M. Luisa Marina

Study of the biodegradation process of polychlorinated biphenyls in liquid medium and soil by a new isolated aerobic bacterium (Janibacter sp.)

We have isolated and characterised a novel aerobic bacterial strain, designated MS3-02, belonging to the genus Janibacter sp. The capability of this new strain to degrade polychlorinated biphenyls (PCBs) in a commercial mixture (Aroclor 1242) in liquid medium and in soil (sterile and non sterile soil), under laboratory scale, has been evaluated. MS3-02 was isolated from the soil around of an incinerator, located in the east of Madrid (Spain). Gas-chromatographic analysis showed that MS3-02 was able to reduce most peaks observed in the chromatogram between 70% and 100% after seven days of incubation in a culture mineral medium containing yeast extract, but without the addition of biphenyl. The presence of biphenyl in the culture medium decreased the rate of PCB degradation by this bacterium. Comparing the performance of the MS3-02 in liquid culture medium and in soil, degradation was less efficient in sterile soil and still less efficient in non sterile soil. Under the best conditions (sterile soil and 20 weeks of incubation) MS3-02 was able to reduce, between 50% and 100%, nine of the main gas-chromatographic peaks in Aroclor 1242.

Development of an ultra-high performance liquid chromatography analytical methodology for the profiling of olive (Olea europaea L.) pulp proteins.

Ultra-high performance liquid chromatography (UHPLC) constitutes an interesting proposal to speed protein separations but it is almost not explored. In this work UHPLC is proposed, for the first time, to separate olive pulp proteins. An important difficulty in the analysis of proteins is related to their extraction. The difficulty in the extraction of proteins from the olive pulp is derived from its high content in lipids and phenolic compounds. Eight different methods for the extraction of pulp proteins were designed and evaluated. The optimized extraction procedure consisted of a cleaning step to remove interfering compounds, followed by the extraction of proteins with a Tris-HCl buffer containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT), precipitation of proteins with acetone, and solubilization in the Tris-HCl buffer. This methodology yielded the most successful isolation of pulp proteins and enabled the optimization of a UHPLC methodology for their separation. The method was applied to the profiling of olive pulp proteins from different olive cultivars observing in all cases a protein that had never been described before.

Isolation and identification of antioxidant peptides from commercial soybean-based infant formulas.

Soybean-based infant formulas (SBIFs) based on soybean protein isolate (90% of proteins) are an interesting alternative to cows milk infant formulas. Different works have demonstrated the presence of bioactive peptides in different soybean-based foodstuffs. The aim of this work was the evaluation, for the first time, of antioxidant peptides in five different commercially available SBIFs. Ultrafiltration through 10 kDa molecular weight cut-off filters was the most suitable extraction method. Despite peptide concentrations ranging between 1.19 and 2.27 mg/mL, similar antioxidant capacities were detected in all SBIF extracts. Extracts were further fractionated according to their molecular weight by ultrafiltration, and fractions from 5 to 10 kDa, 3 to 5 kDa, and below 3 kDa were obtained. The most active fraction was further fractionated by off-gel isoelectrofocusing and reversed-phase chromatography. Antioxidant fractions were also submitted to simulated gastrointestinal digestion (GI) with pepsin and pancreatin to evaluate their antioxidant capacity after digestion. Peptides were identified by HPLC-ESI-Q-ToF-MS/MS. At least 120 peptides were identified in every antioxidant fraction, with 42 peptides common to all SBIFs.

Fast detection of added soybean proteins in cow's, goat's, and ewe's milk by perfusion reversed-phase high-performance liquid chromatography

In order to detect possible adulterations in milk due to addition of soybean proteins a a perfusion reversed-phase chromatographic column has been used with a linear acetonitrile-water-0.1% trifluoroacetic acid binary gradient for the determination of soybean proteins in cows, goats, and ewes milk spiked with such proteins. The method permitted the simultaneous separation of soybean and milk proteins in less than 2 minutes at a flow-rate of 5 mL/min and a temperature of 60°C. The method also enabled the quantitative analysis of soybean proteins in spiked milk, detecting up to 30 μg of soybean proteins/mL of milk. The method was validated by evaluation of its linearity, precision, and robustness and the possible existence of matrix interferences was also investigated for some of the milks. Although the external standard calibration method enabled the quantitation of soybean proteins in most of the milks examined, there were some cases in which the standard addition method was required.

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals.

A simple and robust solid‐phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed‐phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis‐HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis‐HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused‐silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.

Influence of alcohol organic modifiers upon the association constants and retention mechanism for aromatic compounds in micellar liquid chromatography

Abstract Solute-micelle association constants and partition coefficients between stationary, aqueous, and micellar phases have been determined for a group of benzene derivatives and polycyclic aromatic hydrocarbons with sodium dodecyl sulphate and hexadecyltrimethylammcnium bromide by Micellar Liquid Chromatography (MLC) with an octylsilica column using micellar mobile phases in absence and in the presence of organic modifiers (methanol, n-propanol, and n-butanol). The retention mechanism of these compounds in the chromatographic system has been studied by comparing experimental capacity factors and selectivity coefficients with those theoretically calculated assuming a direct transfer mechanism. When the surfactant concentration in the mobile phase is increased, a tendency to a change from a three-partition equilibrium mechanism to a direct transfer of solutes from micellar to stationary phase is observed for both surfactants. This change was favored when: (1) the hydrophobic character of the solute incr...

Identification of native angiotensin-I converting enzyme inhibitory peptides in commercial soybean based infant formulas using HPLC-Q-ToF-MS

This work evaluates, the presence of native antihypertensive peptides in five soybean-based infant formulas (SBIFs). SBIFs peptide extracts (<10 kDa) and their sub-fractions (5-10 kDa, 3-5 kDa, and <3 kDa) from a variety of samples were obtained by ultrafiltration and ACE inhibitory activity was determined. The highest activities were observed in the smaller (<5 kDa) peptide fractions. A set of peptides present in various SBIFs were studied, and identified using HPLC-Q-ToF-MS. Despite ACE inhibitory activity decreasing after in vitro gastrointestinal digestion, it still remained at a high value (IC50 values of 18.2±0.1 and 4.9±0.1 μg/mL). Peptides resisting the action of gastrointestinal enzymes were identified and compared to previously identified peptides, highlighting the presence of peptide RPSYT. This peptide was synthesised, its antihypertensive and antioxidant activity were evaluated, and its resistance to in vitro gastrointestinal digestion and to high processing temperatures were studied.

Identification by hydrophilic interaction and reversed-phase liquid chromatography-tandem mass spectrometry of peptides with antioxidant capacity in food residues

HILIC- and RP-HPLC-ESI-Q-TOF identification of bioactive peptides with antioxidant capacity in peach by-products was carried out. Peach seeds contain more than 40% of proteins (as dried and defatted basis) and could constitute a cheap source of bioactive peptides. Extracted proteins were digested using four different commercial enzymes. Five assays based on different antioxidant mechanisms were employed for a reliable evaluation of the antioxidant capacity of the extracts. Thermolysin enzyme originated the extract with the most favorable antioxidant capacity. Probably due to a synergic effect among antioxidant peptides, it was not possible to find a peptide fraction with a higher antioxidant capacity than the whole extract. Eighteen peptides were identified in the whole hydrolysate when combining HILIC- and RP-HPLC-ESI-Q-TOF. A high percentage of hydrophobic amino acids were observed within their sequences which is a characteristic feature of the antioxidant nature of peptides.

Retention of organic ligands on anionic and nonionic resins: Application to the separation and preconcentration of metal ions

Abstract Complexing resins obtained by retention of an organic compound on anionic and nonionic conventional exchangers have been reviewed. The influence of different parameters—nature of fixed ligand, kind and chemical form of the conventional resin, and pH of the aqueous solution—on characteristics and applications of this type of resin has been discussed. Results from the literature have been tabulated to simplify their presentation.

Development of a reversed-phase high-performance liquid chromatography analytical methodology for the determination of antihypertensive peptides in maize crops

The aim of this work was to estimate the content of three highly antihypertensive peptides (LQP, LSP, and LRP) in different maize crops. For that purpose, a method consisting of the extraction of the protein containing these peptides (α-zeins), releasing of peptides by thermolysin digestion, and separation and detection of peptides was designed. The rapid and efficient ultrasound assisted extraction of α-zeins proteins from whole maize kernels was achieved using 70% of ethanol followed by precipitation with acetone. A 10 mM Tris-HCl (pH 8.0) buffer containing 8M urea enabled to dissolve the precipitated α-zeins. This buffer was diluted to reach a 6 M urea concentration before digestion to keep active the enzyme. Other digestion parameters that were optimized were: enzyme to substrate ratio (5:100 was selected), digestion temperature (50°C) and digestion time (6 h). The RP-HPLC separation in a fused-core column was also optimized allowing the separation of the three peptides extracted from maize kernels in 6 min. The presence of the three antihypertensive peptides in the digested extract was confirmed using HPLC-Q-TOF-MS analysis and by comparison with peptide standards. Clear differences were observed in the content of the three antihypertensive peptides and, thus, in the antihypertensive activity of the analyzed crops. The content of LRP peptide was very low regardless of the maize variety while the content of LQP and LSP significantly varied among studied maize lines.