María Vergara-Barberán

Capillary electrophoresis of free fatty acids by indirect ultraviolet detection: application to the classification of vegetable oils according to their botanical origin.

A method for the determination of fatty acids in vegetable oils by capillary electrophoresis with indirect UV-vis detection has been developed. The separation of fatty acids was optimized in terms of Brij surfactant nature and concentration and organic modifier (2-propanol) percentage. The optimal background electrolyte consisted of 10 mM p-hydroxybenzoate, 5 mM Tris at pH 8.8, 80 mM Brij 98, 40% acetonitrile, and 10% 2-propanol. Under these conditions, vegetable oils from five botanical origins (avocado, corn, extra virgin olive, hazelnut, and soybean) were analyzed and the fatty acid contents established. Linear discriminant analysis (LDA) models were constructed using fatty acid peak areas as predictors. An excellent resolution among all category pairs was obtained, and all samples were correctly classified with assignment probabilities of >95%.

Solid-phase extraction based on ground methacrylate monolith modified with gold nanoparticles for isolation of proteins.

In this study, a novel polymeric material functionalized with gold nanoparticles (AuNPs) was prepared as solid-phase extraction (SPE) sorbent for isolation of proteins. The sorbent was synthesized from a powdered poly(glycidyl-co-ethylene dimethacrylate) monolith, and modified with ammonia, followed by immobilization of AuNPs on the pore surface of the material. To evaluate the performance of this SPE support, proteins were selected as test solutes, being the extraction conditions and other parameters (loading capacity and regenerative ability of sorbent) established. The results indicated that this sorbent could be employed to selectively capture proteins according to their pI, on the basis of the strong affinity of these biomacromolecules towards to AuNPs surface. The applicability of this sorbent was demonstrated by isolating protein species of interest (bovine serum albumin, cytochrome c and lectins in European mistletoe leaves), followed by SDS-PAGE analysis.

Use of an enzyme-assisted method to improve protein extraction from olive leaves

The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64mgg(-1)) were analysed and compared by SDS-PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature.

Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils.

A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV-Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v) acetonitrile/2-propanol mixture containing 5 mM ammonium acetate. The method was applied to the analysis of TAGs of vegetable oil samples. Using linear discriminant analysis of the CEC TAG profiles, the vegetable oils belonging to six different botanical origins (corn, extra virgin olive, hazelnut, peanut, soybean and sunflower) were correctly classified with an excellent resolution among all the categories.

Efficient Extraction of Olive Pulp and Stone Proteins by using an Enzyme-Assisted Method

An efficient protein extraction protocol for proteins from olive pulp and stone by using enzymes was developed. For this purpose, different parameters that affect the extraction process, such as enzyme type and content, pH, and extraction temperature and time, were tested. The influence of these factors on protein recovery was examined using the standard Bradford assay, while the extracted proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The best extraction conditions were achieved at pH 7.0 and 5% (v/v) Palatase® 20000 L (lipase) for pulp and Lecitase® Ultra (phospholipase) for stone proteins. The optimal extraction temperature and time were 30 and 40 °C for 15 min for pulp and stone tissues, respectively. Under these conditions, several protein extracts coming from olive fruits of different genetic variety were analyzed, their profiles being compared by SDS-PAGE. The developed enzyme-assisted extraction method showed faster extraction, higher recovery, and reduced solvent usage than the nonenzymatic methods previously described in the literature. In the case of stone proteins, different electrophoretic profiles and band intensities were obtained that could be helpful to distinguish samples according to their genetic variety.

Use of triacylglycerol profiles established by HPLC–UV and ELSD to predict cultivar and maturity of Tunisian olive oils

The usefulness of triacylglycerol (TAG) profiles established by high-performance liquid chromatography using both UV and evaporative light scattering (ELSD) detectors as a tool to discriminate between seven cultivars of Tunisian extra virgin olive oils (EVOOs) was evaluated in this work. Moreover, the discrimination of EVOOs from the cultivars Chemchali, Fouji and Zarrazi, characterized with different maturity indexes, was also studied. With both detectors, a total of 19 peaks, which were common to all the EVOOs studied, were observed. However, ELSD peaks, which provided a higher signal-to-noise ratio than the UV peaks, were selected to construct linear discriminant analysis models for cultivar and maturity index prediction. In all cases, an excellent resolution between all category pairs was achieved, which demonstrated that TAG profiles are a good marker of both cultivar and maturity index of EVOOs.

Use of protein profiles established by CZE to predict the cultivar of olive leaves and pulps

Intact protein profiles established by CZE have been used to predict the cultivar of olive leaves and pulps. For this purpose, proteins were extracted using a mild enzyme‐assisted extraction method, which provided higher protein recoveries and a lower environmental impact than other previously described methods. These extracts were subjected to CZE determination under basic conditions using a BGE composed of 50 mM phosphate, 50 mM tetraborate and 0.1% PVA at pH 9. Nine and 14 common peaks, for leaf and pulp samples, respectively, were identified in the nine cultivars studied in this work. In addition, and using linear discriminant analysis of the CZE data, olive leaf and pulp samples belonging to nine cultivars from different Spanish regions were correctly classified with an excellent resolution among all categories, which demonstrated that intact protein profiles are characteristic of each cultivar.

Classification of olive leaves and pulps according to their cultivar by using protein profiles established by capillary gel electrophoresis

AbstractA method to classify olive leaves and pulps according to their cultivar using protein profiles obtained by capillary gel electrophoresis (CGE) has been developed. For this purpose, proteins were extracted using an enzyme-assisted method, which provided higher protein recoveries than other previously described methods. Ten and nine common peaks, for leaf and pulp samples, respectively, were identified in the 12 cultivars studied in this work. In addition, and using linear discriminant analysis of the CGE data, olive leaf and pulp samples belonging to 12 cultivars from different Spanish regions were correctly classified with an excellent resolution among all the categories, which demonstrated that protein profiles were characteristic of each cultivar. FigureClassification of olive leaves and pulps according to their cultivar by using protein profiles established by CGE

Poly(ethylene glycol) diacrylate based monolithic capillary columns for the analysis of polar small solutes by capillary electrochromatography

Monolithic stationary phases based on poly(ethylene glycol) diacrylates for capillary electrochromatography were developed. Several poly(ethylene glycol) diacrylates (Mn 250, 575, and 700) were used as single monomers and the resulting columns were carefully compared. Methanol and ethyl ether were selected as porogenic solvents, and in all cases ultraviolet radiation was selected as initiation method to prepare polymeric monoliths. The influence of the monomer chain length and ratio monomer/porogen on the morphological and electrochromatographic properties of the resulting monoliths was investigated. Several families of compounds with different polarity (alkyl benzenes, organophosphorous pesticides, benzoic acid derivatives, and sulfonamides) were selected to evaluate the performance of the fabricated monolithic columns. The best results were obtained for poly(ethylene glycol) diacrylate 700 monoliths affording efficiencies of 144 000 plates/m for retained polar aromatic small molecules and excellent reproducibility in column preparation (RSD values below 2.5%).

New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers

A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significant differences in farrowing rate (n = 13) and litter size (n = 13) after the AI of 10 526 sows were further analyzed (Experiment 2). A total of 679 SP proteins were then quantified by the SWATH approach, where the penalized linear regression LASSO revealed differentially expressed SP proteins for farrowing rate (FURIN, AKR1B1, UBA1, PIN1, SPAM1, BLMH, SMPDL3A, KRT17, KRT10, TTC23, and AGT) and litter size (PN-1, THBS1, DSC1, and CAT). This study extended our knowledge of the SP proteome and revealed some SP proteins as potential biomarkers of fertility in AI boars.