Enriquecimiento nutricional de aceites vegetales con ácidos grasos n-3 de cadena larga mediante interesterificación enzimática con una nueva lipasa vegetal

The aim of the present work was to produce vegetable oils enriched with long-chain n-3 fatty acids of nutraceutical interest, through an enzyme-catalyzed interesterification with a new lipase, from physic nut (Jatropha curcas L.). The Vegetable Lipase Powder (biocatalyst) called VLP, which has never been applied in functional foods, was obtained from the physic nut seed, and efficiently hydrolyzed the 95% of waste fish oil in 24 h. Urea precipitation was used to concentrate polyunsaturated fatty acids (PUFA) and was further interesterified with oils of different sources by means of enzymatic catalysis. After the interesterification reaction, which was also catalyzed by the VLP, the PUFA content in coconut oil increased almost ten-fold from 1.8% to 17.7%. In palm oil, the PUFA content increased two-fold from 10.5% to 21.8%, while in olive oil the level of PUFA increased from 8.6% to 21.3%. The mixture of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (3.7% to 3.9%) was incorporated into the triacylglycerol fraction of each of the coconut, palm and olive oils. Through the hydroesterification (hydrolysis followed by interesterification) all the interesterified vegetable oils tested presented sufficient EPA and DHA levels to satisfy the levels recommended for intake by human adults in one tablespoon.El objetivo del presente trabajo fue producir aceites vegetales enriquecidos con ácidos grasos n-3 de cadena larga de interés nutraceutico, por interesterificación catalizada mediante una nueva lipasa, una enzima de semilla de Jatropha curcas L. La lipasa vegetal en polvo (biocatalizador) llamada VLP, nunca ha sido aplicada en alimentos funcionales, se obtuvo mediante procedimientos físicos con semillas de nueces, e hidrolizó eficientemente el 95% de aceites de residuos de pescado en 24 h. La precipitación con urea se utilizó para concentrar los ácidos grasos poliinsaturados (PUFA) que fueron posteriormente interesterificados con aceites de diferentes fuentes mediante catálisis enzimática. Tras la reacción de interesterificación, que también fue catalizada por la VLP, el contenido de PUFA en el aceite de coco aumentó casi diez veces de 1,8% a 17,7%. En el aceite de palma, el contenido de PUFA aumentó dos veces desde 10,5% a 21,8%, mientras que en el aceite de oliva el nivel de PUFA incrementó de 8,6% a 21,3%. La mezcla de ácido eicosapentaenoico (EPA) y ácido docosahexaenoico (DHA) (3,7% a 3,9%) fué incorporada a la fracción de triacilgliceroles de cada uno de los aceites, coco, palma y oliva. Mediante hidroesterificación (hidrólisis seguido de interesterificación) todos los aceites vegetales interesterificados ensayados contienen en una cucharada suficientes niveles de EPA y DHA para cubrir los niveles recomendados de ingesta de humanos adultos

Multi-target analysis of cytostatics in hospital effluents over a 9-month period

The consumption of cytostatics, pharmaceuticals prescribed in chemotherapy, is increasing every year and worldwide, along with the incidence of cancer. The presence and the temporal evolution of cytostatics in wastewaters from a Portuguese hospital center was evaluated through a 9-month sampling campaign, comprising a total of one hundred and twenty-nine samples, collected from May 2019 to February 2020. Eleven cytostatics out of thirteen pharmaceuticals were studied, including flutamide, mycophenolate mofetil and mycophenolic acid, which have never been monitored before. Target analytes were extracted and quantified by solid-phase extraction coupled to liquid-chromatography-tandem mass spectrometry analysis; the method was fully validated. All pharmaceuticals were detected in at least one sample, bicalutamide being the one found with higher frequency (detected in all samples), followed by mycophenolic acid, which was also the compound detected at higher concentrations (up to 5340 ± 211 ng/L). Etoposide, classified as carcinogenic to humans, was detected in 60% of the samples at concentrations up to 142 ± 15 ng/L. The risk from exposure to cytostatics was estimated for aquatic organisms living in receiving bodies. Cyclophosphamide, doxorubicin, etoposide, flutamide, megestrol and mycophenolic acid are suspected to induce risk. Long-term and synergic effects should not be neglected, even for the cytostatics for which no risk was estimated

Pristine Multi-walled carbon nanotubes for a rapid and efficient plasmid DNA clarification

Therapeutic approaches based on nucleic acids to modulate cell activity have recently gained attention. These molecules arise from complex biotechnological processes, requiring effective manufacturing strategies, high purity, and precise quality control to be used as biopharmaceuticals. One of the most critical and time-consuming steps for nucleic acids-based biotherapeutics manufacturing is their purification, mainly due to the complexity of the extracts. In this study, a simple, efficient, and reliable method to isolate and clarify plasmid DNA (pDNA) from complex samples is described. The method is based on the selective capture of RNA and other impurities, using pristine carbon nanotubes (CNTs). Multi-walled CNTs (MWCNTs) with different diameters were studied to determine their adsorption capacity and to address their ability to interact and distinguish between nucleic acids. The results revealed that MWCNTs preferentially interact with RNA and that smaller MWCNTs present a higher adsorption capacity, as expected by the higher specific surface area. Overall, this study showed that MWCNTs significantly reduce the levels of impurities, namely RNA, gDNA, and proteins, by approximately 83.6 % compared to their initial level, enabling the recovery of clarified pDNA in solution while maintaining its stability throughout the recovery process. This method facilitates the pre-purification of pDNA for therapeutic applications.publishe

Kinetic resolution of (R,S)-1,2-isopropylidene glycerol (solketal) ester derivatives by lipases

AbstractA study on the enantioselective hydrolysis of (R,S)-1,2-isopropylidene glycerol (4-hydroxymethyl-2,2-dimethyl-1,3-dioxolane, solketal) octanoate catalyzed by different lipases was carried out. Among them, Pseudomonas sp. lipase proved to be the most effective. It was shown that the ester bearing the longer octanoyl acyl chain is a more suitable substrate for this lipase compared to the acetate counterpart. By properly combining enzyme load, temperature and reaction time, either the (S)-alcohol or the remaining ester could be obtained in moderate to high selectivities. Ethyl acetate was found to be the best solvent for the kinetic resolutions effected by such lipase but our results show that toluene may prove useful

Pulsar Timing and its Application for Navigation and Gravitational Wave Detection

Pulsars are natural cosmic clocks. On long timescales they rival the precision of terrestrial atomic clocks. Using a technique called pulsar timing, the exact measurement of pulse arrival times allows a number of applications, ranging from testing theories of gravity to detecting gravitational waves. Also an external reference system suitable for autonomous space navigation can be defined by pulsars, using them as natural navigation beacons, not unlike the use of GPS satellites for navigation on Earth. By comparing pulse arrival times measured on-board a spacecraft with predicted pulse arrivals at a reference location (e.g. the solar system barycenter), the spacecraft position can be determined autonomously and with high accuracy everywhere in the solar system and beyond. We describe the unique properties of pulsars that suggest that such a navigation system will certainly have its application in future astronautics. We also describe the on-going experiments to use the clock-like nature of pulsars to "construct" a galactic-sized gravitational wave detector for low-frequency (f_GW ~1E-9 - 1E-7 Hz) gravitational waves. We present the current status and provide an outlook for the future.Comment: 30 pages, 9 figures. To appear in Vol 63: High Performance Clocks, Springer Space Science Review

Do Instantons Like a Colorful Background?

We investigate chiral symmetry breaking and color symmetry breaking in QCD. The effective potential of the corresponding scalar condensates is discussed in the presence of non-perturbative contributions from the semiclassical one-instanton sector. We concentrate on a color singlet scalar background which can describe chiral condensation, as well as a color octet scalar background which can generate mass for the gluons. Whereas a non-vanishing singlet chiral field is favored by the instantons, we have found no indication for a preference of color octet backgrounds.Comment: 25 pages, 7 figure

Novel downstream process and analytical tools developed for Influenza VLP vaccine

Vaccination remains the most effective way to prevent the infection with Influenza viruses. However, their constant antigenic drift implies that current human Influenza vaccines need to be annually updated with high inherent costs. Virus-like particles (VLPs) have become widely used as vaccine candidates because of their versatility, immunogenicity, and safety profile. In this iBET project we are attempting to produce a candidate for a universal vaccine for which 35 different VLPs (mono, trivalent and pentavalent) were purified. Here we describe three recent advances on Influenza VLPs bioprocessing: two new analytical tools and the development of an integrated all filtration purification process, inserted in the “anything but chromatography” concept. The first method is a label-free tool that uses Biolayer interferometry technology applied on an Octet platform to quantify Influenza VLPs at all stages of DSP. Human and avian sialic acid receptors were used, in order to quantify hemagglutinin (HA) content in several mono- and multivalent Influenza VLP strains. The applied method was able to detect and quantify HA from crude sample up to final VLP product with high throughput, real-time results and improved detection limits, when compared to traditional approaches, crucial for in-line monitoring of DSP. Using a click-chemistry approach that involves Azidohomoalanine incorporation and functionalization, Influenza VLPs were selectively and fluorescently tagged. Taking into account that this chemical tag does not affect particle size, charge and biological activity we report here a valuable tool to online/at-line product monitoring during DSP optimization of virus related biopharmaceuticals. Moreover, using this tool coupled with FACS we were able to discriminate between VLPs and baculovirus, the major impurity of the system. The proposed all-filtration process will be described, with special focus on the clarification stage, followed by multiple ultrafiltration and diafiltration steps to achieve the needed concentration and purity specifications. Using this all-filtration platform, we are able to speed up the time process, to improve the scale-up and to reduce costs due to the removal of chromatographic steps