Extraction and purification of theobromine using ionic liquids

Theobromine is an alkaloid present in cocoa and it is used in the treatment of atherosclerosis, hypertension, angina, among others. Due to its importance, the aim of this work consists on the development of an efficient and sustainable technology for the extraction of theobromine from cocoa beans. For the development of a purification technique for theobromine extracted from cocoa, aqueous biphasic systems (ABS) composed of ionic liquids (ILs) were initially studied to infer on the most promising systems. Cholinium-based ILs, based on a non-toxic and biocompatible cation, were used combined with two polymers (PPG 400 and PEG 400) and an inorganic salt (K3PO4). The respective phase diagrams at 298 K and atmospheric pressure were determined, as well as their extraction efficiencies for theobromine. The results obtained indicate that K3PO4 has a greater ability to induce the formation of ABS compared to PEG 400 and PPG 400. ABS consisting of K3PO4 also have a high potential for the extraction of theobromine, with extraction efficiencies ranging between 96.4 and 99.9 %. Based on the most promising ILs for the purification step, they were further used in aqueous solution to extract theobromine from cocoa beans, with extraction yields ranging between 4.5% and 6.5 wt%. Finally, ABS were applied to the aqueous solutions containing theobromine from the cocoa extract, with extraction efficiencies ranging between 96.7 and 99.0%.

Extraction and purification of immunoglobulin G with aqueous biphasic systems

The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG.

Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent.

Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay

Rotavirus double-stranded RNA was detected directly in sewage treatment plant samples over a 1-year period by reverse transcription followed by PCR amplification of the VP7 gene and Southern blot hybridization. The presence of naturally occurring rotaviruses was demonstrated in 42% of raw sewage samples and in 67% of treated effluent samples, Amplified viral sequences were analyzed bg restriction enzymes. Ten different restriction profiles were characterized, most of which were found in treated effluent samples. A mixture of restriction profiles was observed in 75% of contaminated effluent samples, The profiles were compared with those obtained from human rotavirus isolates involved in infections in children from the same area (six different profiles were detected), Five identical viral sequences were detected in both environmental and clinical samples, Restriction profiles sere also compared io profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely.

CERAMIC MATERIAL FROM SEWAGE SLUDGE AS SUPPORT MATERIAL SUPPLY WATER FILTRATION

This study shows a possibility of using municipal sewage sludge after thermal treatment in the production of a filtering material to water treatment. Due to the fast urbanization and implementation of high standards for effluent in many countries in recent years, the sewage sludge is being produced in an ever increasing amount. Therefore, the use of sludge is a suitable solution for the expected large quantity of sludge. Dehydration of sludge was performed by controlled heating at temperatures of 1100 degrees C, 850 degrees C, 650 degrees C, 350 degrees C for 3 hours. After thermal treatment the sludge was characterized by X-ray fluorescence, TG/DTG/DTA, residue solubilization and residue lixiviation tests. The aim of the present work was to observe, thought the characterization techniques, if the treated sewage sludge is or not adequate to be used as filter material to water treatment. It will be verified which treatment temperature of the sludge offer possibility to its use in water treatment without carrying pollutants in concentrations out of the standards.

Sintered Sewage Sludge As Support Material Used In Drinking Water Filtration

The processing of industry and domestic effluents in wastewater treatment plants reduces the amount of polluted material and forms reusable water and dehydrated sludge. the generation of hazardous municipal sludge can be decreased, as well as the impact on surface and underground water and the risk to human health. The aim this study is to verify the possibility to use sintered sewage sludge as support material after thermal treatment in the production of a filtering material to water supply systems. After thermal treatment the sewage sludge ash was characterized by X-ray fluorescence (XRF), leaching test and water solubilization. Dehydration of sludge was performed by controlled heating at temperatures of 180 degrees C, 350 degrees C, 600 degrees C, 850 degrees C and 1000 degrees C for 3 hours.

Influence of a Brazilian sewage outfall on the toxicity and contamination of adjacent sediments

The submarine sewage outfall of Santos (SSOS) is situated in the Santos Bay (São Paulo, Brazil) and is potentially a significant source of contaminants to the adjacent marine ecosystem. The present study aimed to assess the influence of SSOS on the sediment toxicity and contamination at Santos Bay. At the disposal site, sediments tended to be finer, organically richer and exhibited higher levels of surfactants and metals, sometimes exceeding the Threshold Effect Level values. The SSOS influence was more evident toward the East, where the sediments exhibited higher levels of TOC, total S and metals during the summer 2000 sampling campaign. Sediment toxicity to amphipods was consistently detected in four of the five stations studied. Amphipod survival tended to correlate negatively to Hg, total N and % mud. This work provides evidence that the SSOS discharge affects the quality of sediments from Santos Bay, and that control procedures are warranted. (c) 2005 Elsevier Ltd. All rights reserved.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Control of Fusarium in chrysanthemum with sewage sludge, biofertilizer, hydrolyzed fish, chitosan and Trichoderma.

2009

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 mu g/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regius

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002

Chemical changes and heavy metal partitioning in an oxisol cultivated with maize (Zea mays, L.) after 5 years disposal of a domestic and an industrial sewage sludge.

The need for solutions to minimize the negative environmental impacts of anthropogenic activities Fhas increased. Sewage sludge is composed of predominantly organic matter and can be used to improve soil characteristics, such as fertility. Therefore, its application in agriculture is an adequate alternative for its final disposal. However, there is a lack of information on its long-term effects on soil changes in tropical areas. Thus, the objectives of this study were to determine (i) the effect of sewage sludge application on heavy metal build-up in soil and maize grains and leaves, and (ii) the effects of soil amendment with sewage sludge on the chemical properties of a Brazilian oxisol. Besides the increasing levels of Zn, Cu, Ni, and Cr, amending soil with sewage sludge also alters the distribution of these metals by increasing the mobile Phases, which correlated significantly with the increase in metal extraction with two single extractants, Mehlich 1 and DTPA (Diethylene triamine pentaacetic acid). The levels of Fe, Mn, Zn, and Cu in maize grains and leaves increased with the type and rate of sewage sludge application. Nevertheless, metal build-up in soil and plants was within the allowed limits. Significant differences were also found in soil characteristics like humic fractionation with the applied sewage doses. The data obtained does not indicate any expressive drawbacks in the use of sewage sludge as a soil amendment, as the heavy metal concentrations observed are unlikely to cause any environmental or health problems, even overestimated loadings, and are in accordance with the Brazilian regulations on farming land biosolid disposal.