Abstract This study aimed to evaluate the effects of variables on the process of lipases production by Aspergillus niger C by submerged fermentation (SmF). The production assays were performed in shake flasks for 72 hours at 150 rpm and 32°C. First, a fractional factorial design 25-1 (FFD) was carried out to evaluate the effect of the following process variables: sucrose, ammonium sulphate, soybean oil, yeast extract concentration and pH. After the selection of the variables that significantly influenced the lipase production, a central composite rotational design 22 (CCRD) was used, aiming to find the most favorable operational conditions. The selected assay condition (15.0 g.L-1 sucrose, 4.0 g.L-1 ammonium sulphate, 4.0 g.L-1 soybean oil and 1.0 g.L-1 yeast extract at pH 5.0) was the one that presented a lipase activity of 27.46 U.mL-1. It was very close to that best assay (30.76 U.mL-1), but using half of the inducer concentration, consequently reducing process cost. The kinetics of lipase production showed that the highest specific activity was 57.17 U.mg-1. The pH and temperature effects on lipase activity produced in this study was investigated. The optimum activity was found in a more acidic pH (5.0-6.0) and 55°C.

ABSTRACT The aim of this study was to produce bioethanol from enzymatic hydrolysates of cassava pulp, a by-product of cassava flour manufacturing, using an alcohol-tolerant Saccharomyces cerevisiae strain. First, the best operational condition of the starch hydrolysis process was determined through a complete factorial design (24), with triplicates at the central point. The independent variables evaluated were: the concentrations of α-amylase (Termamyl 2X) and glucoamylase (AMG 300L) and both liquefaction and saccharification times. The most favorable hydrolysis condition in the assay was achieved using 0.517 mL AMG.g starch-1 and 0.270 mL Termamyl.g starch-1, with liquefaction and saccharification times of 1 and 2 h, respectively. The broth obtained at this hydrolysis condition contained a high glucose concentration (160 g.L-1). Once the best reaction conditions were determined, fermentation tests were carried out in a 3 L bioreactor, in a batch system, at 30 °C, 100 rpm and pH 5.5, using 3 g.L-1 (dry biomass) of yeast as inoculum. After 24 h of fermentation, an ethanol concentration of 68 g.L-1 was obtained, with 0.48 ethanol yield and 2.83 g.L-1.h-1 productivity. These results indicate the potential use of cassava pulp, a by-product of cassava flour industries in Brazil, as a raw material for bioethanol production.

The aim of the present work was to select filamentous fungi isolated from diverse substrates to obtain the strains with potential to produce the hydrolytic enzymes. From a total of 215 strains, seven strains from the soils, six from the plants and one from sugarcane bagasse were selected and identified as belonging to the Trichoderma, Penicillium and Aspergillus genera. The best hydrolytic activities obtained by semi-solid fermentation using these strains were approximately: 35; 1; 160; 170 and 120 U/gdm (CMCase, FPase, β-glucosidase, xylanase and polygalacturonase, respectively), demonstrating their potential to synthesize the enzymes compared with the results reported in the literature.

The influence of pre-processing of arabica coffee beans on the composition of volatile precursors including sugars, chlorogenic acids, phenolics, proteins, aminoacids, trigonelline and fatty acids was assessed and correlated with volatiles formed during roasting. Reducing sugars and free aminoacids were highest for natural coffees whereas total sugars, chlorogenic acids and trigonelline were highest for washed coffees. The highest correlation was observed for total phenolics and volatile phenolics (R= 0.999). Experimental data were evaluated by Principal Components Analysis and results showed that washed coffees formed a distinct group in relation to semi-washed and natural coffees.

Permeado de soro de queijo foi usado como um substrato para a fermentação de Propionibacterium freudenreichi PS1 para produção de ácidos graxos de cadeia curta, constituintes do bioaroma de queijo suíço. O bioaroma líquido foi encapsulado por secagem por atomização sob diferentes condições de temperatura de entrada de ar e vazão de alimentação. Um estudo foi realizado sobre a estabilidade do bioaroma durante estocagem em embalagens laminadas a 30 °C por 90 dias, usando o produto que mostrou maior retenção de ácido acético e propiônico. Os resultados mostraram que a melhores condições de secagem foram uma temperatura do ar de entrada de 180 °C e taxa de alimentação de 24 g/min, resultando em partículas com superfície lisa e poucas invaginações e microfissuras. No entanto, 72% do ácido acético e 80% do ácido propiônico foram perdidos durante a estocagem, mostrando que o material de parede usado foi inadequado para garantir a estabilidade do produto.

Cheese whey permeate was used as a substrate for the fermentation of Propionibacterium freudenreichi PS1 for the production of short chain fatty acids, components of the bio-aroma of Swiss cheese. The liquid bio-aroma was encapsulated by spray drying under different conditions of air inlet temperature and feed rate. A study was carried out on the stability of the bio-aroma during storage in laminated packages at 30 °C for 96 days using the product showing the greatest retention of acetic and propionic acids. The results showed that the best drying conditions were an air entrance temperature of 180 °C and a feed rate of 24 g/min resulting in particles with a smooth surface and few invaginations and micro-fissures. However, 72% of the acetic acid and 80% of the propionic acid were lost during storage showing that the wall material used was inadequate to guarantee product stability.

O objetivo deste trabalho foi avaliar a produção enzimática de celulases pelo fungo filamentoso Aspergillus niger por fermentação em estado sólido de diferentes substratos. Foram avaliados os substratos sólidos bagaço de cana-de-açúcar, farelo de soja, farelo de trigo e misturas entre os substratos. Em substrato com 90% de bagaço e 10% de farelo de soja, avaliaram-se os efeitos do conteúdo de umidade (60, 70 e 80%, base úmida) e da suplementação com os meios indutores da atividade enzimática: sacarose, Mandels & Weber básico, Mandels & Weber modificado, com acréscimo de carboximetilcelulose, e Czapeck Dox. As maiores atividades de celulase total e endoglucanase, em farelo de trigo, foram obtidas após 72 horas de cultivo: 0,4 e 21,0 UI g-1, respectivamente. Observou-se expressivo aumento nas atividades enzimáticas na medida em que se aumentou a proporção de farelos no substrato, em comparação à fermentação com bagaço de cana apenas. O conteúdo de umidade de 50% foi insuficiente para conseguir completa hidratação do bagaço de cana, e a umidade ideal varia de acordo com o meio utilizado para suplementação e encontra-se entre 70 e 80%. O meio de Mandels & Weber modificado apresenta o melhor resultado como indutor da atividade enzimática.

The objective of this work was to evaluate cellulase enzyme production by the filamentous fungus Aspergillus niger by solid-state fermentation of different substrates. The solid substrates sugarcane bagasse, wheat bran, soybean meal, and their mixtures were evaluated. The effects of substrate moisture contents (60, 70, and 80% in humid basis) and of the supplementation with culture media: sucrose, basic Mandels & Weber, modified Mandels & Weber, with the addition of carboxymethyl cellulose, and Czapeck Dox, were evaluated in substrate with 90% bagasse and 10% soybean meal. The highest total cellulose and endoglucanase activities, in wheat bran, were obtained after 72 hours: 0.4 and 21.0 IU g-1, respectively. There was an expressive increase in enzymatic activities as the proportion of bran in the substrate increased, in comparison to fermentation with sugarcane bagasse only. The 50% moisture content was insufficient to fully hydrate sugarcane bagasse, and the ideal humidity varies according to the medium used for supplementation and is between 70 and 80%. The modified Mandels &Weber medium shows the best result as an inductor of enzymatic activity.

A fruit chemical composition reflects its maturation stage. For coffee, it is also the reflex of the post-harvesting processing type, dry, semi-wet and wet. The object of this work was to verify if headspace solid phase microextraction coupled to gas chromatography (HS-SPME-GC) could be used to discriminate between samples harvested in different maturation stages and treated by different processes. With application of principal component analysis to the area of 117 compounds extracted by SPME, using divinylbenzene/Carboxen/polydimethylsiloxane fiber, it was possible to discriminate, in the roasted and ground coffee, the maturity stage and processing type used .

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37°C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

O presente estudo tem como objetivo comparar o rendimento e a qualidade do óleo da polpa de pequi obtido por dois processos distintos: no primeiro, a secagem da polpa, conduzida em secador de bandeja a 60ºC, foi combinada com tratamento enzimático e prensagem para extração de óleo, no segundo, um processo mai simples foi realizado combinando-se a secagem da polpa ao sol seguida da prensagem a frio. Neste estudo, frutos de pequi in natura foram coletados no estado do Mato Grosso, Brasil. Os frutos foram auto-clavados a 121º C e armazenados sob refrigeração. Um extrato enzimático com atividades pectinase e CMCase foi utilizado para a hidrólise da polpa de pequi antes da extração do óleo. O processo de extração foi realizado por prensagem hidráulica, com ou sem incubação enzimática. O teor de óleo na polpa do pequi (45% w/w) e as características físico-químicas do óleo foram determinados de acordo com métodos analíticos oficiais. O teor de ácidos graxos livres, os índices de peróxido, de iodo e de saponificação foram respectivamente de 1,46 mgKOH/g, 2,98 meq/kg, 49,13 e 189,40. A acidez e os valores de peróxido foram inferiores aos valores obtidos em amostras comerciais de óleo vegetal, respectivamente, 2,48 mgKOH/g, 5,22 meq/kg. A extração aquosa apresentou menor eficiência e promoveu uma maior oxidação dos ácidos graxos insaturados. Por outro lado, a polpa de pequi prensada, após secagem ao sol, produziu um óleo de melhor qualidade. No entanto, sua eficiência ainda é menor do que no processo combinado onde se aplicou o tratamento aquoso enzimático seguido da prensagem. O processo combinado promove a hidrólise da parede celular e a redução da viscosidade da polpa, contribuindo com pelo menos 20% de aumento na produtividade da etapa de extração do óleo por prensagem.

The present study aims to compare yield and quality of pequi pulp oil when applying two distinct processes: in the first, pulp drying in a tray dryer at 60ºC was combined with enzymatic treatment and pressing to oil extraction; in the second, a simple process was carried out by combining sun-drying pulp and pressing. In this study, raw pequi fruits were collected in Mato Grosso State, Brazil. The fruits were autoclaved at 121ºC and stored under refrigeration. An enzymatic extract with pectinase and CMCase activities was used for hydrolysis of pequi pulp, prior to oil extraction. The oil extractions were carried out by hydraulic pressing, with or without enzymatic incubation. The oil content in the pequi pulp (45% w/w) and the physicochemical characteristic of the oil was determined according to standard analytical methods. Free fatty acids, peroxide values, iodine and saponification indices were respectively 1.46 mgKOH/g, 2.98 meq/kg, 49.13 and 189.40. The acidity and peroxide values were lower than the obtained values in commercial oil samples, respectively 2.48 mgKOH/g and 5.22 meq/kg. Aqueous extraction has presented lower efficiency and higher oxidation of unsaturated fatty acids. On the other hand, pequi pulp pressing at room temperature has produced better quality oil. However its efficiency is still smaller than the combined enzymatic treatment and pressing process. This combined process promotes cellular wall hydrolysis and pulp viscosity reduction, contributing to at least 20% of oil yield increase by pressing.

O objetivo deste trabalho foi produzir lipases por fermentação em estado sólido (FES) utilizando, como substrato, resíduo agroindustrial enriquecido com subprodutos do processo de refino do óleo de milho ou óleo de oliva. Para um conjunto de dez linhagens de fungos selecionadas nas primeiras etapas, a atividade lipásica obtida por FES variou de 7,7 a 58,6 U/g de substrato seco (gss). Dentre as linhagens avaliadas, o mutante Aspergillusniger 11T53A14 foi selecionado por apresentar a melhor produção enzimática.Para os testes de fermentação, dois substratos foram investigados: farelo de trigo e sabugo de milho, ambos enriquecidos com óleo de oliva. Nestes testes, os melhores resultados foram obtidos com farelo de trigo. Adicionalmente, três subprodutos industriais do refino do óleo de milho (borra, estearina e ácidos graxos) foram avaliados como substitutos do óleo de oliva na função de indutor para a produção de lipases. Dentre eles, borra e estearina demonstraram ser melhores indutores, enquanto ácidos graxos apresentaram um efeito inibidor. As mais altas atividades lipásicas utilizando borra, estearina e ácidos graxos foram 62,7 U/gss, 37,7 U/gss e 4,1 U/gss, respectivamente.

The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively.

A palmeira Euterpe oleracea (Mart.), conhecida como açaizeiro, é nativa da Amazônia e tem-se destacado pelo potencial dos seus produtos, em especial pela importância econômica para a fruticultura regional. A polpa de açaí contém alto teor de óleo vegetal, cerca de 53% em base seca, e é uma fonte de ácidos graxos essenciais. Neste trabalho, a composição em ácidos graxos da fração lipídica da polpa de açaí foi determinada por cromatografia gasosa de alta resolução. O óleo foi obtido pelo processo enzimático ou por extração com n-hexano. A extração enzimática, em meio aquoso, tem sido indicada como uma alternativa para a obtenção de óleos vegetais derivados de polpas de frutas. Os resultados apresentaram diferenças não significativas na composição em ácidos graxos dos óleos provenientes das diferentes técnicas de extração. Em ambos os casos, houve predominância dos ácidos graxos monoinsaturados (68% a 71%) e de ácidos graxos poliinsaturados (7,7% a 10,6%).

Açaí (Euterpe oleracea Martius) is a typical palm tree from the Amazon, growing spontaneously in states of Pará, Amazonas, Amapá and Maranhão. It has been highlighted by the potential of its products, especially because of the economic importance for the regional fruit growing. The açaí fruits are very appreciated by their pulp flavour. In the açaí pulp, there is a significant amount of lipids, around 53%, in a dry basis, turning out to be an excellent source of essential fatty acids. The main purpose of the present work was to establish the fatty acid content in the lipid fraction of açaí pulp using high resolution gaseous chromatography. The açaí oil was obtained by enzymatic technology in water has been indicated as an alternative for vegetable oils extraction from pulp of fruits. A significant difference was not observed in the composition of the fatty acids present in açaí oil obtained from either process. In both cases, the oil is high in monounsaturated fatty acids (68% to 71%) and in polyunsaturated fatty acids (7.8% to 10.6%).

O efeito da adição de cátions na morfologia do agregado e na síntese de poligalacturonase (PG) por Aspergillus niger 3T5B8 foi estudado e comparado com um sistema controle. Fe(II), Cu(II), Zn(II) and Mn(II) foram adicionados ao meio de fermentação, tanto separadamente, como em grupos de cátions. A adição dos íons Fe2+ e/ou Zn2+ foi positivamente significante para a produção da enzima. Um efeito similar no teor de biomassa, contudo, só pode ser observado quando os mesmos íons foram adicionados isoladamente. Por outro lado, os íons Cu2+ e Mn2+ não afetaram significativamente estes parâmetros. Utilizando-se técnicas de processamento de imagens para o estudo da morfologia dos pellets, observou-se que pequenos aglomerados, com pequenos núcleos, foram geralmente obtidos quando os cátions Fe2+ e Zn2+ foram adicionados ao meio, seja isolada ou simultaneamente. Os aglomerados produzidos em meios contendo íons Fe2+ ou Zn2+ eram compactos, enquanto que aqueles produzidos em meios contendo ambos os cátions foram considerados difusos. A autólise do núcleo foi observada para os grandes agregados produzidos no meio controle, devido ao transporte deficiente de nutrientes para o seu interior. Os agregados produzidos em meio contendo os íons Fe2+ e Zn2+ foram grandes produtores de enzima, provavelmente devido a uma morfologia mais "frouxa", induzida pela presença combinada destes metais no meio, favorecendo o transporte de nutrientes.

The effects of cations addition on pellet morphology and polygalacturonase (PG) synthesis by Aspergillus niger 3T5B8 were studied and compared with a control system. Fe(II), Cu(II), Zn(II) and Mn(II) were added to the fermentation medium separately, and also as combined groups of cations. The addition of Fe2+ and/or Zn2+ ions was significantly positive to the enzyme production. A positive effect in the biomass content, however, was only obtained when the same metal ions were added separately. On the other hand, Cu2+ and Mn2+ ions had almost no effect on these parameters. The morphology of the pellets was studied by image processing techniques. Small pellets with small cores were usually obtained when Fe2+ and Zn2+ ions were individually or collectively added to the medium. The pellets produced in media containing Fe2+ or Zn2+ ions were compact, while the ones produced in a medium containing both cations were considered diffuse. Autolysis of the core was observed for large control pellets, due to the deficient nutrient transfer to the interior of the pellet. The pellets obtained in a medium containing both Fe2+ and Zn2+ ions were high enzyme producers, probably due to a loose morphology, induced by the presence of combined groups of metal ions in the medium, favoring the nutrient transfer.

O objetivo deste trabalho foi selecionar linhagens de Aspergillus niger para síntese de tanase. O crescimento das colônias em meio contendo ácido tânico indicou capacidade de produção de tanase. A atividade enzimática foi determinada em fermentação semi-sólida. A. niger 11T25A5 foi o melhor produtor (67.5 U.g-1/72 horas de fermentação).

The aim of this work was to select strains of Aspergillus niger for tannase production. Growth of colonies in plates with tannic acid-containing medium indicated their ability to synthesize tannase. Tannase activity was also measured in solid-state fermentation. A. niger 11T25A5 was the best tannase producer (67.5 U.g-1/72 hours of fermentation).

No processamento tradicional de extração de óleo de palma, uma quantidade substancial do produto é perdida na fibra - durante a etapa de prensagem do fruto - e na borra - durante a etapa de clarificação do óleo cru. A viscosidade deste constitui a principal dificuldade de separação do óleo dos demais componentes da mistura. A eficiência de recuperação do óleo é usualmente melhorada por adição de água, no tanque de clarificação. Neste trabalho avaliou-se os efeitos da diluição e do tratamento enzimático no comportamento reológico do óleo de palma cru. Estes dados são importantes para estimar a velocidade de separação do óleo nos tanques de clarificação e/ou nos decanters das usinas comerciais. O óleo cru utilizado neste estudo foi recolhido em uma unidade comercial sendo composto de 65% de água, 30% de óleo e 5% de sólidos. A hidrólise enzimática foi conduzida usando como agentes hidrolisantes uma enzima comercial (viscozyme) da Novo Nordisk e um preparado enzimático produzido no CTAA. Pode-se constatar que quanto mais diluída a amostra maior é a redução na viscosidade após a hidrólise enzimática. Para uma taxa de deformação fixa em 6s-1, o tratamento enzimático do óleo cru a 60°C reduziu a viscosidade do mesmo em cerca de 29% enquanto a adição de 20% de água em cerca de 35%. A combinação da diluição com 20% de água seguida de tratamento enzimático reduziu a viscosidade do óleo cru em cerca de 75%.

In conventional extraction of palm oil, a substantial loss of product occurs in the stages of the fruit pressing and clarification of the "crude" oil. The high viscosity of "crude" oil is the main factor that affects the separation of the oil from the other components in the mixture. This is usually overcome by the addition of water in the clarification stage. This work addresses the study of the influence of dilution and enzymatic treatments on the rheological behaviour of crude oil. Data obtained were found to be useful for determining the separation velocities in the clarification and centrifugation stages. The palm oil employed in this work was supplied by a commercial plant and had the following composition: water (65%), oil (30%) and solids (5%). This feed was enzymatically hydrolysed at 60°C using a commercial enzyme (viscozyme) and a laboratory enzyme preparation. For a fix shear rate of 6s-1, the enzymatic treatment decreased the viscosity in about 29 % while with the addition of 20% of water the viscosity reduction was 35%. The combined effect of enzymatic and dilution treatments leads to viscosity reduction of 75 % approximately.