Maria Gabriela Bello Koblitz

Compostos fenólicos, carotenóides e atividade antioxidante em produtos vegetais

The respiratory process and several aerobic cells oxidative reactions lead to the formation of free radicals which contribute to the appearance of different diseases. The human cells depend on their antioxidant ability to provide protection against the prejudicial effects of free radical and reactive oxygen species that are inevitable consequences of aerobic life. Several epidemiologic studies indicate that high plant products ingestion is associated to a reduction in the hazard of a variety of cronical diseases such as arteriosclerosis and cancer. These effects have been specifically attributed to the plant compounds that have antioxidant activity: vitamin C and E, phenolic compounds, specially flavonoids, and carotenoids. 1

Purification and biochemical characterization of an extracellular lipase produced by a new strain of Rhizopus sp.

The present study had as a goal to purify and characterize the lipolytic fraction secreted by a strain of Rhizopus sp. Only 3 steps of purification were necessary to achieve SDS-PAGE homogeneity. One band with 37.5 KDa molecular mass and with 1446 U/mg specific activity was obtained. The purified fraction presented 2 lipase isoforms; both showed optimum activity at 50oC, and were stable between 6.5 and 7.5 pH values and at temperatures below 50oC and also kept their activity in hexane. The lipase was inactivated by Hg+2 and by n-bromosuccinimide and activated by Na+.

Partial characterization and inactivation of peroxidases and polyphenol-oxidases of umbu-cajá (Spondias spp.)

A crude extract of Spondias spp. was evaluated for the influence of pH and temperature on the activity and stability of its peroxidases and polyphenol-oxidases. In order to evaluate the conditions for the inactivation of the enzymes by heat treatment and by addition of a reducing agent, a factorial experimental design (n = 3) was employed using the Statistica (6.0) software package for data analysis. The optimal conditions found for peroxidases were: pH = 5.0 and temperature = 40 °C, and for polyphenol-oxidases they were pH = 7.0 and temperature = 40 °C. The peroxidases and polyphenol-oxidases were stable at all pH values tested (3.0 – 10.0) and maintained more than 60% of their activity at temperatures above 30 and 40 °C, respectively. To achieve the total inactivation of these enzymes, two alternatives can be suggested: incubation at 92 °C for 3.15 minutes with 200 mg.L –1 of ascorbic acid or incubation at 96 °C for 2.80 minutes with 100 mg.L –1 of ascorbic acid.

Substrate and enzyme concentration dependence of the Henri–Michaelis–Menten model probed by numerical simulation

The use of the classic Henry–Michaelis–Menten (HMM) model (or simply, Michaelis–Menten model) to study the substrate and enzyme concentration dependence of enzyme catalysis is a very important step in understanding many biochemical processes, including microbial growth. Although the HMM model has been extensively studied, the conditions in which the substrate concentration is not in excess have still not been adequately defined mathematically. This lack of definition occurs despite at the cellular and molecular levels most systems generally do not operate in a state of substrate excess. In the present work, we describe an approach for studying enzyme reactions in which substrate concentrations are not in excess. Our results show that the use of extent of reactions and numerical simulation of the velocities of reaction provides an important advance in this field and furnishes results not obtained in previous studies involving these aspects. This approach, in association with knowledge of the rate constants, provides a direct and easy means of examining the single substrate–enzyme profile during product formation at any enzyme–substrate ratio. This approach is more direct than previous models that required the use of empirical equations with arbitrary constants.

Antioxidant Activity and Cytotoxicity Effect of Cocoa Beans Subjected to Different Processing Conditions in Human Lung Carcinoma Cells

Lung cancer is a common malignancy in men and the second leading cause of cancer-related mortality in men in the western world. Phenolic cocoa ingredients have a strong antioxidative activity and the potential to have a protective effect against cancer. In the present study, we have evaluated the influence of cocoa beans subjected to different processing conditions on cell viability and apoptosis of human lung cancer cells (A549). We measured the viability of lung cells treated with cocoa beans, unroasted slates (US), roasted slates (RS), unroasted well fermented (UWF) cocoa, and roasted well fermented (RWF) cocoa for 24 h. Using an MTT assay, we observed a decrease in the viability of A549 cells after treatment with cocoa bean extracts. Flow cytometer analysis revealed that cocoa beans increased the percentage of cells in sub-G1 phase and promoted up to twofold increase of apoptotic cells when compared to the control group. Taken together, the present study suggests that cocoa beans may have a protective effect against lung cancer.

Lytic enzyme production optimization using low‐cost substrates and its application in the clarification of xanthan gum culture broth

Lytic enzymes are widely used in industrial biotechnology as they are able to hydrolyze the bacterial cell wall. One application of these enzymes is the clarification of the culture broth for the production of xanthan gum, because of its viability in viscous media and high specificity. The screening process for filamentous fungi producing lytic enzymes, the optimization of production of these enzymes by the selected microorganism, and the optimization of the application of the enzymes produced in the clarification of culture broth are presented in this article. Eleven fungal isolates were tested for their ability to produce enzymes able to increase the transmittance of the culture broth containing cells of Xanthomonas campestris. To optimize the secretion of lytic enzymes by the selected microorganism the following variables were tested: solid substrate, initial pH, incubation temperature, and addition of inducer (gelatin). Thereafter, secretion of the enzymes over time of incubation was assessed. To optimize the clarification process a central composite rotational design was applied in which the pH of the reaction medium, the dilution of the broth, and the reaction temperature were evaluated. The isolate identified as Aspergillus tamarii was selected for increasing the transmittance of the broth from 2.1% to 54.8%. The best conditions for cultivation of this microorganism were: use of coconut husk as solid substrate, with 90% moisture, at 30°C for 20 days. The lytic enzymes produced thereby were able to increase the transmittance of the culture broth from 2.1% to 70.6% at 65°C, without dilution and without pH adjustment.

Trametes villosa lignin peroxidase (TvLiP): genetic and molecular characterization.

White-rot basidiomycetes are the organisms that decompose lignin most efficiently, and Trametes villosa is a promising species for ligninolytic enzyme production. There are several publications on T. villosa applications for lignin degradation regarding the expression and secretion of laccase and manganese peroxidase (MnP) but no reports on the identification and characterization of lignin peroxidase (LiP), a relevant enzyme for the efficient breakdown of lignin. The object of this study was to identify and partially characterize, for the first time, gDNA, mRNA, and the corresponding lignin peroxidase (TvLiP) protein from T. villosa strain CCMB561 from the Brazilian semiarid region. The presence of ligninolytic enzymes produced by this strain grown in inducer media was qualitatively and quantitatively analyzed by spectrophotometry, qPCR, and dye fading using Remazol Brilliant Blue R. The spectrophotometric analysis showed that LiP activity was higher than that of MnP. The greatest LiP expression as measured by qPCR occurred on the 7th day, and the ABSA medium (agar, sugarcane bagasse, and ammonium sulfate) was the best that favored LiP expression. The amplification of the TvLiP gene median region covering approximately 50% of the T. versicolor LPGIV gene (87% identity); the presence of Trp199, Leu115, Asp193, Trp199, and Ala203 in the translated amplicon of the T. villosa mRNA; and the close phylogenetic relationship between TvLiP and T. versicolor LiP all indicate that the target enzyme is a lignin peroxidase. Therefore, T. villosa CCMB561 has great potential for use as a LiP, MnP, and Lac producer for industrial applications.

Influência da concentração de NaCl e pH na extração de ricina em torta de mamona (Ricinus communis L.) e sua caracterização por eletroforese.

Castor bean (Ricinus communis L.) is an oilseed crop of high economic value due to the fact of presenting a clearly defined market for the oil extracted from its seeds. Castor cake, which is a residue of oil extraction, is at the moment receiving special attention because of its high protein content. However, among the proteins found in this cake it is observed the presence of ricin, a cytotoxin, which turns this seed dangerous to be used as an alternative protein source for animal feed. The present research has the objective of identifying the best experimental treatment for extraction of ricin from castor cake, with the aim of future studies of loss of ricin integrity, which would ensure the safety of the product. With this aim, it was initiated the search for a buffer of higher capacity of proteins extraction, using the response surface methodology. A central composite design was developed in order to determine the best pH and NaCl concentration for extraction. Of the five different pH values (4.0, 4.6, 6.0, 7.4, 8.0) and NaCl (0.0M, 0.3M, 1.0M, 1.7M, 2.0M) used, the treatment containing 0.2M potassium phosphate / 1.7M NaCl pH 7.4 was chosen as the best. The amount of protein extracted in this treatment reached values four times larger than the minimum found in other treatment studied. The electrophoresis analysis showed that there was no preferential extraction of ricin in the treatments; however purification steps using dialysis and precipitation with ammonium sulfate led to a better resolution of the two polypeptide chains presents in ricin.

ESTUDO DOS FATORES DE INFLUÊNCIA PARA RECUPERAÇÃO DE ÁCIDOS FENÓLICOS DE TORTA DE GIRASSOL POR ADSORÇÃO

O girassol (Helianthus annuus L.) é uma planta oleaginosa, cultivada em várias partes do mundo, com produção anual de 40 milhões de toneladas. A torta de girassol é um resíduo da extração do óleo de girassol, que contém cerca de 32% de proteína e até 4% de compostos fenólicos, majoritariamente utilizada como ração animal. O girassol apresenta, em sua fração fenólica, os ácidos cafeico, ferúlico e, sobretudo, clorogênico, que permanecem na torta, após a extração do óleo, e que apresentam atividade antioxidante, podendo ser utilizados como conservantes naturais em alimentos sujeitos à peroxidação lipídica. A viabilidade técnica e econômica da exploração dessa fração fenólica depende da otimização da recuperação dos compostos fenólicos presentes na torta do girassol. Diversos estudos vem se dedicando a extrair os ácidos fenólicos da torta, porém a recuperação desses compostos da solução de extração ainda apresenta desafios. O objetivo deste trabalho foi, portanto, estudar, através de planejamento fatorial multivariável, a influência do tipo e concentração do adsorvente sólido, do pH inicial, da temperatura e do tempo de contato na adsorção dos fenólicos obtidos por extração aquosa da torta de girassol. Para tanto, foi utilizado o software Statistica (7.0) e foram avaliadas misturas de celite e carvão ativado, em concentrações variando entre 1 e 5 g de adsorvente por litro de solução extratora, pH entre 4,0 e 10,0, temperatura de 20 a 40oC e tempo de contato entre 1 e 4 horas, num total de vinte ensaios. Como resposta foi avaliada a porcentagem de adsorção, calculada pela relação entre o teor de fenólicos totais, determinado pelo método espectrofotométrico de FolinCiocalteau, da solução extratora inicial e do resíduo de cada ensaio de recuperação. Os resultados foram avaliados por análise de variância, considerando um nível de significância de 95%. Todas as variáveis testadas e a maior parte das interações entre elas se provaram estatisticamente significativas, sendo as variáveis pH, tempo de contato e tipo de adsorvente as de maior influência. Foi possível alcançar adsorção de até 86,88% dos fenólicos totais presentes na solução de extração, quando foram aplicadas as seguintes condições de recuperação: 3 g/L de adsorvente (50% carvão ativado e 50% celite), em pH=7,0, a 30oC, com 2,5h de contato. Os resultados indicaram que a adsorção em sorvente sólido é eficiente e pode ser tecnicamente viável para recuperação de compostos fenólicos extraídos de farelo de girassol. Um novo experimento está sendo desenvolvido com a finalidade de otimizar as variáveis de maior influência, de modo a atingir a maior adsorção possível.

Production of Basidiomata and Ligninolytic Enzymes by the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Licuri (Syagrus coronata) Wastes in Brazil

Ganoderma lucidum is a medicinal mushroom with different forms of bioactivity that has been used in popular medicine for centuries. This study aimed to test the application of agricultural wastes (fruit shells, leaves, and bracts) from the endemic Brazilian palm tree Syagrus coronata (licuri) as substrates for the production of G. lucidum basidiomata and ligninolytic enzymes via solid-state fermentation. The best culture conditions were the same for all substrates (pH 6.5, carbon-to-nitrogen ratio = 40, and temperature 30°C) and were established from preliminary assays. The yield was not significantly different for bracts (33.53 g/kg) and leaves (37.48 g/kg), nor for the biological efficiency in these same substrates: bracts, 3.35%; leaves, 3.75%. The highest laccase (13.80 U/L) and manganese peroxidase (14.92 U/L) activities were achieved after 14 and 28 days of incubation, respectively, using bracts as the substrate. Licuri residues are then potential substrates to be used in the bioconversion process for mycelia, basidiomata, and ligninolytic enzyme production by G. lucidum.