RESUMEN
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.
Asunto(s)
Dendrímeros/química , Nanopartículas de Magnetita/química , Proteínas/aislamiento & purificación , Silanos/química , Animales , Humanos , Magnetismo/métodos , Nanopartículas de Magnetita/ultraestructura , Extracción en Fase Sólida/métodosRESUMEN
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.
Asunto(s)
Hipercolesterolemia/prevención & control , Hipolipemiantes/química , Olea , Péptidos/química , Hidrolisados de Proteína/química , Humanos , Hipolipemiantes/farmacología , Hipolipemiantes/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Hidrolisados de Proteína/farmacología , Hidrolisados de Proteína/uso terapéutico , SemillasRESUMEN
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.
Asunto(s)
Hipolipemiantes/química , Olea/química , Péptidos/química , Extractos Vegetales/química , Semillas/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colesterol/biosíntesis , Colesterol/metabolismo , Cromatografía Líquida de Alta Presión , Dieta , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Hidrólisis , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hipolipemiantes/farmacología , Masculino , Ratones , Péptidos/farmacología , Extractos Vegetales/farmacología , Hidrolisados de Proteína/química , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Cromatografía Liquida/métodos , Hipolipemiantes/análisis , Olea/química , Péptidos/análisis , Semillas/química , Espectrometría de Masas en Tándem/métodos , Hipolipemiantes/química , Péptidos/químicaRESUMEN
The concentration of polyunsaturated fatty acids by formation of urea adducts from three different sources was studied to elucidate the formation of ethyl carbamates in the course of these procedures. Two different methodologies were performed: with ethanol at high temperature and with hexane/ethanol mixtures at room temperature. It was proved that the amount of urethanes generated at high temperature was higher than at room temperature. Besides, subsequent washing steps of the PUFA fraction with water were efficient to remove the urethanes from the final products. The methodology at room temperature with 0.4mL ethanol and 3g urea provided good relationship between concentration and yield of the main bioactive PUFA, with the lowest formation of ethyl carbamates in the process.