransferase (CGTase) from Bacillus macerans with ?-cyclodextrin as acyl donor provided maltoligosaccharides esters. CGTase from Bacillus macerans converted these products from the first step and ?-cyclodextrin into oligosaccharide esters. HPLC coupled to charged aerosol detector and amperometric exchange and thin layer chromatography were used to identify the oligosaccharide esters. About 75% or 55% of ?-cyclodextrin was converted under consumption of 40.5% of glucose propionate (G1P) or 86.3% of maltose propionate (G2P) by CGTase activity. The composition of the final solution was since 2 to 14 glucose units with one acrylate or propionate moiety / Doutorado / Engenharia Química / Doutora em Engenharia Quimica

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar-cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono- and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high-performance liquid chromatography (HPLC) and the products were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The main products are mono- and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D-glucose and D-fructose, followed by D-xylose and D-maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross-linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Immobilization of enzymes provides advantages such as increased in: stability, level of process control, yield and purity of the final product, in addition to allowing the use of different reactor configurations. In this study, two types of support, an organic polymer (polimetilmetacrilato- PMMA) and other inorganic (silica) were used for the immobilization of lipase Candida antarctica type B (CALB). Different alcohols (ethanol, iso-propanol, n-butanol) were evaluated as agents for optimization of the immobilization process, the concentrations of 5, 10 and 15% (volume of alcohol à 100 / solution volume immobilization). Immobilization of PMMA was conducted using two different protocols here denominated first and second protocol generation. The best results of hydrolytic activity and esterification to the derivative of the first generation was 3,91  0,09 UpNPB/g. of catalyst e 409,5  19,7 Uoleic acid/g. of catalyst, respectively. When using the protocol of the second generation, the best results were obtained when aa immobilization was carried out in the presence of 5% Triton X-100 2 mM, rising rate of the esterification reaction between the derivatives studied was highly significant with a higher ratio of 3,81 times to the derivative prepared with Triton X-100. The CALB was also immobilized for the macroporous silica support (IB S60S - Chiralvision), the activate agent used was Triethoxy(octyl)silane (C8-TEOS). The results observed for immobilization in the presence of 5% n-butanol were the best for both hydrolytic activity, being four times higher than the activity for the immobilized derivative without alcohol, as for the esterification activity was 985,1  5,6 Uoleic acid/g. of catalyst . All observed derivatives supported on silica and PMMA reached a level conversion for equilibrium esterification reaction for a reaction time of 24 h the best results varying between 80 to 90%. / A imobilizaÃÃo das enzimas traz vantagens como: aumento da estabilidade, do nÃvel de controle do processo, do rendimento e da pureza do produto final, alÃm de possibilitar o uso de reatores de diferentes configuraÃÃes. Neste trabalho, dois tipos de suporte, um polÃmero orgÃnico (polimetilmetacrilato- PMMA) e outro inorgÃnico (sÃlica), foram utilizados para a imobilizaÃÃo da lipase do tipo B de Candida antarctica (CALB). Diferentes Ãlcoois (etanol, iso-propanol, n-butanol) foram avaliados como agentes de otimizaÃÃo do processo de imobilizaÃÃo, nas concentraÃÃes de 5, 10 e 15 % (volume de Ãlcool Ã100 / volume da soluÃÃo de imobilizaÃÃo). A imobilizaÃÃo em PMMA foi conduzida atravÃs de dois protocolos diferentes, aqui denominados de protocolo de primeira e segunda geraÃÃo. Os melhores resultados de atividade hidrolÃtica e de esterificaÃÃo para o derivado de primeira geraÃÃo foi de 3,91  0,09 UpNPB/g. de catalisador e 409,5  19,7 UÃc. olÃico/g. de catalisador, respectivamente. Quando se utilizou o protocolo de segunda geraÃÃo, melhores resultados foram obtidos quando a a imobilizaÃÃo foi conduzida na presenÃa de n-butanol 5 % e Triton X-100 2mM; o aumento da velocidade da reaÃÃo de esterificaÃÃo entre os derivados estudados foi bastante significativo, sendo 3,8 vezes maior para o derivado preparado com Triton X-100. A CALB foi imobilizada tambÃm para o suporte sÃlica macroporosa (IB S60S - Chiralvision), o agente ativante utilizado foi o octiltrietoxisilano (C8-TEOS). Os resultados observados para imobilizaÃÃo na presenÃa de n-butanol 5 % foram os melhores tanto para a atividade hidrolÃtica, sendo quatro vezes maior que a atividade para o derivado imobilizado sem Ãlcool, quanto para a atividade de esterificaÃÃo que foi de 985,1  5,6 UÃc. olÃico/g. de catalisador. Todos os derivados observados suportados em PMMA e sÃlica atingiram um patamar de conversÃo do equilÃbrio para reaÃÃo de esterificaÃÃo por um perÃodo de reaÃÃo de 24 h com os melhores resultados variando entre 80 a 90%.

L’induction de la synthèse d’une acide férulique estérase (AFE) a été étudiée chez Streptomyces ambofaciens ATCC 23877. L’activité la plus élevée a été détectée en présence de son de blé désamidonné ou de xylane d’avoine (0,22, 0,21 mU/mg protéine, respectivement). Des productions d’AFE en bioréacteur ont également été réalisées en utilisant 1% (p/v) de son de blé comme inducteur. Le niveau de production de l’AFE a été trois fois plus important en bioréacteur qu’en fiole d’Erlenmeyer. L’AFE de Streptomyces ambofaciens ATCC 23877 et celle de Humicola sp., présente dans un mélange enzymatique commercial (DepolTM 740L), ont été partiellement purifiées et caractérisées. A l’issue de la purification, l’activité AFE de Streptomyces ambofaciens ATCC 23877 a été trop faible pour pouvoir être utilisée ultérieurement en synthèse. Par contre, le potentiel de l’AFE de Humicola sp., concentrée par précipitation à l’acétone, pour la synthèse de différents esters phénoliques a été testé. Les meilleurs rendements de conversion ont été observés lors de l’absence de substitutions sur le cycle aromatique de l’acide phénolique ou en présence de groupements hydroxyles. Les synthèses en milieu non aqueux (M2B2) se sont montrées infructueuses en raison, peut-être, d’un effet néfaste du solvant sur l’enzyme / The induction of the ferulic acid esterase (FAE) synthesis was studied with Streptomyces ambofaciens ATCC 23877. The highest activity was detected in the presence of either destarched wheat bran or oat spelt xylan (0,22, 0,21 mU/mg protein, respectively). FAE productions in bioreactor were also carried out using 1% (w/v) of wheat bran as inducer. The FAE production level was three times higher in bioreactor than in Erlenmeyer flask. FAE of Streptomyces ambofaciens ATCC 23877 and that of Humicola sp., present in an enzymatic commercial mixture (DepolTM 740L), were partially purified and characterised. At the end of the purification, FAE activity of Streptomyces ambofaciens ATCC 23877 was too weak to be used later in synthesis. However, the FAE potential of Humicola sp., concentrated by acetone precipitation, for the synthesis of various phenolic esters was tested. The best conversion yields were observed in the absence of substitution on the phenolic acid aromatic cycle or in the presence of hydroxyl groups. The synthesis in non-aqueous medium (M2B2) were unsuccessful maybe because of an harmful effect of the solvent on the enzyme

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study aimed to evaluate the efficiency of supplementation in the diet of silver catfish with blend of buruti and murumuru oils, in natura or enzimatically esterified on biological responses, chemical composition, oxidative stability and sensory characteristics of fillets. Silver catfish females were fed the experimental diets for 8 weeks. The fish growth parameters were evaluated. Subsequent to slaughter, the fillets were frozen for 12 months. After slaughter, were evaluated chemical composition, volatile compounds and sensory analysis of cooked steaks. Was also evaluated influence of power silver catfish on lipid oxidation and protein of frozen fillets within 3, 6 and 12 months of storage. In addition, we assessed levels of vitamin E from the fillets. The fatty acid profile of steaks was assessed after slaughter. We evaluated degradation of fatty acid C22:6n3. Supplementation esterified Amazon oils showed the best results in growth, total length and total biomass. The different treatments did not result in changes in the chemical composition of the fillets. Fatty acids in the diet leads reflect in the composition of fillets for all supplementations. Amazon oils diet had higher MUFA fatty acids. As for sensory analysis, supplementation with Amazon oils no difference in preference for the odor of cooked fillets. A few volatile compounds were detected and the higher amount hexanal was supplemented with soybean oil. Content of conjugated dienes increased three months of storage for all treatments. For amount of peroxides, esterified Amazon oils showed lower values in comparison with other treatments. An increase in TBARS values occurred in 12 months to freeze fillets and interesterified oils showed the lowest values. After 12 months of storage, degradation was approximately 40% C22:6n3 for SO and for BM. There was a reduction in amount of carbonylated proteins after 3-6 months of storage. Sulphydryl proteins no changes over storage for all treatments. There was a decrease in hardness of fillets 12 months of storage for all treatments. In short, supplementation with blends oils (buriti+murumuru) decreases formation of lipid oxidation compounds and volatile compounds leads to lower lipid and protein oxidation. / Este trabalho teve como objetivo avaliar a eficiência da suplementação na dieta de jundiás com mistura de óleos de buruti e murumuru, in natura ou esterificado enzimaticamente, sobre respostas biológicas, composição química, estabilidade oxidativa e características sensoriais de filés. Fêmeas de jundiás foram alimentadas com as dietas experimentais durante 8 semanas. Os parâmetros de crescimento dos peixes foram avaliados. Posteriormente ao abate, os filés foram congelados pelo período de 12 meses. Após o abate, foram avaliados a composição centesimal, os compostos voláteis e análise sensorial por ordenação de preferência dos filés cozidos. Avaliou-se também a influência da alimentação dos jundiás sobre a oxidação lipídica e proteica de filés congelados no período de 3, 6 e 12 meses de congelamento. Além disso, avaliou-se o teor de vitamina E dos filés. O perfil de ácidos graxos dos filés foi avaliado após o abate. Foi avaliada a degradação do ácido graxo C22:6n3. A suplementação com óleo esterificado apresentou os melhores resultados quanto ao crescimento, comprimento total, biomassa total e crescimento específico. Os diferentes tratamentos não acarretaram em alterações na composição centesimal dos filés. Os ácidos graxos presentes na dieta se refletiram na composição dos filés para todas as suplementações. Dietas contendo óleos da Amazônia apresentaram maiores valores de ácidos graxos monoinsaturados. Quanto à análise sensorial, a suplementação com óleos da Amazônia não mostrou diferença na preferência para o odor dos filés cozidos. Poucos compostos voláteis foram detectados e hexanal teve maior quantidade na suplementação com óleo de soja. O conteúdo de dienos conjugados aumentou em três meses de congelamento para todos os tratamentos. Para o valor de peróxidos, suplementação com óleos Amazônia esterificado apresentou menores valores em comparação com os outros tratamentos. Um aumento nos valores de TBARS ocorreu em 12 meses de congelamento dos filés e BME apresentou os menores valores. Após 12 meses de congelamento, houve degradação de aproximadamente 40% de C22:6n3 para suplementações com óleo de soja e buriti+murumuru esterificado. Houve uma redução na quantidade de proteínas carboniladas após 3-6 meses de congelamento. Proteínas sulfidrílicas não alteraram ao longo do armazenamento para todos os tratamentos. Houve uma diminuição da dureza dos filés em 12 meses de congelamento para todos os tratamentos. Em suma, a suplementação com misturas de óleos (buriti+murumuru) diminui a formação de compostos de oxidação lipídica e leva a menores compostos voláteis de oxidação lipídica e proteica.

Un procédé enzymatique sans solvant a été développé permettant la synthèse d'un ester phénolique de DHA. L'optimisation des paramètres réactionnels a permis d'atteindre des rendements élevés (440 g/L) d'ester de DHA et d'alcool vanillique (DHA-VE), dont les activités biologiques et le potentiel applicatif ont été évalués. L'activité inhibitrice du DHA-VE vis-à-vis des radicaux ABTS, DPPH et hydroxyle a été démontrée. Un effet neuroprotecteur de l'ester a également été mis en évidence sur des neurones primaires de rat, exposés aux oligomères du peptide [bêta]-amyloïde. Une étude in vivo a permis de montrer que le greffage d'alcool vanillique conduit à une augmentation du taux de DHA au niveau des globules rouges et des neurones, indiquant une biodisponibilité accrue du DHA lorsque celui-ci est couplé au composé phénolique. Aucune toxicité visible de l'ester n'a été constatée. Par ailleurs, l'incorporation de DHA-VE dans divers systèmes émulsionnés a permis d'accroître leur stabilité à l'oxydation, quelles que soient les conditions de stockage. Ceci montre le potentiel de cet ester pour enrichir diverses matrices alimentaires en DHA, tout en améliorant leur stabilité à l'oxydation. Le procédé enzymatique développé a été appliqué à de l'huile de saumon, utilisée comme source d'acides gras polyinsaturés de la série oméga-3. L'incorporation totale de l’alcool vanillique (50 g/L) a été obtenue après 24 h de réaction, conduisant à la production d'une grande variété d'esters, représentatifs de la composition initiale de l'huile en acides gras. Le milieu réactionnel brut issu de l'alcoolyse de l'huile présente une grande stabilité et des propriétés antioxydantes importantes par rapport à l'huile de saumon native. En conclusion, l'approche consistant à assembler des composés phénoliques et des lipides polyinsaturés au sein d'une même structure semble prometteuse pour renforcer le potentiel applicatif de ces deux familles de biomolécules et produire de nouveaux ingrédients bioactifs stables / An efficient solvent-free bioprocess was developed for the synthesis of DHA phenolic ester, using the lipase B from Candida antarctica. The protocol developed here led to high-level production (440 g/L) of DHA vanillyl ester (DHA-VE) that exhibits interesting application potential as food ingredient. DHA-VE was characterized by a high stability and a high radical scavenging activity towards DPPH, ABTS and hydroxyl radicals. Neuroprotective properties of DHA-VE were also demonstrated in rat primary neurons exposed to amyloid-[beta] oligomers. Enzymatic esterification of DHA with vanillyl alcohol (VA) led to increased DHA levels in erythrocytes and brain tissues of mice fed DHA-VE-supplemented diet comparing with DHA. No visible toxicity of the ester was found. Enrichment of emulsions with DHA-VE improved significantly their oxidative stability whatever the conditions of storage, showing the potential of DHA-VE to enrich various food matrices with DHA while protecting them against oxidation. The enzymatic process was applied to salmon oil as a source of omega-3 polyunsaturated fatty acids (PUFA). The total conversion of VA (50 g/L) was achieved after 24 h of reaction, leading to the production of a wide variety of esters that mirror the initial composition of the oil. The crude reaction medium recovered from salmon oil alcoholysis exhibited a high stability together with high antioxidant properties in comparison with native salmon oil. In conclusion, the approach that consists in bringing phenolic compounds and PUFA-rich lipids together within a single structure is expected to provide stable bioactive ingredients that should broaden the scope of application of omega-3 PUFAs whose health benefits are increasingly sought