María D. Busto

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.

Kinetics of thermal inactivation of pea seed lipoxygenases and the effect of additives on their thermostability

Abstract Mature pea seeds contain two major lipoxygenases (LOX) isoenzymes designated LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recombinant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts of pea LOX were linear from which thermodynamic activation parameters, ΔH # , ΔS # sand ΔG # have been estimated. The enzymatic activity was relatively stable with a respective half-life ( t 1/2 ) at 60 °C of 54.2 min for LOX from pea ( Pisum sativum L. cv. Birte) or 18.4 min for a mutant line lacking LOX-2. At 50°C the thermostability of LOX-3 present in crude extracts of the mutant strain ( t 1/2 =66.8 min) was 90% greater than purified recombinant LOX-3 (rLOX-3; t 1/2 =34.6 min). However, rLOX-3 was more heat-stable than rLOX-2. Both rLOX-3 and pea mutant line lacking LOX-2 possessed considerable thermostability at 60°C ( t 1/2 =16.5 min and 18.4 min, respectively). Even at the higher temperatures of 70°C the t 1/2 values were 84 and 51, respectively. It is suggested that LOX in crude enzyme extracts was stabilised at 50°C due to protection by other constituents, possibly including starch and proteins. Separate tests at 70°C in the presence of additives (polyols, detergents and small ions) showed that sucrose was the most effective stabiliser and increased the stability of pea LOX by 400–600%.

Application of factorial design and response surface methodology to the analysis of bovine caseins by capillary zone electrophoresis

Central composite design and response surface methods were applied to optimise and assess the robustness of a capillary zone electrophoresis method for the separation of bovine caseins. Buffer pH, running voltage and polymeric additive buffer concentration were the parameters selected for this study. Successful results were obtained at 18.5 kV, with 10 mM phosphate buffer at pH 3.0 with 0.05% (w/v) hydroxypropyl methyl cellulose. Under these optimised conditions αs-casein (CN), β-CN and κ-CN and the various phosphorylation states of the αs1-CN and αs2-CN, as well as, some genetic variants of β-CN (A1, A2 and B) were well separated.

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.

Barley seed coating with free and immobilized alkaline phosphatase to improve P uptake and plant growth

SUMMARY Coating barley seeds with free and immobilized alkaline phosphatase was investigated as a potential means to enhance plant utilization of accumulated soil phosphorus (P). Two coating techniques were studied: film-coating and pelleting. The highest phosphatase activity retention in the coating layer, ranging from 0·48 to 0·67, was observed when seeds were film-coated with phosphatase–polyresorcinol complex (PPC). The germination of seeds film-coated or pelleted with alkaline phosphatase ranged from 0·84 to 0·97 or 0·14 to 0·25, respectively. Low germination of the pelleted seeds was attributed to freezing the seeds in liquid nitrogen (N) for the layer coating formation. Pelleted seeds were not used in the remainder of the studies. Under pot culture conditions, an increase in the soil inorganic P was detected when the seeds were film-coated with phosphatase. Moreover, the film-coating significantly increased the P uptake by plants (between 25 and 31% after 35 days after planting (DAP)). The present study showed that the seed film-coating with free and immobilized phosphatase increased the phosphatase activity in the rhizosphere and the P uptake by plants.

Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

Enzymatic Saccharification of Pretreated Wheat Straw by T. Reesei Cellulases and A. Niger β-Glucosidase

Three methods of wheat straw treatment (with NaOH, H2O2 and butylamine) and its subsequent saccharification by Trichoderma reesei cellulases and Aspergillus niger β-glucosidase are reported. The treatment of straw with NaOH for 1 h in the autoclave (120°C) caused a great loss of the hemicellulose content and a partial removal of lignin, provoking an increase of the cellulose content (from 24% to 69%) in the residue. When the straw was pre-treated with H2O2 at 25°C for 20 h, the relative content of cellulose in the straw increased (from 24% to 52%) due to the solubilisation of hemicellulose. The effect of varying the hydrolysis parameters (enzyme and substrate concentration, temperature and pH) was studied in order to maximise the yield of sugars. Under the best conditions and after 48 h with a mixture of 2:1 (w/w) cellulase/β-glucosidase (with a concentration of 7 and 0.1 U ml-1, respectively) the native, NaOH-treated and H2O2-treated straw material were degraded to reducing sugars for 28%, 89% and 97% respectively.

Novel qPCR systems for olive (Olea europaea L.) authentication in oils and food.

The traceability of olive oil is an unresolved issue that remains a challenge. In this field, DNA-based techniques are very powerful tools for discrimination that are less negatively influenced by environmental conditions than other techniques. More specifically, quantitative real time PCR (qPCR) achieves a high degree of sensitivity, although the DNA that it can directly isolate from these oils presents drawbacks. Our study reports the analysis of eight systems, in order to determine their suitability for olive detection in oil and oil-derived foodstuffs. The eight systems were analyzed on the basis of their sensitivity and specificity in the qPCR assay, their relative sensitivity to olive DNA detection and DNA mixtures, their sensitivity and specificity to olive in vegetable oils and the detection of olive in commercial products. The results show that the PetN-PsbM system, designed in this study, is a suitable and reliable technique in relation to olive oil and olive ingredients in both food authentication and food safety processes.