Clara Esteve

Vegetable foods: A cheap source of proteins and peptides with antihypertensive, antioxidant, and other less occurrence bioactivities

Despite less explored than foods from animal origin, plant derived foods also contain biologically active proteins and peptides. Bioactive peptides can be present as an independent entity in the food or, more frequently, can be in a latent state as part of the sequence of a protein. Release from that protein requires protein hydrolysis by enzymatic digestion, fermentation or autolysis. Different methodologies have been used to test proteins and peptides bioactivities. Fractionation, separation, and identification techniques have also been employed for the isolation and identification of bioactive proteins or peptides. In this work, proteins and peptides from plant derived foods exerting antihypertensive, antioxidant, hypocholesterolemic, antithrombotic, and immunostimulating capacities or ability to reduce food intake have been reviewed.

Novel strategy for the revalorization of olive (Olea europaea) residues based on the extraction of bioactive peptides

This work proposes a new strategy for the revalorization of residual materials from table-olive and olive oil production based on the extraction of bioactive peptides. Enzymatic hydrolysates of olive seed protein isolate were prepared by treatment with five different proteases: Alcalase, Thermolysin, Neutrase, Flavourzyme and PTN. Although all hydrolysates presented antioxidant properties, Alcalase was the enzyme that yielded the hydrolysate with the highest antioxidant capacity. All hydrolysates showed antihypertensive capacity, obtaining IC50 values from 29 to 350 μg/ml. Thermolysin was the enzyme which yielded the hydrolysate with the highest ACE-inhibitory capacity. Hydrolysates were fractionated by ultrafiltration showing a high concentration of short chain peptides, which exhibited significantly higher antioxidant and antihypertensive capacities than fractions with higher molecular weights. Peptides in most active fractions were identified by LC-MS/MS, observing homologies with other recognized antioxidant and antihypertensive peptides. Finally, their antioxidant and antihypertensive capacities were evaluated after in vitro gastrointestinal digestion.

In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries

Musa ssp. is among the worlds leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low‐abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in‐depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system.

Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries.

Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.

Identification of avocado (Persea americana) pulp proteins by nano‐LC‐MS/MS via combinatorial peptide ligand libraries

Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse‐phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low‐abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin‐like protein, a glucanase, and an isoflavone reductase like protein.

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.

Analysis of olive allergens

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Besides this, some cases of contact dermatitis and food allergy to the olive fruit and olive oil have been also described. Several scientific studies dealing with olive allergens has been reported, being the information available about them constantly increasing. Up to date, twelve allergens have been identified in olive pollen while just one allergen has been identified in olive fruit. This review article describes considerations about allergen extraction and production, also describing the different methodologies employed in the physicochemical and immunological characterization of olive allergens. Finally, a revision of the most relevant studies in the analysis of both olive pollen and olive fruit allergens is carried out.

Identification of Olive (Olea europaea) Pulp Proteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Nano-Liquid Chromatography Tandem Mass Spectrometry

Proteins in the pulp of olive ( Olea europaea ) constitute a minor fraction. They have been sparsely studied despite their suggested role in oil stability and olive allergenicity. The analysis of a pulp protein extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a major band at 24 kDa that was subjected to tryptic in-gel digestion. Peptide extracts were analyzed by MALDI-TOF MS and nanoLC-MS/MS. The use of different search engines enabled the assignment of a number of fragmentation spectra to peptide sequences, identifying a major band as a thaumatin-like protein and other low-abundant proteins such a drought-induced protein SDi-6-like, an acyl carrier protein, Cu/Zn and Mn superoxide dismutases, a small heat shock protein, and an ATP-dependent protease subunit. Many of the produced spectra did not give good matches in the database searches, due to the scarce presence of O. europaea entries in protein databases. Nevertheless, a huge number of spectra corresponded to peptides, which showed a high degree of homology with others from sequenced organisms. These results proved that database searching with MS/MS spectra constitutes a promising approach for the characterization of olive pulp proteins.

A sarabande of tropical fruit proteomics: Avocado, banana, and mango

The present review highlights the progress made in plant proteomics via the introduction of combinatorial peptide ligand libraries (CPLL) for detecting low‐abundance species. Thanks to a novel approach to the CPLL methodology, namely, that of performing the capture both under native and denaturing conditions, identifying plant species in the order of thousands, rather than hundreds, is now possible. We report here data on a trio of tropical fruits, namely, banana, avocado, and mango. The first two are classified as “recalcitrant” tissues since minute amounts of proteins (in the order of 1%) are embedded on a very large matrix of plant‐specific material (e.g., polysaccharides and other plant polymers). Yet, even under these adverse conditions we could report, in a single sweep, from 1000 to 3000 unique gene products. In the case of mango the investigation has been extended to the peel too, since this skin is popularly used to flavor dishes in Far East cuisine. Even in this tough peel 330 proteins could be identified, whereas in soft peels, such as in lemons, one thousand unique species could be detected.

Proteins in Olive Fruit and Oil

This paper is a comprehensive review grouping the information on the extraction, characterization, and quantitation of olive and olive oil proteins and providing a practical guide about these proteins. Most characterized olive proteins are located in the fruit, mainly in the seed, where different oleosins and storage proteins have been found. Unlike the seed, the olive pulp contains a lower protein content having been described a polypeptide of 4.6 kDa and a thaumain-like protein. Other important proteins studied in olive fruits have been enzymes which could play important roles in olives characteristics. Part of these proteins is transferred from the fruit to the oil during the manufacturing process of olive oil. In fact, the same polypeptide of 4.6 kDa found in the pulp has been described in the olive oil and, additionally, the presence of other proteins and enzymes have also been described. Protein profiles have recently been proposed as an interesting strategy for the varietal classification of olive fruits and oils. Nevertheless, there is still a lot of knowledge without being explored requiring new studies focused on the determination and characterization of these proteins.