Enhancing nutrient recovery and bioactive compound extraction from spirulina through supercritical fluid extraction : implications for SH-SY5Y cell viability

This work explores the efficiency of supercritical fluid extraction (SFE) to recover minerals, pigments, and antioxidant compounds from the spirulina microalgae. Moreover, the fatty acids and phenolic profiles of the extracts obtained were also investigated, and the effect of the extracts on SH-SY5Y cell viability was tested. The extraction of phycocyanin was improved by SFE compared to conventional extraction, from 2.838 ± 0.081 mg/g dry matter (DM) (control) to 6.438 ± 0.411 mg/g DM (SFE). SFE treatment also improved chlorophyll a and carotenoid recoveries increasing from 5.612 ± 0.547 to 8.645 ± 0.857 mg/g DM and from 0.447 ± 0.096 to 0.651 ± 0.120 mg/g DM, respectively. Regarding minerals, the SFE improved Mg recovery with 77% more than the control extraction. Moreover, palmitoleic, stearic, γ-linolenic, eicosadienoic and eicosatrienoic acids recovery was improved by SFE. Phenolic profiles were identified via triple-TOF-LC-MS-MS. Considering heavy metals, a higher rate of Pb extraction was observed for the SFE extract, while no significant differences were observed for Hg between both extractions. Finally, SFE extract improved cell viability compared to the control extract. Thus, SFE constitutes an interesting tool to sustainably extract high-added-value compounds; however, potential contaminants such as Pb need to be controlled in the resulting extracts

Influence of temperature, solvent and pH on the selective extraction of phenolic compounds from tiger nuts by-products: Triple-TOF-LC-MS-MS characterization.

The aim of this study was to assess the effect of temperature, solvent (hydroethanolic mixtures) and pH on the recovery of individual phenolic compounds from 'horchata' by-products. These parameters were optimized by response surface methodology and triple-TOF-LC-MS-MS was selected as the analytical tool to identify and quantify the individual compounds. The optimum extraction conditions were 50% ethanol, 35 °C and pH 2.5, which resulted in values of 222.6 mg gallic acid equivalents (GAE)/100 g dry matter and 1948.1 µM trolox equivalent (TE)/g of dry matter for total phenolic content (TPC) and trolox equivalent antioxidant capacity (TEAC), respectively. The extraction of phenolic compounds by the conventional solvent method with agitation was influenced by temperature (p = 0.0073), and more strongly, by the content of ethanol in the extraction solution (p = 0.0007) while the pH did not show a great impact (p = 0.7961). On the other hand, the extraction of phenolic acids was affected by temperature (p = 0.0003) and by ethanol amount (p < 0.0001) but not by the pH values (p = 0.53). In addition, the percentage of ethanol influenced notably the extraction of both 4-vinylphenol (p = 0.0002) and the hydroxycinnamic acids (p = 0.0039). Finally, the main individual phenolic extracted with hydroethanolic mixtures was 4-vinylphenol (303.3 μg/kg DW) followed by spinacetin3-O-glucosyl-(1→6)-glucoside (86.2 μg/kg DW) and sinensetin (77.8 μg/kg DW)

Development and implementation of an "escape room" gamification methodology as an innovative teaching tool for students of the degree in Gastronomic Sciences

[EN] Various international organisations have emphasized the importance of establishing both health-based as well as adequate sanitary food guidelines to avoid possible food contamination and potential danger due to improper handling of food. Thus, students of gastronomic sciences must be able to detect hazards (chemical and biological) in the consideration and preparation of menus. The growing interest of students in this field of study also requires a more dynamic and practical method of teaching which can be carried out in seminars. Multidisciplinary case studies oriented towards hazard detection and nutritional objectives are proposed. Due to the current Covid-19 health crisis, hosting and participication in these seminars has been adversely affected. The creation of a virtual reality learning resource in which to interact while reducing risk could be a very interesting alternative. The aim of this resource was to create a task using a gamification approach ("escape room") in a virtual laboratory, in which to pass related tests of both a nutritional and food safety background, and thus save their patient from a specific ailment. As indicated by the high percentage of correct answers in the activity this "escape room" resource was shown to positively reinforce students´ knowledge.[ES] Los diferentes organismos internacionales hacen hincapié en la necesidad de establecer pautas dietéticas saludables y seguras, evitando posibles intoxicaciones por manipulación incorrecta en restauración o alimentos preparados para reducir el posible peligro. Así el estudiante de ciencias gastronómicas debe ser capaz de detectar peligros (químicos y biológicos) en la elaboración de menús. El creciente interés de los estudiantes en este campo de estudio, requiere también de una docencia más dinámica y práctica que puede llevarse a cabo en los seminarios. Se proponen casos de estudio multidisciplinar orientados a la detección de peligros y al cumplimiento de objetivos nutricionales. Debido a la situación actual de pandemia, la asistencia a estos seminarios ha sido difícil. La creación de un recurso didáctico de realidad virtual en el que interactuar pero sin poner en riesgo su salud sería una alternativa muy interesante. El objetivo de este recurso fue crear una tarea utilizando un enfoque de gamificación ("escape room") en un laboratorio virtual, en el que pasar pruebas relacionadas, tanto nutricionales como de seguridad alimentaria, y así salvar a su paciente de una dolencia específica. El “escape room” reforzó positivamente los conocimientos del estudiante ante el gran porcentaje de aciertos en la actividad.Este trabajo ha contado con el apoyo del Vicerectorat d’Ocupació i Programes Formatius de la Universitat de València – a través del proyecto concedido en el marco de las “Ajudes per al Desenvolupament de Projectes d’Innovació Educativa per al curs 2021-2022” con nombre “Sherlock-Salud: Utilización de metodología de escape room para casos de estudio de Nutrición y Seguridad Alimentaria” (NOU-PID, UV-SFPIE_PID-1640423) así como el proyecto AQUABIOPRO-FIT “Aquaculture and agriculture biomass side stream proteins and bioactives for feed, fitness and health promoting nutritional supplements” (Grant Agreement no. 790956) financiado por la Comisión Europea a través del programa H2020-BBI-JU.Barba Orellana, F.; Martí Quijal, F.; Pallarés Barrachina, N.; Juan García, C.; Berrada Ramdani, H. (2022). Desarrollo e implementación de una metodología de gamificación "escape room" como herramienta docente innovadora para los estudiantes del grado en Ciencias Gastronómicas. En In-Red 2022 - VIII Congreso Nacional de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 231-238. https://doi.org/10.4995/INRED2022.2022.1585323123

Obtaining antioxidants and natural preservatives from food by-products through fermentation: A review

Industrial food waste has potential for generating income from high-added-value compounds through fermentation. Solid-state fermentation is promising to obtain a high yield of bioactive compounds while requiring less water for the microorganism’s growth. A number of scientific studies evinced an increase in flavonoids or phenolics from fruit or vegetable waste and bioactive peptides from cereal processing residues and whey, a major waste of the dairy industry. Livestock, fish, or shellfish processing by-products (skin, viscera, fish scales, seabass colon, shrimp waste) also has the possibility of generating antioxidant peptides, hydrolysates, or compounds through fermentation. These bioactive compounds (phenolics, flavonoids, or antioxidant peptides) resulting from bacterial or fungal fermentation are also capable of inhibiting the growth of commonly occurring food spoilage fungi and can be used as natural preservatives. Despite the significant release or enhancement of an-tioxidant compounds through by-products fermentation, the surface areas of large-scale bioreactors and flow patterns act as constraints in designing a scale-up process for improved efficiency. An in-process purification method can also be the most significant contributing factor for raising the overall cost. Therefore, future research in modelling scale-up design can contribute towards mitigating the discard of high-added-value generating residues. Therefore, in this review, the current knowledge on the use of fermentation to obtain bioactive compounds from food by-products, emphasizing their use as natural preservatives, was evaluated

Sequential extraction of almond hull biomass with pulsed electric fields (PEF) and supercritical CO2 for the recovery of lipids, carbohydrates and antioxidants

This work reports the first example of combined sequential extraction by pulsed electric fields (PEF) (3 kV/cm, 100 kJ/kg, 2 Hz, 100 ms) and supercritical (SC) fluid extraction (SFE) (15 MPa, 25 mL/min, 50ºC, 60 min) with CO2 (SC-CO2) for the valorisation of almond hull (AH) biomass. PEF+SFE boosted the efficiency of the protocol up to 77% for total antioxidant capacity and 20% in terms of polyphenols recovery compared to the traditional soaking. Triple-TOF-LC-MS-MS analysis provided the phenolic profiles for the PEF and SC-CO2 extracts, observing significant differences in the polyphenol profile according to the technology applied. Additionally, NMR analysis detected the presence of the carbohydrate soluble (mainly glucose, fructose and sucrose) and lipidic fractions, both selectively extracted by PEF or SC-CO2, respectively. Finally, the post-extraction residual solid biomass was characterized by several techniques such as TGA, FT-IR and SEM. For the latter, the formation of surface pores after PEF and a high fibre compaction after SFE was observed. On the other hand, DTG curves allowed to firmly propose concurrent valorisation routes for this solid, in agreement with a zero-waste approach

Influence of Geographical Location of Spirulina (Arthrospira platensis) on the Recovery of Bioactive Compounds Assisted by Pulsed Electric Fields

Spirulina (Arthrospira platensis) has been consumed by humans since ancient times. It is rich in high added-value compounds such as chlorophylls, carotenoids and polyphenols. Pulsed electric fields (PEF) is an innovative non-thermal technique that improves the extraction of bioactive compounds from diverse sources. PEF pre-treatment (3 kV/cm, 100 kJ/kg) combined with supplementary extraction with binary solvents at different times was evaluated to obtain the optimal conditions for extraction. In addition, the results obtained were compared with conventional treatment (without PEF pre-treatment and constant shaking) and different strains of Spirulina from diverse geographical locations. The optimal extraction conditions for recovering the bioactive compounds were obtained after applying PEF treatment combined with the binary mixture EtOH/H2O for 180 min. The recovery of total phenolic content (TPC) (19.76 ± 0.50 mg/g DM (dry matter) and carotenoids (0.50 ± 0.01 mg/g DM) was more efficient in the Spirulina from Spain. On the other hand, there was a higher recovery of chlorophylls in the Spirulina from China. The highest extraction of total antioxidant compounds was in Spirulina from Costa Rica. These results show that PEF, solvents and the condition of growing affect the extraction of antioxidant bioactive compounds from Spirulina. The combination of PEF and EtOH/H2O is a promising technology due to its environmental sustainability

Obtención de nutrientes y compuestos bioactivos a partir de biomasa marina y evaluación de la toxicidad con bioensayos = Nutrients and bioactive compounds recovery from marine biomass and toxicity evaluation using bioassays

Esta Tesis Doctoral se centra en primer lugar en la utilización de la fermentación con bacterias ácido-lácticas aisladas de pescado para la obtención de compuestos antioxidantes y antifúngicos a partir de subproductos de pescado. Se identificaron ácidos fenólicos como el ácido DL-3-fenil-láctico y el ácido benzoico tras fermentar caldos elaborados con carne de pescado y subproductos durante diferentes tratamientos. Los extractos obtenidos de la fermentación de subproductos de pescado por bacterias aisladas del colon y del estómago mostraron la mejor actividad antioxidante. Además, demostraron actividad antifúngica frente a diferentes cepas de hongos de los géneros Penicillium, Aspergillus y Fusarium. Para optimizar la extracción de compuestos bioactivos de subproductos de pescado, se emplearon pulsos eléctricos (PE). Este pretratamiento mejoró la extracción de proteínas y compuestos antioxidantes de la cabeza y la piel del pescado, aunque no mejoró la extracción en el caso de las vísceras. Los PE alteraron la distribución del peso molecular de las proteínas extraídas y de la recuperación de minerales, la cual varió según el subproducto y mineral estudiado. Por otra parte, la exposición de los extractos obtenidos de la cabeza de pescado en cultivos de células SH-SY5Y mostraron un aumento en la viabilidad celular. Además, se estudió el efecto de los PE en la recuperación de compuestos bioactivos de la microalga espirulina (Arthrospira platensis). Se probaron diferentes tiempos de extracción y disolventes, obteniéndose mejores resultados con tiempos de extracción cortos en la extracción de clorofilas, carotenoides y compuestos fenólicos totales en los primeros minutos de extracción. La eficiencia de la extracción con fluidos supercríticos (EFS) en la recuperación de compuestos de la espirulina también fue evaluada. La EFS mejoró la extracción de ficocianina, clorofila, carotenoides y minerales en comparación con la extracción convencional. Además, se observó una mayor recuperación de ácidos grasos específicos. El extracto obtenido con EFS también mostró una mejora en la viabilidad celular cuando se expuso a las células SH-SY5Y. Siguiendo con el estudio de las microalgas, se evaluó cómo la adición de espirulina y chlorella (Chlorella vulgaris), además de diversas legumbres, a productos cárnicos procesados afecta a los parámetros fisicoquímicos y nutricionales. Se observó un cambio significativo en los parámetros de color tras la incorporación de proteínas de microalgas y un aumento del contenido total de aminoácidos con la inclusión de proteínas de espirulina. Finalmente, se estudió la actividad citoprotectora de los extractos obtenidos en cultivos de células SH-SY5Y expuestos a la micotoxina citrinina (CIT). Se evaluaron los efectos citotóxicos de la CIT expuesta de forma individual y combinada con los extractos. Se compararon los resultados en dos modelos in vitro, cultivos de células SH-SY5Y en monocapa (2D) y esferoides (3D) y un modelo in vivo, embriones de pez cebra (Danio rerio). Los resultados demostraron que la CIT afectó el potencial de membrana mitocondrial, indujo apoptosis, detuvo el ciclo celular y causó alteraciones en el retículo endoplásmico en células SH-SY5Y. En el modelo in vivo de embriones de pez cebra, la exposición a altas concentraciones de CIT provocó malformaciones. La exposición combinada de CIT con los extractos optimizados de cabeza de lubina obtenidos mediante PE y extracto etanólico de espirulina, mostró un aumento de la viabilidad celular y una reducción en la apoptosis en comparación con la exposición a CIT sola en células SH-SY5Y.This doctoral thesis focuses first of all on the use of fermentation with lactic acid bacteria isolated from fish to obtain antioxidants and antifungals from fish side streams. Phenolic acids such as DL-3-phenyl-lactic acid and benzoic acid were identified after fermenting broths made with fish meat and side streams during different treatments. The extracts obtained from the fermentation of fish side streams by bacteria isolated from the colon and stomach showed the best antioxidant activity. In addition, they demonstrated antifungal activity against different strains of fungi from the Penicillium, Aspergillus, and Fusarium genera. To optimise the extraction of bioactive compounds from fish side streams, pulsed electric fields (PEF) were used. This pre-treatment improved the extraction of proteins and antioxidant compounds from the head and skin of the fish, although it did not improve extraction in the case of the viscera. The PEF treatment altered the molecular weight distribution of the extracted proteins and the recovery of minerals, which varied according to the by-product and mineral studied. On the other hand, the exposure of extracts obtained from the fish head in SH-SY5Y cell cultures showed an increase in cell viability. In addition, the effect of PEF on the recovery of bioactive compounds from the microalgae spirulina (Arthrospira platensis) was studied. Different extraction times and solvents were tested, with better results obtained with short extraction times in the extraction of chlorophylls, carotenoids, and total phenolic compounds in the first minutes of extraction. The efficiency of extraction with supercritical fluids (SFE) in the recovery of compounds from spirulina was also evaluated. The SFE improved the extraction of phycocyanin, chlorophyll, carotenoids, and minerals compared to conventional extraction. In addition, a higher recovery of specific fatty acids was observed. The extract obtained with SFE also showed an improvement in cell viability when exposed to SH-SY5Y cells. Continuing with the study of microalgae, it was evaluated how the addition of spirulina and chlorella (Chlorella vulgaris), in addition to various legumes, to processed meat products affects the physicochemical and nutritional parameters. A significant change in colour parameters was observed after the incorporation of microalgae proteins and an increase in the total amino acid content with the inclusion of spirulina proteins. Finally, the cytoprotective activity of the extracts obtained in SH-SY5Y cell cultures exposed to the mycotoxin citrinin (CIT) was studied. The cytotoxic effects of CIT exposed individually and combined with the extracts were evaluated. The results were compared in two in vitro models, SH-SY5Y cell cultures in monolayer (2D) and spheroids (3D), and an in vivo model, zebrafish embryos (Danio rerio). The results showed that CIT affected the mitochondrial membrane potential, induced apoptosis, stopped the cell cycle, and caused alterations in the endoplasmic reticulum in SH-SY5Y cells. In the in vivo model of zebrafish embryos, exposure to high concentrations of CIT caused malformations. The combined exposure of CIT with the optimised extracts of sea bass head obtained by PEF and ethanolic extract of spirulina, showed an increase in cell viability and a reduction in apoptosis compared to exposure to CIT alone in SH-SY5Y cells

Extraction of Antioxidant Compounds and Pigments from Spirulina (Arthrospira platensis) Assisted by Pulsed Electric Fields and the Binary Mixture of Organic Solvents and Water

The application of pulsed electric fields (PEF) is an innovative extraction technology promoting cell membrane electroporation, thus allowing for an efficient recovery, from an energy point of view, of antioxidant compounds (chlorophylls, carotenoids, total phenolic compounds, etc.) from microalgae. Due to its selectivity and high extraction yield, the effects of PEF pre-treatment (3 kV/cm, 100 kJ/kg) combined with supplementary extraction at different times (5–180 min) and with different solvents (ethanol (EtOH)/H2O, 50:50, v/v; dimethyl sulfoxide (DMSO)/H2O, 50:50, v/v) were evaluated in order to obtain the optimal conditions for the extraction of different antioxidant compounds and pigments. In addition, the results obtained were compared with those of a conventional treatment (without PEF pre-treatment but with constant shaking). After carrying out the different experiments, the best extraction conditions to recover the different compounds were obtained after applying PEF pre-treatment combined with the binary mixture EtOH/H2O, 50:50, v/v, for 60–120 min. PEF extraction was more efficient throughout the study, especially at short extraction times (5–15 min). In this sense, recovery of 55–60%, 85–90%, and 60–70% was obtained for chlorophylls, carotenoids, and total phenolic compounds, respectively, compared to the maximum total extracted amount. These results show that PEF improves the extraction yield of antioxidant bioactive compounds from microalgae and is a promising technology due to its profitability and environmental sustainability

Sea bass side streams extracts obtained by pulsed electric fields: nutritional characterization and effect on SH-SY5Y cells

Fish side streams are an environmental and economic problem. In this work, pulsed electric fields (PEF) extraction was optimized and used as a new method for their valorization. Sea bass head, skin, viscera, and backbone were used. PEF technology (123-300 kJ/kg, 1-3 kV/cm) improved the extraction of proteins and antioxidant compounds from head and skin, but was not successful for viscera. SDS-PAGE showed that the protein molecular weight distribution was affected by the extraction process, revealing differences between the control and PEF extraction conditions. In addition, the extraction of macro-minerals and micro-minerals were also evaluated. The effect of PEF differed according to the matrix and the mineral studied. Heavy metals were also taken into account, studying the presence of As, Cd, Hg, and Pb in the extracts. PEF pre-treatment reduced the presence of As in skin, viscera, and backbone, ranging from 18.25 to 28.48% according to the matrix evaluated. The analysis of potential antioxidant bioactive peptides showed that the treatment of the sample directly influenced their variety. Additionally, the extracts obtained from the head were found to increase cell viability when tested on SH-SY5Y cells. In conclusion, PEF extraction can be a useful tool for the valorization of fish side streams

Improved Extraction Efficiency of Antioxidant Bioactive Compounds from Tetraselmis chuii and Phaedoactylum tricornutum Using Pulsed Electric Fields

Pulsed electric fields (PEF) is a promising technology that allows the selective extraction of high-added value compounds by electroporation. Thus, PEF provides numerous opportunities for the energy efficient isolation of valuable microalgal bioactive substances (i.e., pigments and polyphenols). The efficiency of PEF-assisted extraction combined with aqueous or dimethyl sulfoxide (DMSO) solvents in recovering pigments and polyphenols from microalgae Tetraselmis chuii (T. chuii) and Phaeodactylum tricornutum (P. tricornutum) was evaluated. Two PEF treatments were applied: (1 kV/cm/400 pulses, 3 kV/cm/45 pulses), with a specific energy input of 100 kJ/kg. The total antioxidant capacity (TAC) was positively influenced by the use of DMSO. The highest TAC in the T. chuii culture was achieved at a lower extraction time and electric field than for P. tricornutum. The use of DMSO only improved the polyphenols′ extraction for P. tricornutum, whereas the PEF and extraction time were more important for T. chuii. Carotenoids and chlorophyll a were more efficiently extracted using DMSO, while chlorophyll b levels were higher following aqueous extraction for both microalgae. In P. tricornutum, the TAC and pigment extraction efficiency were in general higher at lower extraction times. It can be concluded that PEF may be a promising alternative for the enhancement of the selective extraction of antioxidant bioactive compounds from microalgaepublishedVersio