Extraction of Ascorbate Oxidase from Cucurbita maxima by Continuous Process in Perforated Rotating Disc Contactor Using Aqueous Two-Phase Systems

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Fed-Batch Production of Tetanus Toxin by Clostridium Tetani

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield (similar to 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration (similar to 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial,scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 88-92, 2010

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5-76.0% and 13.2-19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m(biogas)(3)/kgCOD(i)) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO(2) was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 +/- 0.006 g(DM)/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%. (C) 2009 Elsevier Ltd. All rights reserved.

Sterilization of Medical Devices by Ethylene Oxide, Determination of the Dissipation of Residues, and Use of Green Fluorescent Protein as an Indicator of Process Control

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (131) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/- 3.2)% to 7.5 (+/- 2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/- 3.0)% to 21.5 (+/- 6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/- 3.2)% to 23.9 (+/- 3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 9113: 626-630, 2009

Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060

Clavulanic acid (CA) is a potent inhibitor of beta-lactamases, produced by some resistant pathogenic microorganisms, which allows efficient treatment of infectious diseases. The kinetic and thermodynamic parameters of CA production by a new isolate of Streptomyces DAUFPE 3060 and its degradation were evaluated. The effect of temperature on the system was investigated in the range 24-40 degrees C adopting an overall model accounting for (a) the Arrhenius-type formation of CA by fermentation, (b) the hypothetical reversible unfolding of the enzyme limiting the overall metabolism, and (c) the irreversible first-order degradation of CA. The higher rates of CA formation (k(CA) = 0,107 h(-1)) and degradation (k(d) = 0.062 h(-1)) were observed at 32 and 40 degrees C, respectively. The main thermodynamic parameters of the three above hypothesized events were estimated. In particular, the activation parameters of degradation (activation energy = 39.0 kJ/mol; Delta H(d)* = 36.5 kJ/mol; Delta S(d)* = -219.7 J/(mol K); Delta G(d)* = 103.5 kJ/mol) compare reasonably well with those reported in the literature for similar system without taking into account the other two events. (C) 2009 Elsevier Inc. All rights reserved.

Liquid-liquid extraction by mixed micellar systems: A new approach for clavulanic acid recovery from fermented broth

This work is the first attempt to apply aqueous two-phase mixed micellar systems (ATPMS) of the nonionic surfactant Triton X-114 and the anionic one AOT to extract clavulanic acid (CA) from broth fermented by Streptomyces clavuligerus. Cloud points were determined in McIlvane buffer pH 6.5 with or without NaCl, and diagram phases/coexistence curves were constructed. CA partition was investigated following a 2(4)-full factorial design in which AOT (0.022, 0.033 and 0.044% w/w), Triton X-114 (1.0, 3.0 and 5.0% w/w) and NaCl (0, 2.85 and 5.70% w/w) concentrations and temperature (24,26 and 28 degrees C) were selected as independent variables, and CA partition coefficient (K(CA)) and yield in the top phase (eta(CA)) as responses. CA partitioned always to the top, micelle-poor phase. The regression analysis pointed out that NaCl concentration and interaction between temperature and Triton X-114 concentration had statistically significant effects on K(CA), while eta(CA) was mainly influenced by temperature, Triton X-114 concentration and their interaction. Different ATPMS compositions were then needed to maximize these responses, specifically 0.022% (w/w) AOT, 5% (w/w) Triton X-114 for K(CA) (2.08), and 0.044% (w/w) AOT, 1% (w/w) Triton X-114 for eta(CA) (98.7%), both at 24 degrees C without NaCl. Since at 0.022% (w/w) AOT, 1% (w/w) Triton X-114 and 28 degrees C without NaCl the system was able to ensure satisfactory intermediate results (K(CA) = 1.48; eta(CA) = 86.3%), these conditions were selected as the best ones. These preliminary results are of concern for possible industrial application, because CA partition to the dilute phase can simplify the subsequent purification protocol. (C) 2011 Elsevier B.V. All rights reserved.

The behavior of key enzymes of xylose metabolism on the xylitol production by Candida guilliermondii grown in hemicellulosic hydrolysate

A variety of raw materials have been used in fermentation process. This study shows the use of rice straw hemicellulosic hydrolysate, as the only source of nutrient, to produce high added-value products. In the present work, the activity of the enzymes xylose reductase (XR); xylitol dehydrogenase (XD); and glucose-6-phosphate dehydrogenase (G6PD) during cultivation of Candida guilliermondii on rice straw hemicellulosic hydrolysate was measured and correlated with xylitol production under different pH values (around 4.5 and 7.5) and initial xylose concentration (around 30 and 70 g l(-1)). Independent of the pH value and xylose concentration evaluated, the title of XD remained constant. On the other hand, the volumetric activity of G6PD increased whereas the level of XR decreased when the initial xylose concentration was increased from 30 to 70 g l(-1). The highest values of xylitol productivity (Q (P) a parts per thousand 0.40 g l(-1)) and yield factor (Y (P/S) a parts per thousand 0.60 g g(-1)) were reached at highest G6PD/XR ratio and lowest XR/XD ratio. These results suggest that NADPH concentrations influence the formation of xylitol more than the activity ratios of the enzymes XR and XD. Thus, an optimal rate between G6PD and XR must be reached in order to optimize the xylitol production.

Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L-2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes. (c) 2007 Elsevier B.V. All rights reserved.

Contribution of peripheral vanilloid receptor to the nociception induced by injection of spermine in mice

Polyamines (putrescine, spermidine and spermine) are important endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, we have investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception in vivo. The subcutaneous (s.c.) injection of capsaicin (as positive control), spermine, spermidine or putrescine produced nociception with ED(50) of 0.16 (0.07-0.39) nmol/paw, 0.4 (0.2-0.7) mu mol/paw, 0.3 (0.1-0.9) mu mol/paw and 3.2 (0.9-11.5) mu mol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81 +/- 10 and 68 +/- 9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82 +/- 9% and 67 +/- 11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 mu M) enhanced the specific binding of [(3)H](center dot)-RTX to TRPV1 receptor. Altogether, polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors. (C) 2011 Elsevier Inc. All rights reserved.

Determination of chlorpheniramine in human plasma by HPLC-ESI-MS/MS: application to a dexchlorpheniramine comparative bioavailability study

In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether-dichloromethane, 80:20, v/v) and analyzed by HPLC-ESI-MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH(4)OH on a Gemini Phenomenex C(8) 5 mu m column (50 x 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05-10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra-batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter-batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine (R), Schering-Plough). The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and AUC ratios were all within the 80-125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption. Copyright (C) 2009 John Wiley & Sons, Ltd.

Antimicrobial compounds produced by Lactobacillus sakei subsp sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

One-pot synthesis of alpha,beta-epoxy ketones by palladium-catalyzed epoxidation-oxidation of terminal allylic alcohols

Described herein is a one-pot synthesis of a,p-epoxy ketones using a palladium-catalyzed epoxidation-oxidation sequence. Functionalized terminal allylic alcohols are treated with m-CPBA Under mild reaction conditions to obtain the alpha,beta-epoxy ketones. The main benefit of this approach is that the epoxidation of the terminal double bond and the oxidation of the secondary alcohol occured in the same reaction under mild reactions and both electron-donating and electron-withdrawing functionalities are tolerated in the reaction sequence. (C) 2010 Elsevier Ltd. All rights reserved.

Vegetative and fruit characteristics of papaya trees obtained by mass selection

The present work had as purpose to evaluate some characteristics of papaya trees (Carica papaya L.), Golden cultivar, obtained trough plant mass selection, regarding plant and fruit quality in the first months of production. The samples were evaluated in a commercial crop at: 0, 20, 40, 70, 130, 180, 230, 260, 280, 310 and 340 days after the planting (DAP) and the first fruits were harvested at 230 DAP. The results showed the low height (199cm in 340 DAP) and low first flowering`s heigth (71cm), which is important to facilitate the harvest process. The plants presented good yield with high number of leafs (allowing a great area of fruit cover) and about 60 fruits per plant. The fruits kept similar features to cv. Golden. The fruit`s fresh weight ranged from 302.4 to 467.5g, which is in the range of the Brazilian market. The pulp thickness was 2.35cm, which is a feature of great economic interest. The pulp thickness showed close relation with climatic factors, and great variations of temperature and precipitation accelerated the pulp loss of thickness.

Synergism on antioxidant activity between natural compounds optimized by response surface methodology

Despite the increase in the use of natural compounds in place of synthetic derivatives as antioxidants in food products, the extent of this substitution is limited by cost constraints. Thus, the objective of this study was to explore the synergism on the antioxidant activity of natural compounds, for further application in food products. Three hydrosoluble compounds (x(1) = caffeic acid, x(2) = carnosic acid, and x(3) = glutathione) and three liposoluble compounds (x(1) = quercetin, x(2) = rutin, and x(3) = genistein) were mixed according to a ""centroid simplex design"". The antioxidant activity of the mixtures was analyzed by the ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAL) methodologies, and activity was also evaluated in an oxidized mixed micelle prepared with linoleic acid (LAOX). Cubic polynomial models with predictive capacity were obtained when the mixtures were submitted to the LAOX methodology ((y) over cap = 0.56 x(1) + 0.59 x(2) + 0.04 x(3) + 0.41 x(1)x(2) - 0.41 x(1)x(3) - 1.12 x(2)x(3) - 4.01 x(1)x(2)x(3)) for the hydrosoluble compounds, and to FRAP methodology ((y) over cap = 3.26 x(1) + 2.39 x(2) + 0.04 x(3) + 1.51 x(1)x(2) + 1.03 x(1)x(3) + 0.29 x(1)x(3) + 3.20 x(1)x(2)x(3)) for the liposoluble compounds. Optimization of the models suggested that a mixture containing 47% caffeic acid + 53% carnosic acid and a mixture containing 67% quercetin + 33% rutin were potential synergistic combinations for further evaluation using a food matrix.