Magnetic nanoparticles coated with carboxylate-terminated carbosilane dendrons as a reusable and green approach to extract/purify proteins.

Extraction/purification of proteins, at both analytical and industrial levels, is a limiting step that usually requires the use of organic solvents and involves tedious work and a high cost. This work proposes a more sustainable alternative based on the use of magnetic nanoparticles (MNPs) coated with carboxylate-terminated carbosilane dendrons. MNPs coated with first- and second-generation carbosilane dendrons and bare MNPs were employed for the extraction of proteins with different molecular weights and charges. Interaction of proteins with MNPs significantly varied with the pH, the protein, and the dendron generation (different sizes and number of charges in the periphery). Optimal dendron:protein molar ratios and suitable conditions for disrupting interactions after protein extraction were also researched. Second-generation dendron-coated MNPs showed 100% retention capability for all proteins when using acidic conditions. They were reused without losing magnetism or interaction capacity after a disruption of protein-dendron interactions with 0.2% SDS at 100 °C for 10 min. The capacity of dendron-coated MNPs was successfully applied to the recovery/purification of proteins from two food by-products, olive seeds and cheese whey.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

Evaluation of the relationship between the peptide profiles and the lipid-lowering properties of olive seed hydrolysates as a tool for tuning hypocholesterolemic functionality.

Olive processing generates large amounts of stones with high protein contents. Previous studies have demonstrated that Manzanilla variety olive seed proteins release peptides with lipid-lowering capacity. However, no work has demonstrated their roles in the overall hypolipidemic activity. Moreover, further studies using different olive varieties are required to propose a solid method for the exploitation of olive seeds. Twenty different olive varieties were employed in this work. Proteins were extracted using high-intensity focused ultrasound and digested with Alcalase. The released peptides were identified using proteomic techniques, and their capabilities to reduce the absorption of dietary cholesterol (by inhibiting cholesterol esterase enzyme, binding bile acids, and reducing micellar cholesterol solubility) or the biosynthesis of endogenous cholesterol were evaluated. Peptides with different lipid lowering capacities were obtained from all varieties although the genotype significantly affected the hypolipidemic characteristics. Univariate and multivariate statistical analyses showed strong correlations, positive and negative, between the presence of certain peptides in the hydrolysates and their capacity to reduce exogenous cholesterol absorption and endogenous cholesterol synthesis. Therefore, the selection of the olive seed genotype can direct its lipid-lowering properties, e.g., by promoting the reduction of dietary cholesterol absorption or the inhibition of cholesterol biosynthesis.

Identification of Peptides Potentially Responsible for In Vivo Hypolipidemic Activity of a Hydrolysate from Olive Seeds.

Previous studies demonstrated that peptides produced by the hydrolysis of olive seed proteins using Alcalase enzyme showed in vitro multifunctional lipid-lowering capability. This work presents a deeper insight into the hypolipidemic effect of olive seed peptides. The capability of olive seed peptides to inhibit endogenous cholesterol biosynthesis through the inhibition of HMG-CoA reductase enzyme was evaluated observing a 38 ± 7% of inhibition. Two in vivo assays using different peptides concentrations (200 and 400 mg/kg/day) were designed to evaluate the hypolipidemic effect of olive seed peptides in male and female mice. A low concentration of hydrolysate reduced total cholesterol in male mice in a 20% after 11 weeks compared to the mice feeding with hypercholesterolemic diet. A higher hydrolysate concentration showed a greater reduction in total cholesterol (25%). The analysis of the olive seed hydrolysate by reverse phase high-performance liquid chromatography mass spectrometry (RP-HPLC-MS) enabled the identification of peptides that could be responsible for this hypolipidemic effect.

Isolation and Characterization of Angiotensin Converting Enzyme Inhibitory Peptides from Peach Seed Hydrolysates: In Vivo Assessment of Antihypertensive Activity.

A peptide fraction with molecular masses below 3 kDa (PSH-3 kDa) from a peach seed hydrolysate demonstrated high angiotensin converting enzyme (ACE) inhibitory activity (concentration to inhibit 50% ACE (IC50) = 16.4 µg/mL) in our previous work. This work proposes a further study of this highly active fraction. RP-HPLC enabled two fractions (F3 and F4) with high inhibitory activity (IC50 = 2.0 ± 0.5 and 1.2 ± 0.2 µg/mL, respectively) to be isolated. Peptide analysis by LC-Q-TOF-MS/MS using reverse-phase and hydrophilic interaction chromatography enabled 33 peptides within both fractions to be identified. Among them, peptide isoleucine-tyrosine-serine-proline-histidine (IYSPH) showed the highest capacity. The lack of cytotoxicity of peptides was demonstrated in three different cell lines (HeLa, HT-29, and HK-2). Oral administration of PSH-3 kDa fraction or peptide IYSPH caused a significant systolic blood pressure reduction (-30 mmHg) on spontaneously hypertensive rats after 3-6 h treatment.

Relevance of Gender and Social Support in Self-Rated Health and Life Satisfaction in Elderly Spanish People.

Background: Gender and social support are important social determinants of health, but the relevance of such variables in older people's health has raised less scholarly attention than in younger age groups. This study examines the relevance of gender and social support in the self-rated health and life satisfaction of elderly Spanish people. A cross-sectional study with a sample of 702 men and 754 women aged between 60 and 94 years was conducted. All participants were evaluated through questionnaires that assess gender role traits, social support, and life satisfaction. Results: Men scored higher than women in masculine/instrumental trait and in life satisfaction whereas women scored higher than men in feminine/expressive trait. Results from multiple regression analyses indicated that women and men presenting higher social support had better self-rated health and higher life satisfaction. High scores in masculine/instrumental trait also proved to be an important predictor of men's and women's high life satisfaction and of women's better self-rated health, whereas the high feminine/expressive trait predicted better self-rated health in the men group. A high educational level was associated in the women's group with better self-rated health and higher life satisfaction. Conclusions: We conclude that gender and social support are important social determinants of health among older people.

Gold nanoparticles coated with carbosilane dendrons in protein sample preparation.

The feasibility of using carbosilane dendronized gold nanoparticles (GNPs) for protein sample preparation was evaluated. Three different dendrons with three different generations (1G, 2G, and 3G) were employed to modify the GNPs, viz. sulfonate terminated (STC-GNPs), carboxylate terminated (CTC-GNPs), and trimethylammonium terminated (ATC-GNPs) dendrons. The synthesis of the CTC-GNP is described. The other dendronized GNPs were synthesized using previously described routes. Bovine serum albumin, lysozyme, and myoglobin were employed to study the potential of GNPs to interact with proteins. The interaction between the GNPs and the proteins was evaluated using fluorescence spectroscopy and polyacrylamide gel electrophoresis. The CTC-GNPs and STC-GNPs under acidic and neutral conditions, respectively, promoted the establishment of electrostatic interactions with positively charged proteins. Proteins from 10 to 75 kDa molecular weights interacted with GNPs at protein: nanoparticle ratios of 1:0.25. The GNPs were applied to the extraction of proteins from a peach seed. In the authors' perception, the method is a clean alternative to established extraction methods based on the use of organic or polluting chemicals. Graphical abstract Schematic representation of the interaction of peach seeds proteins and carbosilane dendron coated gold nanoparticles, and the electrophoretic profiles of extracted proteins.

Psychological Distress and Social Functioning in Elderly Spanish People: A Gender Analysis.

Psychological distress has been considered a key component in the psychosocial functioning and functional disability of the elderly, but the determining factors of social functioning and psychological distress in the elderly people are not yet fully known. The aim of this study is to perform a gender analysis of the relevance of psychological distress and psychosocial factors in the social functioning of the elderly. A cross-sectional study with a sample of 589 men and 684 women from the general Spanish population aged between 65 and 94 years was conducted. All participants were evaluated through questionnaires and scales that assess psychological distress, social functioning, stress, coping styles, self-esteem and social support. Results: Women scored higher than men in psychological distress, chronic stress, emotional coping and instrumental social support, whereas men scored higher than women in self-esteem and rational coping. Psychological distress was significantly associated in women and men with worse social functioning, which was also lower in older people and in women with lower self-esteem. Psychological distress has a considerable impact on the social functioning of the elderly, and gender is a relevant factor in the psychological distress experienced and its predictors.

Isolation and identification by high resolution liquid chromatography tandem mass spectrometry of novel peptides with multifunctional lipid-lowering capacity.

This work describes the isolation, characterization, and identification by RP-HPLC-ESI-Q-TOF of novel peptides that interfere in the fat digestion and absorption mechanisms by multiple pathways. Peptides were ultrafiltrated and peptides in the most active fraction were further separated by semipreparative RP-HPLC. Nine different subfractions were obtained observing a high amount of peptides in subfraction F3. Peptides in subfraction F3 could simultaneously reduce the solubility of cholesterol in micelles and inhibit pancreatic cholesterol esterase and pancreatic lipase, even after a simulated gastrointestinal digestion. The identification of lipid-lowering peptides has been scarcely performed and when done, low selectivity or sensitivity of employed identification techniques or conditions did not yield reliable results. Separation and detection of peptides by RP-HPLC-ESI-Q-TOF-MS was optimized and most favorable conditions were employed for the identification of peptides using de novo sequencing. Ten different peptides with 4-9 amino acids were identified. Main feature of identified peptides was the high acidity derived from a high presence of amino acids glutamic acid and aspartic acid in their sequences.

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.

Development of a capillary high performance liquid chromatography-ion trap-mass spectrometry method for the determination of VLIVP antihypertensive peptide in soybean crops.

Soybean peptide VLIVP presents a very high antihypertensive activity (IC50 value 1.69µM), even higher than extensively studied IPP and VPP peptides from milk. Nevertheless, no much attention has been paid to this peptide and there is no method enabling its determination in soybeans. The aim of this work was the development of an analytical methodology for this purpose. A methodology consisting of the extraction of soybean proteins, their digestion with Protease P enzyme, their chromatographic separation using capillary-HPLC, and IT-MS detection was optimized. Protein extraction was performed by the use of high intensity focused ultrasounds to obtain a reduced extraction time. Optimization of chromatographic and mass spectrometry parameters enabled the separation of VLIVP peptide within just 7min and its sensitive detection. The analytical characteristics of the capillary-HPLC-IT-MS method were evaluated through the study of linearity, LOD, LOQ, study of the presence of matrix interferences, precision, and recovery. The method enabled to detect as low as 3.6ng of peptide and to determine as low as 12ng of peptide in 1g of soybean (as dry basis). Finally, the developed method was applied to the determination of the antihypertensive peptide VLIVP in different soybean varieties. The results showed the highest yield of VLIVP peptide in variety Mazowiecka II from Poland.

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 cow's 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.

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.

Fast determination of the functional peptide soymetide in different soybean derived foods by capillary-high performance liquid chromatography.

The determination of bioactive peptides derived from food sources is gaining special attention in last years, due to their ability to promote health and their potential to reduce the risk of chronic diseases. In this work, a new analytical methodology has been developed enabling for the first time the determination of soymetide, a new immunostimulating peptide derived from soybean, in different soybean derived foodstuffs. Capillary-HPLC was employed for peptide separation after accelerated tryptic digestion of soybean proteins. Two different capillary-HPLC columns were tested and chromatographic separation was optimized in each case. The use of a 300 µm fused-core technology C18 column enabled a suitable separation of soymetide from the other peptides in less than 18 min. Different analytical characteristics of the method were evaluated: selectivity, linearity, accuracy, precision, limit of detection and quantitation, and stability. The developed method was applied to the determination of soymetide content in different soybean dairy-like products for human consumption (powdered milks and infant formulas).

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.

Simultaneous and rapid determination of the anticarcinogenic proteins Bowman-Birk inhibitor and lectin in soybean crops by perfusion RP-HPLC.

There are numerous studies demonstrating a direct association between the ingestion of soybean and low cancer incidence. This fact has been related to the presence of Bowman-Birk inhibitor (BBI) and lectin in soybean. The simultaneous and fast determination of BBI and lectin in soybean is proposed, for the first time, in this work. Two different strategies were designed for the extraction of BBI and lectin: extraction of soybean proteins using a Tris-HCl buffer followed by isolation of BBI and lectin by the isoelectric precipitation of other soybean proteins (method I) or by the direct extraction of BBI and lectin using an acetate buffer (method II). The effect of the previous soybean defating on the extraction of BBI and lectin was also studied. Moreover, the possibility of using a high-intensity focalized ultrasonic probe for accelerating the extraction was explored and an optimization of the extraction time and ultrasound amplitude was performed. The extracts obtained were analysed by RP-HPLC-ESI-MS for the correct identification of BBI and lectin in soybean. Moreover, a fast chromatographic methodology using a perfusion column and UV detection was optimized for the rapid determination of BBI and lectin in soybean. After evaluating its analytical characteristics (linearity, precision, and recovery), the method was applied to the quantitation of BBI and lectin in different soybean varieties.

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins.

Methods for the analysis of maize proteins using HPLC and CE are reviewed. Most of the references cited in this review concern HPLC methods. Size-exclusion HPLC and especially RP-HPLC methods have been developed for characterization of normal and genetically modified maize, cultivar differentiation, and prediction of quality. Few CE methods for the analysis of maize proteins were found in the existing literature. Most of these methods focus on optimization of the separation of maize proteins using CZE and SDS-capillary gel electrophoresis.