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In the present work, borage oil (Borago officinalis) was used as the main source of gamma linolenic acid (GLA) to obtain ethyl esters by enzymatic ethanolysis using immobilized enzymes for its application in the food industry. Commercial Thermomyces lanuginosus lipase (TLL) was compared to chemical ethanolysis in alkaline medium. In addition, TLL was immobilized by adsorption on hydrophobic porous support (Octadecyl-Sepabeads®) to compare the results. Fatty acid ethyl ester (FAEE) yields of both reactions were compared under the same conditions (25 °C and 200 rpm) and analyzed by GC-MS. Moreover, the conversion yield for borage oil ethanolysis catalyzed by TLL immobilized on C18-Sepabeads® supports was similar to the chemical pathway (93.4% and 99.5%, respectively). When this biocatalyst was used in a solvent-free system (at 40 °C and 200 rpm), it was possible to obtain a high FAEE yield of 84.3% in the first 24 h of reaction. Furthermore, it was possible to re-use the immobilized biocatalyst for the performance of five reaction cycles maintaining 68% of its initial activity. Thus, the use of immobilized enzymes in solvent-free systems is an eco-friendly alternative to obtain GLA ethyl esters for its possible application in cosmetics and food.

Damage to the retinal pigment epithelium, Bruch's membrane and/or tissues underlying macula is known to increase the risk of age-related macular degeneration (AMD). AMD is commonly categorized in two distinct types, namely, the nonexudative (dry form) and the exudative (wet form). Currently, there is no ideal treatment available for AMD. Recommended standard treatments are based on the use of vascular endothelial growth factor (VEGF), with the disadvantage of requiring repeated intravitreal injections which hinder patient's compliance to the therapy. In recent years, several synthetic and natural active compounds have been proposed as innovative therapeutic strategies against this disease. There is a growing interest in the development of formulations based on nanotechnology because of its important role in the management of posterior eye segment disorders, without the use of intravitreal injections, and furthermore, with the potential to prolong drug release and thus reduce adverse effects. In the same way, 3D bioprinting constitutes an alternative to regeneration therapies for the human retina to restore its functions. The application of 3D bioprinting may change the current and future perspectives of the treatment of patients with AMD, especially those who do not respond to conventional treatment. To monitor the progress of AMD treatment and disease, retinal images are used. In this work, we revised the recent challenges encountered in the treatment of different forms of AMD, innovative nanoformulations, 3D bioprinting, and techniques to monitor the progress.

Microalgae are described as a new source of a wide range of bioactive compounds with health-promoting properties, such as omega-3 lipids. This biomass product is gaining attention mainly due to its potential to accumulate different compounds depending on the species and environment, and it has been commonly recognized as a valuable nutraceutical alternative to fish and krill oils. In this work, we obtained the extract of the microalga Nannochloropsis gaditana, selected on the basis of its content of eicosapentaenoic acid (EPA) and glycolipids, which were determined using GC-MS and high-performance liquid chromatography (HPLC), respectively. To develop an oral formulation for the delivery of the extract, we used a 23 factorial design approach to obtain an optimal lipid nanoparticle formulation. The surfactant and solid lipid content were set as the independent variables, while the particle size, polydispersity index, and zeta potential were taken as the dependent variables of the design. To ensure the potential use of the optimum LN formulation to protect and modify the release of the loaded microalga extract, rheological and differential scanning calorimetry analyses were carried out. The developed formulations were found to be stable over 30 days, with an encapsulation efficiency over 60%.

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLLSepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLLDuolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA.

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was performed with several lipases in a solvent-free system and it involves the initial formation of sn-2 docosahexaenyl monoacylglyceride. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp (QL), and Candida antarctica-fraction B (CALB) were tested. In certain cases (CALB), using an excess of DHA ethyl ester and high temperatures the transesterification reaction continues to the formation of triacylglycerides, but in other cases, sn-2 monoacylglyceride (2-MG) is the unique synthetic product even in the presence of high concentrations of DHA ethyl ester. At low temperatures (e.g. 37°C), RML derivatives synthesize only 2-MG in 15 min. These very mild conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid and for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate markers, the formation of the 2-MG was confirmed.

The synthesis of docosahexaenoyl triacylglycerides at low temperature (e.g., 50°C) using biocatalysts of 6 commercial lipases adsorbed on hydrophobic supports was studied. In general, the triacylglyceride yields were very low with the exceptions of those produced with the enzymes from Candida antarctica fraction B, CALB (82%), and those produced with the enzyme from Pseudomonas fluorescens, PFL (57%). The reactions were performed under vacuum to remove the released ethanol. The yields varied widely when different derivatives of CALB were used, and they were higher when CALB adsorbed on hydrophobic supports was used (82%). One interesting by-product (18% of sn-2 monoacylglyceride of DHA) remained at the end of the synthetic process. CALB adsorbed on Sepabeads exhibited better activity and stability than did the commercial derivative Novozym 435. The best CALB biocatalyst preserved 90% of the activity after 30 days under the reaction conditions.

A process based on the use of a semi-continuous countercurrent supercritical fluid extraction (CC-SFE) has been optimized to concentrate minor components, with functional properties, from olive oil. The optimization of the main variables involved in the supercritical extraction process (extraction pressure, temperature and sample flow rate) was performed using a surface response methodology (RSM). A central composite circumscribed design (CCCD) was employed to study the responses selected to describe the process, such as the extraction yield, the concentration of the different minor compounds (squalene, sterols and tocopherols) in the CO2-rich upper current stream, and the recovery of these compounds from the olive oil. Parameters of the model, for each response variable, were simultaneously estimated by Partial Least Squares (PLS) regression. The statistical analysis of the results allowed obtaining mathematical models able to predict the behavior of the different responses selected as a function of the main variables involved in the process. The optimum conditions obtained, that maximized all the responses as a whole, were: extraction pressure, 234 bar; extraction temperature, 35°C and sample flow rate, 82 mL/h, working with a solvent flow rate equal to 2000 mL/h (S/F equal to 28.4 kg CO2/kg oil). The optimum of the model was experimentally confirmed allowing around 90% recovery of squalene without significant changes on the nutritional and physicochemical properties of the oil. © 2014 Bentham Science Publishers.

[Background]: This study focuses on obtaining a highly purified lysophosphatidylcholine from phosphatidylcholine powder via hydrolysis, catalyzed by a phospholipase A2 in a solvent-free medium, and subsequent purification either by organic solvent or by supercritical fluid extraction. [Results]: A solvent-free hydrolysis reaction of phosphatidylcholine powder to produce lysophosphatidylcholine with residual phosphatidylcholine content lower than 10% has been described and scaled-up. Supercritical fluid mixtures of carbon dioxide, ethanol and formic acid were studied at 250, bar 50 °C to selectively extract the free fatty acids fraction from hydrolyzed phosphatidylcholine. Finally, a solid product with lysophosphatidylcholine content of c. 95% (w/w) and with free fatty acids content of c. 5% (w/w) was attained. Under optimum conditions, the content of the original phosphatidylcholine in the lysophosphatidylcholine product was lower than 5% (w/w). [Conclusions]: An efficient hydrolysis of phosphatidylcholine by phospholipase A2 in a solvent-free reaction was developed. In addition, three novel strategies for the purification of lysophosphatidylcholine have been developed: (1) hexane/formic acid extraction process, (2) supercritical CO2 extraction with ethanol as modifier, and (3) supercritical CO2 extraction with ethanol/formic acid as modifier. © 2012 Society of Chemical Industry.

Type 1 diabetes mellitus is one of the most common chronic disorders of childhood. The metabolic control is lost due to the lack of insulin, which is the main treatment for the disease. Nevertheless, long-term complications appear even under good glycemic control. Metabolomics, an emerging strategy, can help in diagnosis, prognosis, and monitoring of metabolic disorders. The objective of the present study was to investigate the alterations in plasma (by LC-MS) and urine (CE-MS) of type 1 diabetic children that were under insulin treatment and good glycemic control. Even without remarkable biochemical differences between the two groups (diabetic and control) except for glucose level and glycosilated hemoglobin, metabolomic tools were able to capture subtle metabolic differences. The main changes in plasma were associated to lipidic metabolism (nonesterified fatty acids, lysophospholipids, and other derivatives of fatty acids), and some markers of the differential activity of the gut microflora were also found (bile acids, p-cresol sulfate). In urine, changes associated to protein and amino acid metabolism were found (amino acids, their metabolites and derivatives), and among them one advanced glycation end product (carboxyethylarginine) and one early glycation end product (fructosamine) were excreted in higher proportion in the diabetic group. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two immobilized lipases from Candida antarctica have been compared for the direct esterification of tyrosol with oleic acid in equimolar conditions and in the absence of organic solvent. Candida antarctica lipase B was immobilized on octyl-silica agglomerates and compared with commercial Novozym 435. Reduction of tyrosol particle size to 0.1 mm significantly increased the reaction rate with both immobilized lipases, and reduced pressure improved the final tyrosyl oleate yield up to 95% (w/w) in both cases. Immobilized lipases were recovered and reutilized in three consecutive trials with negligible inactivation. Under optimum conditions, a product mixture comprising more than 95% of tyrosyl oleate (w/w) was attained in less than 2 hours. Finally, the index of antioxidant activity obtained, according to the Rancimat method, indicated that tyrosyl oleate was slightly more effective than tyrosol as an antioxidant in a low polar matrix. © 2012 Informa UK, Ltd.

Rosmarinusofficinalis (rosemary) extracts were obtained in a supercriticalpilot-scale plant. Based on experimental information available in the literature for analytical or low-scale processes, extraction temperature and pressure were selected to be 313 K and 30 MPa. At these extraction conditions, the kinetic behavior of the pilot-scale overall extraction curve were determined with respect to yield, antioxidant activity and carnosic acid content. The overall extraction curve was represented using Sovova's model; the average deviation between measured and calculated yields was lower than 2%. Mass transfer coefficients in the fluid and solid phases were determined and were compared with previous data reported in the literature for low-scalerosemarysupercriticalextraction. A two-stage depressurization procedure was accomplished and the effect of both on-line fractionation and extraction time on the antioxidant activity of the samples collected was studied. The antioxidant activity of the different fractions could be straight correlated with the carnosic acid content with a regression coefficient of 0.92.

Pressurized liquid extraction (PLE), an environmentally friendly technique, was used to obtain antiviral compounds from the edible seaweed Himanthalia elongata. The antiviral properties of PLE extracts (hexane, ethanol, and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pre-treatment of Vero cells with 75 ¿g mL-1 of ethanol extract inhibited virus infection by approximately 90%, whereas the same concentration of water and hexane extracts reduced the virus infectivity to 78% and 70%, respectively. Moreover, ethanol extract was also more effective against HSV-1 intracellular replication than water and hexane extracts. The antiviral activity of water PLE extract was found to correlate with polysaccharides, since the polysaccharide-rich fraction isolated from this extract showed higher antiviral activity than the original water extract. A GC-MS characterization of the hexane and ethanol extracts showed that the antiviral activity of the hexane extract seemed to be related with the presence of fucosterol; meanwhile, in the case of the ethanol extract, other compounds, besides fucosterol, could be involved in this activity. Results demonstrated that PLE was an appropriate technique to obtain antiviral agents from H. elongata. These antiviral compounds were in addition to polysaccharides, which are the antiviral agents usually proposed when studying seaweeds. © 2010 Springer Science+Business Media B.V.

The rat treated with streptozotocin has been proposed as the most appropriate model of systemic oxidative stress for studying antioxidant therapies. In that sense, rosemary extracts have long been recognized as having antioxidant properties, and folic acid may be able to improve endothelial progenitor cell function. A mixture containing both has been tested as a possible nutraceutical to improve health complications in diabetes. We have developed the methodology to evaluate metabolic changes in the urine of streptozotocin-induced diabetic rats after supplementing their diet with rosemary extract obtained with supercritical fluids (SFE) containing 10% folic acid in an acute but short-term study. It has been done with a metabolomics approach using LC-QTOF as an analytical tool. About 20 endogenous metabolites have been identified by databases and MS/MS showing statistically significant changes. Among them, several amino acids and their metabolites point to changes due to the effect of the gut microbiota. In addition, the comparison between control and streptozotocin-diabetic rats has permitted the showing of some metabolic coincidences between type 1 diabetes and other (possible) autoimmune diseases such as autism and/or Crohn's disease, and the nutraceutical intervention has succeeded in inducing changes in such biomarkers. © 2011 American Chemical Society.

Dispositivo y procedimiento para la extracción y secado en línea de extractos complejos. La presente invención se refiere a un dispositivo y un proceso de extracción y secado en línea de productos naturales. El proceso de extracción y secado de productos naturales se lleva a cabo empleando agua en condiciones subcríticas junto con CO2 en condiciones supercríticas y N2 como gas inerte. El proceso se lleva a cabo en una sola etapa, a diferencia de otros procesos conocidos y que se describen en el estado de la técnica. Además de la obtención de extractos secos, al proceso WEPO permite manipular el tamaño y morfología de las partículas, lo que tiene gran importancia en todo tipo de compuestos bio-activos por la relación entre el tamaño de partícula y la actividad, estabilidad, y biodisponibilidad de los mismos.

[ES]: En este trabajo se presentan los resultados más relevantes obtenidos en el estudio de diferentes procesos para enriquecer, purificar y sintetizar ingredientes lipídicos con propiedades saludables, que tienen un alto valor añadido y pueden emplearse en la elaboración de alimentos funcionales y nutracéuticos. Las dos principales metodologías utilizadas en la presente Tesis son la extracción y fraccionamiento con fluidos supercríticos y la síntesis enzimática, que ofrecen ventajas debido a su alta versatilidad, selectividad, eficacia y respeto por el medio ambiente frente a métodos convencionales, siendo idóneas para la obtención de productos con grado alimentario. La obtención y purificación de compuestos lipídicos minoritarios como fitoesteroles y tocoferoles ha sido abordada partiendo de destilados de desodorización de aceite de soja, empleando procesos de extracción supercrítica en contracorriente (CC-SFE) combinados con transformaciones químicas y enzimáticas de los materiales de partida, dando lugar a extractos y fracciones no extraídas de alto valor añadido. Por otro lado, a partir de grasa láctea se fraccionaron otros lípidos con destacada actividad biológica, como ácidos grasos de cadena corta y cadena media (entre 4 y 12 átomos de carbono). Estos ácidos grasos, junto a otros como los diferentes isómeros del ácido linoleico conjugado (CLA), se emplearon como material de partida en reacciones sin disolventes catalizadas por lipasas, permitiendo la obtención de ésteres de fitoesteroles de elevada pureza y alto potencial. Finalmente conviene destacar que parte de los resultados descritos en la presente Tesis han sido obtenidos a escala de planta piloto, lo que les confiere una mayor viabilidad y aplicabilidad en el sector industrial. De este modo, se demuestra el empleo eficaz de diversas herramientas tecnológicamente avanzadas y limpias para el aislamiento, purificación y síntesis de diversos compuestos lipídicos con contrastada actividad biológica y eleva, [EN]: The present work describes the most important results obtained throughout the study of different processes for the enrichment, purification and synthesis of high added value lipidic ingredients with healthy properties that may be used in nutraceuticals and functional food production. Supercritical fluid extraction and fractionation techniques along with enzymatic synthesis have been the main methodologies employed within this thesis. These techniques are adequate for the production of food-grade ingredients and offer advantages regarding conventional methods, mainly due to the greener chemistry involved, enhanced versatility, selectivity, and efficiency. Minor lipid component production and concentration like phytosterols and tocopherols has been scrutinized from soybean deodorizer distillate by means of countercurrent supercritical fluid extraction processes (CC-SFE) combined with chemical and enzymatic transformations of the starting materials, rendering high added value extracts and rafinates. In addition, other lipids with important biological activity from milk fat, such as short-chain and medium chain fatty acids (4 to 12 carbon atoms) were fractionated. These fatty acids, along with others like the different isomers of conjugated linoleic acid material for solvent-free reactions catalyzed by lipases allowing the collection of phytosterol esters of high purity and high potential. Finally, it should be noted that some of the results described in this Thesis have been obtained at pilot plant scale, which confers greater viability and applicability in the industrial sector. Thus, this Thesis presents an effective use of different advanced and clean technologies for the synthesis, isolation and purification of several lipidic compounds with relevant biological activity and high potential, from which functional food ingredient preparations and nutraceuticals production might result.

In this work, extraction of antioxidant carotenoids from Haematococcus pluvialis microalga, has been studied combining pressurized liquid extraction (PLE), using hexane and ethanol as extracting solvents, and analytical techniques such as thin layer chromatography (TLC) and HPLC with DAD. The effect of the extraction temperature (50, 100, 150 and 200 °C) and the polarity of the solvent have been studied in terms of in vitro antioxidant activity and chemical composition considering two different morphological cells (green vegetative cells and red cysts). Results demonstrate that the extraction temperature had a positive influence in the extraction yield while its effect in the antioxidant activity was negative, lowering the activity of the extracts with an increase of the extraction temperature. The best yields were obtained with ethanol at the higher extraction temperature while the best antioxidant activity was also achieved using ethanol but at lower temperatures. Chemical composition was determined by TLC and HPLC with DAD. Several compounds were identified in the samples and concentration of astaxanthin was obtained. Results pointed out that the extracts contained different carotenoids in both, the green and the red phase, and that depending on its contribution a stronger antioxidant activity would be expected. © 2009 Elsevier Ltd. All rights reserved.

In this work, extraction and characterization of compounds with antioxidant and antimicrobial activity from Haematococcus pluvialis microalga in red phase have been carried out. To do this, subcritical water extraction (SWE) has been combined with analytical techniques such as HPLC-DAD, HPLC-QqQ-MS and GC-MS and in vitro assays (i.e., for antioxidant and antimicrobial activity). The effect of the extraction temperature (50, 100, 150 and 200 °C) and solvent polarity has been studied in terms of yield and activity of the extracts. Results demonstrate that the extraction temperature has a positive influence in the extraction yield and antioxidant activity. Thus, the extraction yield achieved with this process was higher than 30% of dry weight at 200 °C as extraction temperature. Moreover, the extract obtained at 200 °C presented the highest antioxidant activity by far, while temperature does not seem to significatively affect the antimicrobial activity. Chemical composition was determined by HPLC-DAD, HPLC-QqQ-MS and GC-MS. Short chain fatty acids turned out to be responsible of the antimicrobial activity, whereas the antioxidant activity was correlated to vitamin E (present exclusively in the 200 °C extract), together with simple phenols, caramelization products and possible Maillard reaction products obtained during the extraction at high temperatures. © 2009 Elsevier B.V. All rights reserved.

The supercritical removal of ethanol from alcoholic beverages (brandy, wine, and cider) was studied using the GC-EoS model to represent the phase equilibria behavior of the CO2 + beverage mixture. Each alcoholic drink was represented as the ethanol + water mixture with the corresponding ethanol concentration (35 wt% for brandy, 9-12 wt% for different wines and 6 wt% for cider). The thermodynamic modeling was based on an accurate representation of the CO2 + ethanol and CO2 + water binary mixtures, and the CO2 + ethanol + water ternary mixture. The GC-EoS model was employed to simulate the countercurrent supercritical CO2 dealcoholization of the referred beverages; the results obtained compared good with experimental data from the literature. Thus, the model was used to estimate process conditions to achieve an ethanol content reduction from ca. 10 wt% to values lower than 1 wt%. The model results were tested by carrying out several extraction assays using wine, in a 3 m height packed column at 308 K, pressures in the range of 9-18 MPa and solvent to wine ratio between 9 and 30 kg/kg. © 2010 Elsevier B.V. All rights reserved.