Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production.

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40-60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae's potential for tannase and gallic acid production for biotechnological applications.

Multivariate optimization of ultrasound-assisted liquid-liquid microextraction based on two solvents for cadmium preconcentration prior to determination by flame atomic absorption spectrometry.

A method based on ultrasound-assisted emulsification liquid-liquid microextraction (USAEME) for cadmium determination by flame atomic absorption spectrometry (FAAS) was developed in this work. USAEME is based on the use of the mixture of 1,2-dichloroethane and trichloroethylene as an acceptor phase, 2-(2-bromo-5-pyridylazo)-5(diethylamino)phenol (Br-PADAP) as a chelating reagent, and ethanol as a dispersive solvent. The composition of the extraction and dispersive solvents, the volume of the extraction solvent, pH, and the sonication time were optimized using the multivariate strategy. The limits of detection and quantification calculated under optimum conditions were, respectively, 0.39 and 1.33 µg L-1, and the obtained enrichment factor was 21. The accuracy was tested by the analysis of certified reference materials. The method was applied to cadmium determination in bivalve mollusks, water, and urban wastewater from Pontal Bay, Bahia, Brazil. The proposed method is simple, fast, and efficient, and uses small amounts of organic solvents for the determination of cadmium.

Artificial neural network employment for element determination in Mugil cephalus by ICP OES in Pontal Bay, Brazil.

Fish are important sources of protein, making them very significant in the human diet. Although the consumption of this food is beneficial for health, it is essential that the product does not contain inorganic components above the limits recommended by the current legislation. Therefore, a method for determination of elements in fish (Mugil cephalus) samples was optimized. A simplex centroid mixture design with restriction was applied for optimization of the acid digestion of samples in an open system under reflux in order to evaluate the best ratio between the reagents HNO3, H2O2 and H2O. The results indicated that more intense analyte signals were obtained when a mixture containing 3.6 mL of HNO3 (65% v/v), 0.4 mL of H2O2 (30% v/v) and 6.0 mL of H2O was used. The accuracy of the method was assessed with a CRM of oyster tissue (NIST 1566b). The method presented relative standard deviations (RSDs) of 3.54%; 3.82%; 4.81% and 3.50% for Zn, Fe, Cu and S, respectively. The detection limits were 0.002 mg kg-1 for Cu and Zn and 0.02 mg kg-1 for Fe and S. The proposed method was applied for the determination of Zn, Fe, Cu and S in fish samples. A Kohonen Self-Organizing Map (KSOM) with K-means implementation was applied to better delimit the boundary between groups and the spatial and temporal influence on how concentrations of the chemical elements were perceived. To verify the separation, the Davies-Bouldin and Silhouette indices were used, obtaining 0.5374 and 0.8541, respectively, indicating satisfactory separation.

Screening of Mangifera indica L. functional content using PCA and neural networks (ANN).

A method using reversed-phase high-performance liquid chromatography with diode array detection (HPLC-DAD) was applied after extraction with acidified methanol, to determine 12 bioactive phenolic compounds in the peel and pulp of the mango fruit (Mangifera indica L.) cultivated in the Bahia, state of Brazil. The proposed methodology was previously fully validated and proven successful in the analysis of methanolic extracts of lyophilized samples. The limits of quantification ranged between 0.78 and 3.14 mg L-1 and the individual recovery values obtained for the spiked samples ranged from 80% to 120%. The results were evaluated using PCA and ANN. The results indicate that the fruits are rich in polyphenols, mainly: ellagic acid, gallic acid, rutin and catechin, which contribute to their greater use as functional foods, natural antioxidants and in the pharmaceutical industry, as well as other applications.

Development of procedure for sample preparation of cashew nuts using mixture design and evaluation of nutrient profiles by Kohonen neural network.

A procedure using ICP OES for sample preparation for the determination of copper, iron and manganese in cashew nuts was developed. Constrained simplex-centroid design was applied in the optimization of the digestion in microwave oven procedure, and the results evaluated from topological maps of the Kohonen network. The best proportion evaluated for the digestion of the sample with HNO3, H2O2 and H2O was 10:45:45 (%). With optimized conditions, the detection limits were 0.63, 4.3 and 0.37 mg kg-1, and quantification 2.1, 14 and 1.2 mg kg-1 for Cu, Fe and Mg, respectively. The precision (% RSD) was 1.84, 2.31 and 2.73, for Cu, Fe and Mg, respectively. The procedure proposed had the accuracy confirmed using NIST 1568b (at 95% reliability) and was applied in the samples obtaining concentrations in the range of 10.7-19.4, 44.3-67.2 and 11.0-21.4 mg kg-1 for Cu, Fe and Mg, respectively.

Evaluation of macro and micronutrient elements content from soft drinks using principal component analysis and Kohonen self-organizing maps.

This study approaches the determination of nine elements from Brazilian carbonated soft drinks of several flavors and manufactures using inductively coupled plasma optical emission spectrometry (ICP OES). The concentrations of the elements varied as follows: (in µg L-1: Cu: 4.00-78.0; Fe: 74.0-506; Mn: 20.0-66.0; Zn: 104-584) and (in mg L-1: Ca: 4.81-16.2; K: 6.73-260; Na: 26.0-175; S: 1.43-5.41; P: 0.186-219). Principal component analysis has shown some tendencies to form two groups according to the drink flavor (orange and cola), but only cola presented a clear and complete separation. Using Kohonen maps, it was observed a tendency to form three flavor groups: (i) cola, (ii) orange and lemon, and (iii) guarana. However, this last tool proved to be more accurate in the groups' formation.

Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124.

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8 hr (rice husks) and 7.3 hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5-7 (rice hulls) and 6-9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.

Screening of Passiflora L. mineral content using principal component analysis and Kohonen self-organizing maps.

A method using digestion with diluted nitric acid and inductively coupled plasma optical emission spectrometry was applied to determine metal ions in the peel, pulp, and seed of Passiflora edulis f. flavicarpa and Passiflora cincinnata cultivated in the Bahia state, Brazil. Plasma parameters (radio frequency power and argon flow rates) were optimized. The quantification limits (µg g-1) were: Ca (43.0); Co (1.3); Cu (10.0); Fe (10.0); K (20.0); Mg (26.6); Mn (0.33); Na (56.6) and Zn (10.0). The accuracy of the method was evaluated by the analysis of certified reference materials (NIST 1567a Wheat Flour and NIST 1577b Bovine Liver). Recovery studies were carried out and values found were between 89 and 120%. PCA and Kohonen self-organizing maps showed the formation of groups, corresponding to the fruit parts. Thus, passion fruit has great importance and biological potential, due to its high content of K and low Na.

Thermoresistant xylanases from Trichoderma stromaticum: Application in bread making and manufacturing xylo-oligosaccharides.

The enzymes Xyl1 and Xyl2 from T. stromaticum were purified and identified by mass spectrometry (MALDI-TOF/MS). Xyl1 contained three proteins with similarity to xylanase family 10, 62 and anarabinofuranosidase of the Trichoderma genus and Xyl2 contained a protein with similarity to endo-1,4-ß-xylanase. High xylanase activity was found at 50°C for Xyl1 and 60°C for Xyl2 and pH 5.0 for both, retaining more than 80% of activities for one hour at 60°C and pH 5-8. Ag2+ and ß-mercaptoethanol increased while SDS and EDTA inhibited the xylanase activity of both Xyl1 and Xyl2 extracts. The Km and Vmax values for purified Xyl2 were 9.6mg/mL and 28.57µmol/min/mg, respectively. In application tests, both Xyl1 and Xyl2 were effective in degrading beechwood xylan to produce xylo-oligosaccharides. In baking, adding Xyl1 increased the softness and volume of wheat bread and whole grain bread, qualities increasingly desired by consumers in this segment.

Determination and Evaluation of Metallothionein and Metals in Mugil cephalus (Mullet) from Pontal Bay, Brazil.

To assess environmental contamination, studies have been increasingly carried out using biomarkers to diagnose the effects of toxic metal exposure, such as metallothionein (MT). In this context, the aims of the present study were to determine Cd, Cu, Ni and Pb concentrations, by ICP-MS, in liver samples from Mugil cephalus from the municipality of Ilhéus, located in North-Southeastern Brazil, in the state of Bahia, which has been increasingly suffering from anthropogenic pressure, and to quantify total MT in liver and gills. Among essential metals, copper (199.2 µg g-1) presented concentrations approximately 7 times higher than allowed by the World Health Organization (30 µg g-1). Liver samples showed higher concentrations than gills. Metallothionein concentrations indicate differential contamination along the study area. The first sampling showed higher induction of MT synthesis overall. Differences between liver and gill MT concentration trends were relatively minor, which may indicate the adequacy of analyzing gills in an environmental monitoring context.

Statistical designs and response surface techniques for the optimization of chromatographic systems.

This paper describes fundamentals and applications of multivariate statistical techniques for the optimization of chromatographic systems. The surface response methodologies: central composite design, Doehlert matrix and Box-Behnken design are discussed and applications of these techniques for optimization of sample preparation steps (extractions) and determination of experimental conditions for chromatographic separations are presented. The use of mixture design for optimization of mobile phases is also related. An optimization example involving a real separation process is exhaustively described. A discussion about model validation is presented. Some applications of other multivariate techniques for optimization of chromatographic methods are also summarized.