Desarrollo de nuevos sistemas separativos para la determinación de biomoléculas en matrices vegetales

La presente memoria se enmarca dentro de las líneas de investigación del grupo de investigación, entre las que se encuentran: i) el desarrollo de materiales poliméricos con fines separativos, y ii) la puesta a punto de metodologías analíticas rápidas y fiables para el control de calidad en la industria de aceites vegetales y otros productos de interés agroalimentario. En consecuencia, los objetivos principales de esta Tesis Doctoral son: i) el diseño y optimización de soportes poliméricos modificados con nanopartículas metálicas (tales como el oro y la plata) para la extracción de proteínas en matrices vegetales, ii) la preparación y evaluación de sistemas de extracción de proteínas en dichas muestras asistida por enzimas y iii) el desarrollo de metodologías analíticas de separación rápidas y fiables con el fin de poder establecer el origen genético y botánico de diversos productos alimentarios. La presente memoria de Tesis Doctoral se divide en cuatro grandes bloques. El primer bloque consta de una introducción, donde se describen brevemente las muestras vegetales empleadas en esta Tesis Doctoral (fruto, hoja y aceite de olivo, frutos de la variedad Citrus (en concreto naranjas y mandarinas) y muérdago, correspondientes a los Capítulos 1-3, respectivamente). Además, se incluye una revisión actualizada de los métodos de extracción empleados durante la tesis: extracción en fase sólida (SPE), extracción asistida por enzimas y por último, el empleo de las bibliotecas combinatorias de ligandos peptídicos (CPLLs). El bloque de introducción concluye con una breve descripción de las técnicas analíticas empleadas en el desarrollo de la Tesis Doctoral (Capítulo 5). El segundo bloque (que engloba los capítulos 6-11) está dedicado al desarrollo de nuevas técnicas de extracción de proteínas en matrices de origen vegetal. Para ello, se han puesto a punto nuevos sorbentes, basados en materiales poliméricos sintetizados a partir del metacrilato de glicidilo (GMA), que son posteriormente modificados con nanopartículas metálicas (de oro y plata) para su uso como sorbentes en SPE. Por otro lado, la presencia de sistemas reticulares u organizados, presentes en los vegetales, requiere habitualmente de tratamientos relativamente “agresivos” con el fin de poder extraer los analitos diana. En este sentido, se han llevado a cabo estudios de extracción de proteínas asistidos por enzimas, empleándose tanto enzimas individuales como mezclas de las mismas. Gracias a la acción de estas biomacromoléculas, se facilita la ruptura de las membranas celulares, y por ende la extracción de los analitos para su posterior análisis y cuantificación. Estos pretratamientos de la muestra, sin duda, permitirán obtener resultados de mayor calidad. En este caso, se ha empleado dicha tecnología para la extracción de proteínas de diversos productos del olivo (hojas, huesos y pulpa), así como para muestras de naranja y mandarina, incluyendo tanto la piel como la pulpa de las mismas. Por último, se ha llevado a cabo un estudio del proteoma del muérdago mediante la extracción del mismo empleando las CPLLs, que son capaces de extraer y preconcentrar las proteínas de baja abundancia. La identificación del mapa proteico de esta planta ha permitido profundizar en un mayor conocimiento de sus propiedades medicinales. El tercer bloque (que engloba los capítulos 12-20) muestra el desarrollo de una amplia variedad de métodos de análisis para la determinación de diferentes compuestos en matrices de origen vegetal, tales como aceite de oliva, frutos y hojas de olivo, frutos de la variedad Citrus (naranjas y mandarinas), y muérdago). Dichas metodologías abarcan tanto técnicas de carácter cromatográfico (HPLC) y afines (CZE, CGE y CEC) así como de carácter espectrométrico (ATR-FTIR o DIMS). Se han desarrollado metodologías analíticas para la determinación de proteínas, TAGs, esteroles, ácidos grasos y péptidos. Además, en muchos de los capítulos, se han aplicado herramientas quimiométricas de análisis, en concreto, se han construido modelos de análisis discriminante lineal (LDA), para establecer la clasificación de muestras de aceites vegetales en función de su origen botánica, para la discriminación de productos del olivo, incluyendo aceites de oliva, en función de su variedad genética y en caso de los aceites de oliva, también para clasificarlos en función de su índice de madurez. En algunos de ellos, se ha aplicado la regresión lineal múltiple (MLR) para cuantificar mezclas binarias de diferentes variedades genéticasThis PhD thesis reports the development of novel separation systems to determine biomolecules from complex matrices, especially in vegetal samples. In the first part of this thesis, extraction methodologies were designed to isolate proteins, constituting a significant contribution in the sample preparation field. Thus, the development of novel in-house sorbents based on organic polymers modified with Au and AgNPs for SPE with improved properties, the proper selection of enzyme preparations (either alone or in mixtures) for efficient release of these biomacromolecules from plant cells, and the use of CPLLs to preconcentrate LAPs in complex matrices have been accomplished. Significant enhancements such as large protein extraction yield, tunable selectivity, cost-effective, environmentally friendly and other advantages should place the developed methodologies in a competitive position compared to commercial SPE materials or other traditional extraction methodologies (that imply the use of organic solvents). Another part covered by this thesis was the development of methods for the characterization and determination of several compounds in olive products using different chromatographic and spectrometric techniques. For this purpose, different profiles belong to components present in these matrices, such as proteins, TAGs, sterols and fatty acids were considered. These profiles allowed a discrimination of vegetable oils according to their botanical origin, and also the characterization and authentication of Spanish and Tunisian olive oils in relation to their genetic variety. The proposed methodologies are of utmost interest to assure product quality and to investigate fraudulent practices in food science. In this section of the PhD Thesis, and as required by the aforementioned regulation of the University of Valencia, a summary of the results shown in the successive articles jointly with Sections II and III and the most relevant conclusions of each work is briefly presented here

Determination of a lectin protein allergen in food by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg·mL-1 and the limit of detection (LOD) was 0.05 µg·mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (< 0.5 % and 2.4 %, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100 % cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control

Importance of the chemical defenses and sugars in the feeding preference of Paracentrotus lividus over two sympatric template seagrass species

Herbivory is a fundamental process regulating the functioning of the ecosystems both in land and marine systems. Few decades ago, herbivory was thought to play only a minor role in seagrass dominated areas, while currently its importance has risen. However, the complex interrelationships between seagrasses and their consumers are not yet fully understood. For instance, seagrasses can tune up morphological, biomechanical, nutritional, and chemical traits in order to reduce the palatability of their leaves and therefore curtail tissue losses, but the final tradeoff of such changes may depend on herbivore guilds. This work focuses on the relative importance of nutritional versus chemical traits in the feeding behavior of the sea urchin Paracentrotus lividus, a temperate generalist mesograzer, over two sympatric seagrass species (Cymodocea nodosa and Zostera noltei). To do that, a set of no-choice and multiple-choice feeding assays were conducted using freeze-dried plants agarbased diets (i.e. whole nutritional properties), polar extracts of both species (i.e. soluble sugars and phenolic natural products), purified phenolic natural products, and also three different concentrations of soluble sugars. Later, a chemical identification and quantification of the phenolic natural products present in the extracts were performed to assess their ecological role as deterrents. Results clearly indicated that the feeding behavior of this generalist herbivore, once overlooked the structural, morphological and biomechanical traits, is mainly determined by nutritional properties, while the presence of phenolic compounds has only a minimal effect on its feeding behavior. In addition, although this study showed that sugars had a positive effect over P. lividus consumption rates, we demonstrated for the first time the deterrent properties of rosmarinic acid and the sulphated flavonoids produced by Z. noltei, which were able to reduce the attractive effect of sugars in the feeding preference of this generalist herbivore

Preclinical and randomized phase I studies of plitidepsin in adults hospitalized with COVID-19

Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 replication at nanomolar concentrations by targeting the host protein eukaryotic translation elongation factor 1A. Here, we show that plitidepsin distributes preferentially to lung over plasma, with similar potency against across several SARS-CoV-2 variants in preclinical studies. Simultaneously, in this randomized, parallel, open-label, proof-of-concept study (NCT04382066) conducted in 10 Spanish hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg (n = 15), 2.0 mg (n = 16), or 2.5 mg (n = 15) plitidepsin once daily for 3 d. The primary objective was safety; viral load kinetics, mortality, need for increased respiratory support, and dose selection were secondary end points. One patient withdrew consent before starting procedures; 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hypersensitivity and diarrhea). Treatment-related adverse events affecting more than 5% of patients were nausea (42.2%), vomiting (15.6%), and diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive ventilation was required in 8 of 44 evaluable patients (16.0%); six patients required intensive care support (13.6%), and three patients (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety profile in patients with COVID-19.This work was supported by grants from the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. We also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N Izquierdo-Useros has nonrestrictive funding from PharmaMar to study the antiviral effect of Plitidepsin. NJ Krogan was funded by grants from the National Institutes of Health (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory for Genomics Research (LGR), a collaboration between the University of California, San Francisco (UCSF), University of California, Berkley (UCB), and GlaxoSmithKline (GSK) (#133122P); by the Roddenberry Foundation, and gifts from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR0011-19-2-0020. The views, opinions, and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. This research was partly funded by Center for Research for Influenza Pathogenesis and Transmission (CRIPT), a National Institute of Allergy and Infectious Diseases (NIAID) supported Center of Excellence for Influenza Research and Response (CEIRS, contract # 75N93021C00014), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, U19AI135972, and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to A García-Sastre. S Yildiz received funding from a Swiss National Foundation Early Postdoc Mobility fellowship (P2GEP3_184202).Peer reviewe

Determinazione di polifenoli nell'olio di oliva

Determinazione di polifenoli nell'olio di oliva mediante tecniche cromatografiche e di spettrometria di massa. Ottimizzazione delle condizioni di estrazione mediante disegno sperimental

Aptamer-functionalized magnetic supports for sample preparation

In the last few years, aptamer-functionalized materials have been used as promising affinity sorbents for sample preparation purposes. These selective materials have attracted much attention due to their excellent molecular recognition properties, high stability, and the possibility of incorporation onto the surface of different supporting materials, such as nanoparticles, polymers, etc. On the other hand, current trends in extraction techniques encompass the use of miniaturized devices integrated with advanced affinity-based sorbents. Within these sample preparation devices, magnetic nanoparticles have become excellent candidates for the preparation of new functional nanomaterials for the solid-phase extraction processes. In addition to this approach, magnetic elements can be integrated in extraction devices (e.g., stir bars, stir cakes, etc.) to provide efficient extraction/stirring integrated techniques able to simplify the extraction and improve the performance. This review gives an overview of the literature published regarding aptamer-based sorbents using as host supports magnetic nanoparticles and integrated stirred units. For this purpose, the most relevant developments and applications achieved in the last seven years (from 2017 to 2023) in sample preparation by the integration of these affinity-based materials in extraction techniques such as magnetic solid-phase extraction and stir bar sorptive extraction will be reviewed

Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis

Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH 7.2 and at pH 2.2. Finally the biological function of identified proteins was evaluated in order to understand their role on human health and the potential benefits of mistletoe extracts in medicine. Significance Viscum album L. (VA) extracts are recently used as supporting medicine for cancer therapy, improving patients' survival and increasing their quality of life in medicine. These anticancer effects are investigated and they are probably due to mistletoe's capability to favor tumor cell's death and to modulate the immune system. Although the increasing interest in VA medical benefits, the role of its components in human health remains unclear. In order to exploit this aspect, it is important to comprehensively study proteins present in Viscum album L. (VA) extracts. Nevertheless, since plant proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, a total of 648 non-redundant proteins were identified: 56% and 31% of CPLLs species were respectively recognized at pH 7.2 and at pH 2.2. This deep proteome identification was useful to investigate the biological functions of proteins in order to evaluate their potential role in human health