Manuel Perez-Mateos

Antioxidant properties, radical scavenging activity and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study

BACKGROUND This study was designed to evaluate and compare antioxidant capacity and radical scavenging activity of naringin and its aglycone by different in vitro assays. The effects of flavanones on lipid peroxidation, glutathione (GSH) oxidation and DNA cleavage were also assessed. RESULTS The results showed that naringenin exhibited higher antioxidant capacity and hydroxyl and superoxide radical scavenger efficiency than naringin. Our results evidenced that glycosylation attenuated the efficiency in inhibiting the enzyme xanthine oxidase and the aglycone could act like a more active chelator of metallic ions than the glycoside. Additionally, naringenin showed a greater effectiveness in the protection against oxidative damage to lipids in a dose-dependent manner. Both flavanones were equally effective in reducing DNA damage. However, they show no protective effect on oxidation of GSH. CONCLUSION The data obtained support the importance of characterizing the ratio naringin/naringenin in foods when they are evaluated for their health benefits.

Kinetics of cellulose saccharification by Trichoderma reesei cellulases

In order to optimize the enzymatic hydrolysis of three celluloses (carboxymethyl cellulose, powdered cellulose and microgranular cellulose), the effect of varying enzyme and substrate concentration, temperature and pH was studied. The best experimental conditions found to degrade the celluloses were 48 h of incubation, an enzyme concentration of 7 U ml -1 for the cellulases and 0.1 U ml -1 for the β-glucosidase (from A. niger) and a substrate concentration of 0.5%. Under these conditions, carboxymethyl, powdered and microgranular celluloses were degraded 11.2, 40.2 and 15.8%, respectively. Results suggested that cellulases induced in the presence of each substrate were able to hydrolyze more effectively, if substrate concentration was similar to that used in the induction of enzymes. The rate of saccharification of Sigmacell 100 at high substrate concentrations was 93% lower than the maximum rate observed with 0.5% cellulose. Differences in the degree of substrate inhibition when using different substrates were particularly relevant to the experimental design of comparative studies.

Neutrase immobilization on alginate-glutaraldehyde beads by covalent attachment.

Neutrase, a commercial preparation of Bacillus subtilis , was covalently immobilized on alginate-glutaraldehyde beads. Immobilization conditions and characterization of the immobilized enzyme were investigated. Central composite design and response surface methods were employed to evaluate the effects of immobilization parameters, such as glutaraldehyde concentration, enzyme loading, immobilization pH, and immobilization time. Under optimized working conditions (2% alginate, 6.2% glutaraldehyde, 61.84 U mL(-1) Neutrase, pH 6.2, and 60 min) the immobilization yield was about 50%. The immobilized enzyme exhibited higher K(m) compared to the soluble enzyme. The pH-activity profile was widened upon immobilization. The optimum temperature was shifted from 50 to 60 degrees C, and the apparent activation energy was decreased from 47.7 to 22.0 kJ mol(-1) by immobilization. The immobilized enzyme also showed significantly enhanced thermal stability.

Location, kinetics and stability of cellulases induced in Trichoderma reesei cultures

Amorphous cellulose induced the synthesis of endoglucanase in Trichoderma reesei to a greater extent than did cellobiose, lactose, sucrose or other commercial celluloses. In contrast, none of these carbohydrates was able to induce significant levels of endoglucanase activity in Aspergillus niger or Pseudomonas pickettii. Both endoglucanase and β-glucosidase activities were found in cell wall, cell-free extracts and extramycelial fractions of Trichoderma reesei cultures grown on amorphous cellulose. When carboxymethyl cellulose was used as substrate, Km and Vmax values of 1.32% (w/v) and 405.5 μmol glucose ml−1 h−1 for carboxymethyl cellulase were obtained Additionally, the activity was maximum over a pH range of 4.5–5.5, declining sharply beyond 5.5. The optimum temperature was between 50 and 70°C, with maximum activity at 60°C. The half-life of the enzyme appeared to be 9.4 h at 55°C and 4.3 h at 60°C.

Effect of immobilization on the stability of bacterial and fungal β-d-glucosidase

Abstract The thermal and proteolytic stability of free and immobilized β- d -glucosidase, isolated from Pseudomonas pickettii and Aspergillus niger , were determined. The optimal temperatures of soluble and entrapped β-glucosidase extracted from P. pickettii were 40 and 50°C, respectively. In contrast, the optimal temperature of enzyme isolated from A. niger remained unaltered (60°C). Free and immobilized A. niger β-glucosidase showed an unusual discontinuity around 40°C in the Arrhenius plot, suggesting that the enzyme could exist in two (or more) interconvertible forms with different activation energies. The polymeric network influenced the reactivity of both fungal and bacterial β-glucosidases since their E a values changed with respect to their soluble counterparts. Nevertheless, both the thermal stability and the resistance to proteolysis were apparently related to the origin (bacterial or fungal) and location (intracellular or exocellular) of the enzyme. The half-lives of soluble and immobilized β-glucosidases at six different temperatures were also calculated. The properties assayed were compared critically with those reported by other authors.

Optimisation of β-glucosidase entrapment in alginate and polyacrylamide gels

Abstract Immobilisation of β-glucosidase, isolated from Aspergillus niger, by entrapment in both calcium alginate and polyacrylamide gels was studied. A retention of 66% of initial activity was observed in the alginate beads prepared with 3% (w/v) alginate, 0.2 m CaCl 2 and 1 h of treatment. The maximum β-glucosidase activity in polyacrylamide gels (∼55%) was achieved in gels prepared with 20% acrylamide and 1.2% of crosslinking agent (bisacrylamide). β-Glucosidase immobilised in alginate gel did not follow pure Michaelis kinetics, exhibiting substrate inhibition. The K m of this enzyme was larger than that of the free β-glucosidase, suggesting that the alginate network limited the permeation rate of substrate and product. However, β-glucosidase entrapped in polyacrylamide gel showed a similar K m value to that of native enzyme. The pH value for maximum activity of free and immobilised enzymes was 4.0. The pH-activity curves were coincident, except at very low pH values where the enzyme trapped in alginate was more stable.

Studies of microbial β-d-glucosidase immobilized in alginate gel beads

About 50–60% β-d-glucosidase (isolated from Pseudomonas pickettii and Aspergillus niger) was immobilized in calcium alginate gel beads by entrapment, retaining 15–26% of original activity. The pH values for maximum activitiy of the immobilized bacterial and fungal enzymes appeared to be 5.0 and 3.0 respectively, very similar to those of the free enzymes. The pH-activity curves were coincident, except at very low pH values where the fungal immobilized enzymes was more stable. Bacterial β-glucosidase did not follow pure Michaelis kinetics, exhibiting substrate inhibition and showing similar Km values for immobilized and soluble enzymes. The Km of the immobilized fungal enzyme was larger than that of the free enzyme, suggesting that the alginate network limited the permeation rate of substrate and product. The Vmax values of immobilized β-glucosidases from A. niger and P. pickettii were 14.7 and 29.5 smaller than those of the native enzymes, respectively.

Stabilisation of cellulases by cross-linking with glutaraldehyde and soil humates

Abstract β-Glucosidase and carboxymethyl cellulase were induced in and extracted from Aspergillus niger and Trichoderma reesei cultures and cross-linked with glutaraldehyde or complexed with a soil polyphenolic structure (humates). The concentration of the bifunctional reagent (2·5 or 5·0%) determined the activity of the immobilised β-glucosidase while the time and temperature of reaction had no significant influence. Although the catalytic activity of the cross-linked cellulases was 85% smaller than that of the free counterparts, they resisted thermal inactivation to a greater extent. When cellulases were bound to soil humates, their activity was much higher than that of the free enzymes. Furthermore, their thermal stability was enhanced by their complexation to the colloids. In contrast, when cellulases were bound to humates in the presence of glutaraldehyde, the cross-linked humate-enzyme complexes obtained showed a partial deactivation (β-glucosidase 10·4% and CMCase 37·1%). The thermostability of the immobilised CMCase was reduced (9·1%) but the thermostability of the β-glucosidase was increased (36·9%) with respect to its soluble counterpart at 60°C.

Induction of β-glucosidase in fungal and soil bacterial cultures

Abstract The production of β-glucosidase by soil enrichment cultures grown on cellobiose and carboxymethylcellulose and several experimental conditions were investigated in this work. The highest rates of induced enzyme activity were found in the soil cultures in which 0.4% cellobiose at pH 8.0 was used as the sole C and energy source. A bacterial strain, producing significant amounts of enzyme associated with the cell extracts and which was identified as Pseudomonas pickettii , was isolated from these soil cultures. In a comparative way, induction and location of β-glucosidase in Aspergillus niger cultures (using 0.4% cellobiose as C substrate) were also studied. In contrast with the results obtained with the bacterial cultures, only low levels of activity were detected when no N source was used. However, an appreciable amount of enzyme was assayed when 0.2% peptone was included in the culture medium with more than 70% of the total activity located in the extramycelial fraction.

Development of a method to recovery and amplification DNA by real-time PCR from commercial vegetable oils.

This study describes the design of a suitable DNA isolation method from commercial vegetable oils for the application of DNA markers for food safety and traceability. Firstly, a comparative study was made of eight methods for the recovery of high quality DNA from olive, sunflower and palm oils, and a CTAB-based method was selected. In order to optimize this method, the effect of the organic compounds and several components in the lysis buffer and the lysis and precipitation time were evaluated. For the purpose of overcoming the limitations detected in spectrophotometric and PCR DNA yield evaluations, the performance of the extraction protocols during the optimization processes was evaluated using qPCR. The suggested DNA extraction optimized is less time consuming than other conventional DNA extraction methods, uses a reduced oil volume and is cheaper than available commercial kits. Additionally, the applicability of this method has been successfully assayed in ten commercial vegetable oils and derivatives.