Plinho Francisco Hertz

Cellulase and xylanase productions by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation

In Brazil, a large amount of a fibrous residue is generated as result of soybean ( Glycine max) protein production. This material, which is rich in hemicellulose and cellulose, can be used in solid state cultivations for the production of valuable metabolites and enzymes. In this work, we studied the bioconversion of this residue by bacteria strains isolated from water and soil collected in the Amazon region. Five strains among 87 isolated bacteria selected for their ability to produce either celullases or xylanases were cultivated on the aforementioned residue. From strain BL62, identified as Bacillus subtilis, it was obtained a preparation showing the highest specific cellulase activity, 1.08 UI/mg protein within 24 hours of growth. Concerning xylanase, the isolate BL53, also identified as Bacillus subtilis, showed the highest specific activity for this enzyme, 5.19 UI/mg protein within 72 hours of cultivation. It has also been observed the production of proteases that were associated with the loss of cellulase and xylanase activities. These results indicated that the selected microorganisms, and the cultivation process, have great biotechnological potential.

Effect of the support size on the properties of β-galactosidase immobilized on chitosan: advantages and disadvantages of macro and nanoparticles.

The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. β-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. β-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.

High stability of immobilized β-D-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis.

β-D-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37 °C at flow rates up to 2.6 mL min(-1). Maximal GOS concentration of 26 g L(-1) was obtained at a flow rate of 3.1 mL min(-1), with a productivity of 186 g L(-1) h(-1). Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.

Chitosan crosslinked with genipin as support matrix for application in food process: Support characterization and β-D-galactosidase immobilization.

In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested for the immobilization of β-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. β-D-Galactosidase-chitosan-genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan-genipin particles seem to be a good alternative for application in food process.

Characterization and antioxidant potential of Brazilian fruits from the Myrtaceae family.

The objective of this study is to evaluating the Brazilian biodiversity through physicochemical characterization and determination of antioxidant potential of three species from the Myrtaceae family, namely yellow guava (Psidium cattleyanum Sabine), guabiroba (Campomanesia xanthocarpa O. Berg), and uvaia ( Eugenia pyriformis Cambess). Guabiroba had the greater quantity of phenolic compounds (9033 mg chlorogenic acid/100 g) and vitamin C (30.58 mg/g) and showed the best TSS/TTA (total soluble solid/total titratable acid) ratio (45.12). For the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic) method, the guabiroba (507.49 μM Trolox/g) presented the highest antioxidant potential; however, in the DPPH (2,2-diphenyl-1-picrylhydrazyl) method, uvaia (170.26 g/g DPPH) and guabiroba (161.29 g/g DPPH) were not statistically different. The uvaia outranked the other fruits with respect to its high carotenoid (909.33 μg/g) and vitamin A (37.83 μg/g) contents, and the yellow guava, although showing a lower bioactive compound content and antioxidant activity, nevertheless presented much higher values than many traditionally consumed fruits.

Development of active biofilms of quinoa (Chenopodium quinoa W.) starch containing gold nanoparticles and evaluation of antimicrobial activity.

Active biofilms of quinoa (Chenopodium quinoa, W.) starch were prepared by incorporating gold nanoparticles stabilised by an ionic silsesquioxane that contains the 1,4-diazoniabicyclo[2.2.2]octane chloride group. The biofilms were characterised and their antimicrobial activity was evaluated against Escherichiacoli and Staphylococcusaureus. The presence of gold nanoparticles produces an improvement in the mechanical, optical and morphological properties, maintaining the thermal and barrier properties unchanged when compared to the standard biofilm. The active biofilms exhibited strong antibacterial activity against food-borne pathogens with inhibition percentages of 99% against E. coli and 98% against S. aureus. These quinoa starch biofilms containing gold nanoparticles are very promising to be used as active food packaging for the maintenance of food safety and extension of the shelf life of packaged foods.

High operational stability of invertase from Saccharomyces cerevisiae immobilized on chitosan nanoparticles

Invertase (E.C.3.2.1.26) from Saccharomyces cerevisiae was covalently immobilized on chitosan nanoparticles prepared by the ionotropic gelation method and activated with glutaraldehyde. The support was characterized and it was studied its load capacity, the influence of the presence of substrate during immobilization, and determined the biocatalyst kinetic parameters and stabilities. The light scattering analysis (LSA) and transmission electron microscopy (TEM) techniques indicated a mixture of chitosan nano and aggregated nanoparticles, providing high superficial area for enzyme immobilization. The thermal and storage stabilities, the optimal pH and temperature of the enzyme were not altered. K(m) increased 3-fold, while V(max) remained unaltered. The immobilized biocatalyst was reused for 59 batches with maximal invertase activity, the highest operational stability so far described in the literature. These results fulfill some important aspects for the enzyme immobilization: the simplicity of the protocols, the conservation of the enzyme activity, and the high operational stability.

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan.

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.

Fructooligosaccharides synthesis by highly stable immobilized β-fructofuranosidase from Aspergillus aculeatus

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50 °C and 60 °C, being around 100 times higher at 60 °C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100 min per batch at 50 °C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230 Ut/g, which is among the best for FOS production.

Detection of the origin of Brazilian wines based on the determination of only four elements using high-resolution continuum source flame AAS.

A method has been developed to determine 10 elements in Brazilian red wines using high-resolution continuum source flame atomic absorption spectrometry, a technique that allows the fast sequential determination of an essentially unlimited number of elements per sample, each one under previously optimized conditions. All measurements were made without sample preparation, using aqueous standard solutions for calibration. The results were in agreement within 99% of confidence (t-test) with those obtained by inductively coupled plasma optical emission spectrometry. The same grape, Cabernet sauvignon, was used in all experiments, and the wines from each region were prepared especially for this investigation in order to avoid any confusion due to grapes from other regions, which are often used in commercial wines. The elements K, Mn, Rb and Sr were found to be the best indicators for the origin of the wines, based on a Principal Component Analysis.