Valorización de biomasa marina para el desarrollo de materiales biopoliméricos y aditivos con propiedades funcionales

Tesis por compendio[ES] Esta tesis doctoral se centró en la valorización de diferentes fuentes de biomasa marina para desarrollar materiales biopoliméricos e ingredientes destinados a la industria alimentaria. Así pues, en la primera parte de la tesis, se llevó a cabo la valorización de la biomasa de diversas especies de algas con el objetivo de identificar compuestos de interés y evaluar su potencial para la producción de materiales biopoliméricos e ingredientes alimentarios ricos en proteínas. Entre estas especies se encontraba R. okamurae, un alga parda invasora que representa una amenaza para los ecosistemas marinos. No obstante, se demostró que esta especie posee un gran potencial como fuente sostenible de compuestos bioactivos, transformando su papel de amenaza en oportunidad. La biomasa de las algas rojas también destacó por su potencial en diversas aplicaciones, incluyendo el desarrollo de materiales rentables y respetuosos con el medio ambiente obtenidos mediante de un procesado mínimo. En este contexto, la composición y la estructura de las paredes celulares de las algas desempeñaron un papel fundamental para entender cómo se comporta cada especie durante el procesado y en las propiedades de las películas resultantes. Además de la producción de materiales, también se estudiaron las algas rojas como fuente de proteínas para el consumo humano. Aunque las algas nativas presentaron una baja digestibilidad, esta mejoró mediante su procesado. No obstante, a pesar de esta mejora en la digestibilidad, el procesado podía inducir la degradación de los aminoácidos más lábiles, lo que repercutía negativamente en la calidad nutricional de las proteínas. En la segunda parte de esta tesis, se exploraron varias especies de algas rojas para extraer agar de forma más eficiente mediante métodos simplificados, y se evaluó la idoneidad de los extractos obtenidos para desarrollar materiales biopoliméricos e hidrogeles para aplicaciones alimentarias. El grado de purificación del agar tuvo un impacto significativo en las propiedades finales tanto de las películas como de los hidrogeles. En el caso de las películas, las fabricadas con agar puro presentaron propiedades superiores; sin embargo, sufrieron cambios significativos en su estructura semicristalina durante el almacenamiento. Por el contrario, las películas derivadas de extractos de agar menos purificados mostraron una mayor estabilidad en el tiempo, sugiriendo su potencial como aditivo para reducir costes y mejorar la estabilidad de las películas de agar puro. En el caso de los hidrogeles, la purificación del agar condujo a la formación de hidrogeles con una resistencia y dureza más altas. Por otro lado, el protocolo de extracción simplificado produjo fracciones de agar menos purificadas que contenían compuestos adicionales como proteínas o polifenoles. Aunque esto dio lugar a hidrogeles menos rígidos y resistentes, podrían ser de interés para la producción de hidrogeles con propiedades bioactivas. En la tercera y última parte de la tesis, se abordó la valorización de los residuos industriales generados tras la extracción de alginato, con el propósito de obtener fracciones de celulosa mediante métodos simplificados para su aplicación en la producción de materiales biopoliméricos. En este contexto, se observó que los residuos de A. esculenta y S. latissima resultaron adecuados para la extracción de fracciones celulósicas, mientras que A. nodosum podría ser más interesante para la producción de extractos ricos en fucoidano. Las fracciones de celulosa con mayor pureza generaron películas con propiedades mecánicas y aspecto visual más deseables. En cambio, las fracciones menos purificadas presentaron una mayor barrera al vapor de agua. De acuerdo con los resultados, se determinó que la simple aplicación de un tratamiento alcalino puede producir fracciones de celulosa que dan lugar a películas con un equilibrio óptimo entre propiedades funcionales y eficiencia económica y medioambiental.[CA] Aquesta tesi doctoral es va centrar en la valorització de diferents fonts de biomassa marina para desenvolupar materials biopolimèrics i ingredients destinats a la indústria alimentària. Així doncs, en la primera part de la tesi, es va dur a terme la valorització de la biomassa de diverses espècies d'algues amb l'objectiu d'identificar compostos d'interés i avaluar el seu potencial per a la producció de materials biopolimèrics i ingredients alimentaris rics en proteïnes. Entre aquestes espècies es trobava R. okamurae, una alga marró invasora que representa una amenaça per als ecosistemes marins. No obstant això, es va demostrar que aquesta espècie posseeix un gran potencial com a font sostenible de compostos bioactius, transformant el seu paper d'amenaça en oportunitat. La biomassa de les algues roges també va destacar pel seu potencial en diverses aplicacions, incloent-hi el desenvolupament de materials rendibles i respectuosos amb el medi ambient obtinguts mitjançant d'un processament mínim. En aquest context, la composició i l'estructura de les parets cel·lulars de les algues van exercir un paper fonamental per a entendre el comportament cada espècie durant el processament i en les propietats de les pel·lícules resultants. A més, també es van estudiar les algues roges com a font de proteïnes per al consum humà. Encara que les algues natives van presentar una baixa digestibilitat, aquesta va millorar mitjançant el seu processament. No obstant això, malgrat aquesta millora en la digestibilitat, el processament podia induir la degradació dels aminoàcids més làbils, la qual cosa repercutia negativament en la qualitat nutricional de les proteïnes. En la segona part d'aquesta tesi, es van explorar diverses espècies d'algues roges per a extraure agar de forma més eficient mitjançant mètodes simplificats, i es va avaluar la idoneïtat dels extractes obtinguts per a desenvolupar materials biopolimèrics i hidrogels per a aplicacions alimentàries. El grau de purificació de l'agar va tindre un impacte significatiu en les propietats finals tant de les pel·lícules com dels hidrogels. En el cas de les pel·lícules, les fabricades amb agar pur van presentar propietats superiors; no obstant això, van patir canvis significatius en la seua estructura semicristal·lina durant l'emmagatzematge. Per contra, les pel·lícules derivades d'extractes d'agar menys purificats van mostrar una major estabilitat en el temps, suggerint el seu potencial com a additiu per a reduir costos i millorar l'estabilitat de les pel·lícules d'agar pur. En el cas dels hidrogels, la purificació de l'agar va conduir a la formació d'hidrogels amb una resistència i duresa més altes. D'altra banda, el protocol d'extracció simplificat va produir fraccions d'agar menys purificades que contenien compostos addicionals com a proteïnes o polifenols. Encara que això va donar lloc a hidrogels menys rígids i resistents, podrien ser d'interés per a la producció d'hidrogels amb propietats bioactius. En la tercera i última part de la tesi, es va abordar la valorització dels residus industrials generats després de l'extracció d'alginat, amb el propòsit d'obtindre fraccions de cel·lulosa mitjançant mètodes simplificats per a la seua aplicació en la producció de materials biopolimèrics. En aquest context, es va observar que els residus de A. esculenta i S. latissima van resultar adequats per a l'extracció de fraccions cel·lulòsiques, mentre que A. nodosum podria ser més interessant per a la producció d'extractes rics en fucoidans. Les fraccions de cel·lulosa amb major puresa van generar pel·lícules amb propietats mecàniques i aspecte visual més desitjables. En canvi , les fraccions menys purificades van presentar una major barrera al vapor d'aigua. D'acord amb els resultats, es va determinar que la simple aplicació d'un tractament alcalí pot produir fraccions de cel·lulosa que donen lloc a pel·lícules amb un equilibri òptim entre propietats funcionals i eficiència econòmica i mediambiental.[EN] This doctoral thesis focused on the valorization of different sources of marine biomass with the main purpose of developing biopolymeric materials and ingredients for the food industry. Thus, in the first part of the thesis, the valorization of biomass from different algae species was carried out to identify compounds of interest and assess their potential to produce biopolymeric materials and protein-rich food ingredients. Among these species was Ruguloperyx okamurae, an invasive brown seaweed that poses a threat to marine ecosystems. However, this species was shown to have significant potential as a sustainable source of bioactive compounds, transforming its role as a threat into a valuable opportunity. Red seaweed biomass was also highlighted for its potential in a variety of applications, including the development of cost-effective and environmentally friendly materials for food applications obtained through minimal processing. In this context, the composition and structure of the algal cell walls played a crucial role in understanding how each species behaves during processing and in the properties of the resulting films. In addition to the production of biopolymeric materials, red seaweeds were also studied as a source of proteins for human consumption. Although native seaweeds had low digestibility, this was improved through processing. However, it is important to note that, despite the improvement in digestibility, processing could induce the degradation of more labile amino acids, having a negative impact on the nutritional quality of the proteins. In the second part of this thesis, several species of red seaweeds were explored to extract agar more efficiently by simplified methods, and the suitability of the extracts obtained to develop biopolymeric materials and hydrogels for food applications was evaluated. It could be observed that the degree of agar purification had a significant impact on the final properties of both films and hydrogels. In the case of films, those made of pure agar exhibited superior properties; however, they underwent significant changes in their semicrystalline structure during storage. In contrast, those films derived from less purified agar extracts showed greater stability over time, suggesting the potential of less purified agars as additives to reduce costs and improve the stability of pure agar films. Regarding the hydrogels, agar purification led to the formation of hydrogels with significantly higher strength and hardness. On the other hand, the simplified extraction protocol produced less purified agar fractions containing other compounds such as proteins, polyphenols, and minerals. Although this resulted in less rigid and resistant hydrogels, this feature could be advantageous for the production of hydrogels with bioactive properties. In the third and last part of the thesis, the valorization of industrial wastes generated after alginate extraction was addressed, with the purpose of obtaining cellulosic fractions by simplified methods for their application in the production of biopolymeric materials. In this context, it was observed that the residues of Alaria esculenta and Saccharina latissima were suitable for the extraction of cellulosic fractions, while Ascophyllum nodosum might be more interesting for the production of fucoidan-rich extracts. Cellulosic fractions with higher purity produced films with more desirable mechanical properties and visual appearance. Conversely, less purified fractions presented a greater barrier to water vapor. According to the results, it was determined that the simple application of an alkaline treatment could produce cellulosic fractions that resulted in films with an optimal balance between functional properties and economic and environmental efficiency in biopolymeric material production.This work was financially supported by the “Agencia Estatal de Investigación” (PCI2018-092886 Grant) and co-funded by the European Union’s Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2) and by the grant TED2021-129711B-I00, funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. This work was done within the framework of a collaboration with HISPANAGAR S.L (Burgos, Spain). The research was funded by the Spanish Ministry of Science, Innovation and Universities through projects PID2020-114821RB-I00, PID2020-117744RJ-I00, as well as CEX2021-001189-S, funded by MCIN/AEI/10.13039/501100011033. The authors also acknowledge funding through project CIRCALGAE (Horizon Europe) under grant agreement 101060607. In all projects, funding from “ERDF A way of making Europe”, the “European Union” or the “European Union NextGenerationEU/PRTR” is also acknowledged.Cebrián Lloret, V. (2024). Valorization of Marine Biomass for the Development of Biopolymeric Materials and Additives with Functional Properties [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202961Compendi

Estudio de las propiedades fisicoquímicas y sensoriales de esferas de microalgas

[ES] En el presente trabajo se estudia el proceso de elaboración de un nuevo producto alimentario utilizando como base dos tipos diferentes de microalgas -Chlorella vulgaris y Arthrospira platensis-, elegidas debido a los beneficios que proporcionan sus propiedades a la salud humana. Además, estos beneficios se ven incrementados mediante la incorporación de batidos naturales a la formulación final del producto. Estos batidos están compuestos por una combinación de frutas y verduras como: zanahoria, pepino, naranja, manzana y apio que, aparte de elevar su valor nutricional, suponen un incremento en la intensidad de sabor del producto. El objetivo principal es que el producto final tenga las características propias del caviar en cuanto a su forma, textura y tamaño, empleando para ello la técnica de esferificación inversa. Esta técnica parte de una disolución base en la que se encuentra el batido natural, la microalga utilizada, goma xantana al 1’2% y gluconolactato al 2%. Esta disolución se vierte sobre una disolución de alginato al 0,5%, consiguiendo así pequeñas esferas gelificadas de tamaño constante que mantienen en estado líquido su interior. Para concluir, se realizan diferentes pruebas físico-químicas como: textura, color o viscosidad, para comprobar su estabilidad, además de un análisis sensorial con el fin de conocer el grado de aceptabilidad del producto final.[EN] The object of study of this dissertation is the elaboration process of a new alimentary product, using as base two different kinds of microalgae -Chlorella vulgaris y Arthrospira platensis-, chosen because of the great benefits that their properties give to human health. Besides, these benefits are increased due to the incorporation of natural shakes to the final formulation of the product. These shakes are made of some fruits and vegetables like carrot, cucumber, orange, apple and celery that, in addition to raising their nutritional value, increase flavour intensity of the product. The main objective is to obtain a final product to have the characteristics of caviar regarding its texture, shape and size, using for that purpose the reverse spherification technique. This method starts from a base dissolution containing the natural shake, the used microalgae, xantham gum at 1’2% and gluconolactate at 2%. This dissolution is poured over a solution of alginate at 0,5%, getting, thereby, small gelled spheres which maintain their liquid state inside and with a constant size. To conclude, different physicochemical tests are carried out, such as texture, colour and viscosity to check their stability, aside from a sensorial analysis in order to know the degree of acceptance of the final product.Cebrián Lloret, V. (2017). Estudio de las propiedades fisicoquímicas y sensoriales de esferas de microalgas. http://hdl.handle.net/10251/85375.TFG

Hidrogeles de agar para la encapsulación de bacterias probióticas: efecto de la incorporación de polifenoles

[ES] El objetivo de este trabajo de fin de máster fue evaluar distintos agares extraídos a partir del alga Gelidium sesquipedale con el fin de valorar su potencial para encapsular una cepa de probióticos con alto potencial para su uso en alimentación (cepa B. uniformis CECT 7771). Dichos agares fueron obtenidos mediante protocolos simples basados en agua caliente y sonicación y, como se pudo observar, la combinación de sonicación con agua caliente redujo hasta 4 veces el tiempo de extracción sin afectar significativamente a su rendimiento (aproximadamente 10-13%). También se evaluó la aplicación de un tratamiento previo con álcali con el fin de obtener un extracto de agar más puro, pero los rendimientos de extracción obtenidos fueron mucho más bajos (aproximadamente 2,5%), y estos agares no fueron eficaces en la protección de las bacterias encapsuladas. Por el contrario, la presencia de otros compuestos adicionales como las proteínas o los polifenoles en los extractos no purificados mejoraron la protección de las bacterias aumentando su supervivencia hasta 9 días después de la encapsulación. Adicionalmente, otro objetivo del trabajo era comprobar si la presencia de polifenoles en las muestras menos purificadas era la causante de la mejora en la protección de las bacterias, para lo que se adicionó a la matriz un polifenol modelo (una catequina). Los resultados mostraron, sin embargo, que la adición de catequinas tuvo un efecto negativo en la protección de las mismas.[EN] The objective of this master's thesis was to evaluate different agars extracted from Gelidium sesquipedale algae in order to assess their potential to encapsulate a probiotic strain with great potential for functional food applications (strain B. uniformis CECT 7771). These agars were obtained through simple protocols based on hot water and sonication. As it was observed, the combination of sonication with hot water reduced up to 4 times the extraction time without significantly affecting yield (approximately 10-13%). The application of a pre-treatment with alkali in order to obtain a pure agar extract was also evaluated, but the extraction yields obtained were much lower (approximately 2.5%), and these agars were not effective in the protection of encapsulated bacteria. On the contrary, the presence of other additional compounds such as proteins or polyphenols in the unpurified extracts improved the protection of the bacteria increasing their survival up to 9 days after the encapsulation. An additional objective of this study was to evaluate whether the presence of polyphenols in the less purified samples was the cause of the improvement in the protection of bacteria. In order to check this hypothesis, a model polyphenol (specifically a catechin) was added to the encapsulation matrix. However, the results showed that polyphenol addition had a negative impact on probiotic protection.Cebrián Lloret, V. (2019). Hidrogeles de agar para la encapsulación de bacterias probióticas: efecto de la incorporación de polifenoles. http://hdl.handle.net/10251/125066TFG

Valorization of alginate-extracted seaweed biomass for the development of cellulose-based packaging films

Seaweed residues from Alaria esculenta, Saccharina latissima and Ascophyllum nodosum after alginate extraction have been valorized to produce cellulose-based fractions with different purification degrees. The residues were mainly composed of carbohydrates (35–57%) and proteins (12–37%), Alaria and Saccharina being richer in cellulose and Ascophyllum richer in fucoidan. The lower cellulose content in the latter made it unsuitable for the extraction of cellulosic fractions. Self-supporting films were obtained from the cellulosic fractions from Saccharina and Alaria residues. While the higher cellulose purity films presented more desirable characteristics in terms of mechanical properties (with elastic moduli of ca. 5–7 GPa and elongation values of ca. 3–5%) and visual appearance, the presence of non-cellulosic components in the films from less purified fractions reduced their water sensitivity and promoted greater water barrier (with water permeability values of ca. 4–6 kg·m/s·m2·Pa). These results point towards the potential of a simple alkaline extraction to generate cellulose-based films from seaweed residuals with the best compromise between functional properties and economical and environmental efficiency.publishedVersio

Sustainable Bio-Based Materials from Minimally Processed Red Seaweeds: Effect of Composition and Cell Wall Structure

This study reports on the use of whole seaweed biomass to obtain bio-based films for food packaging applications. Specifically, four different species of agarophytes (Gelidium corneum, Gracilaria chilensis, Gracilaria tenuistipitata and Gracilariopsis longissima) were minimally processed by melt blending and compression molding, and the effect of their composition and cell wall structure on the final performance of the films was investigated. The seaweed biomass was mainly composed of carbohydrates (35–50%), but significant amounts of proteins and ashes were also detected. Temperature-resolved SAXS experiments and microscopy analyses evidenced that a higher temperature of 130 ℃ is required to promote the release of agar from the tougher cell walls from G. corneum and G. tenuistipitata. The higher cellulose content of G. corneum (ca. 15%) resulted in films with higher mechanical resistance and water vapor barrier capacity, while the higher agar content of G. chilensis improved the elongation capacity of the films. The results from this work evidence the potential of red seaweed biomass to generate food packaging materials in a cost-effective and environmentally friendly way.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was financially supported by Hispanagar. Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (2020024090 project).Peer reviewe

Improved performance of less purified cellulosic films obtained from agar waste biomass

The residues generated after the extraction of agar from Gelidium sesquipedale by means of a hot-water treatment, with (NaOH+HW residue) and without (HW residue) an alkali pre-treatment have been valorized to produce high performance cellulosic films. Both residues were mainly composed of structural carbohydrates (in particular, agar), ashes and lipids. The residual agar could only be completely removed by applying a two-step process based on bleaching and alkaline treatments. The application of the alkaline pre-treatment for the extraction of agar did not significantly affect the properties of the films produced from the extracted fractions, hence making the HW residue more sustainable and economically viable. The agar remaining in the less purified fractions had a positive effect on the performance of the films, improving their transparency, mechanical properties and water vapour barrier, outperforming benchmark biopolymers; in addition, these materials presented antioxidant capacity inhibiting the degradation of β-carotene.Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (2018022638 project). This work was financially supported by the “Agencia Estatal de Investigación” (PCI2018-092886 Grant) and co-funded by the European Union’s Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2).Peer reviewe

Exploring alternative red seaweed species for the production of agar-based hydrogels for food applications

Three different red seaweed species, Gelidium corneum and two Gracilaria species (Agarophyton chilensis, and Gracilariopsis longissima), were used to produce agar-based fractions through conventional and simplified extraction methods and their composition and gel-forming properties were evaluated. The use of an alkaline pre-treatment was effective in removing impurities such as proteins, lipids, and ash, while the agarose/agaropectin ratio in agar was not affected. This led to the formation of hydrogels with higher stiffness and strength. Surprisingly, the presence of semi-crystalline agaropectin in the agar fractions from the two Gracilaria species, especially G. longissima, promoted the formation of more densely packed and stronger hydrogel networks, with higher gelling temperatures and superior mechanical properties. Thus, these results suggest that G. longissima has the potential to be used as an alternative to the more widespread use of G. corneum for the production of agar hydrogels for food applications.This work was financially supported by Hispanagar. Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (proposal 2022025569). The authors also acknowledge funding from the Spanish ministry of Science and Innovation through project PID 2020-117744RJ-I00 and grant CEX 2021-001189-S as well as the European Commission through project CIRCALGAE (Horizon Europe) under grant agreement 101060607.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S)Peer reviewe

Agar-based packaging films produced by melt mixing: Study of their retrogradation upon storage

The ability of agar with different purification degrees to produce packaging films has been evaluated and the stability of the obtained materials after prolonged storage has been investigated. The less purified agar resulted in films with higher water vapor permeability and lower mechanical performance than pure commercial agar. However, the commercial agar film required the addition of a plasticizer to produce films that could be manipulated. It has also been observed that prolonged storage at 53 % RH results in a change in the semi-crystalline structure of the agar and in water-polysaccharide interactions. As a result, pure agar films undergo a rigidizing effect resulting in unmanageable films after 7 days of storage. The presence of glycerol improved the stability of the films by limiting the structural changes up to 14 days of storage. In contrast, the films from the least purified agar extract, seemed to be less affected by moisture, showing a higher stability during storage. This points to the potential of the less purified extract to be used as an additive to reduce costs and improve the storage stability of pure agar films.This work was financially supported by Hispanagar.Peer reviewe

Valorization of alginate-extracted seaweed biomass for the development of cellulose-based packaging films

Seaweed residues from Alaria esculenta, Saccharina latissima and Ascophyllum nodosum after alginate extraction have been valorized to produce cellulose-based fractions with different purification degrees. The residues were mainly composed of carbohydrates (35–57%) and proteins (12–37%), Alaria and Saccharina being richer in cellulose and Ascophyllum richer in fucoidan. The lower cellulose content in the latter made it unsuitable for the extraction of cellulosic fractions. Self-supporting films were obtained from the cellulosic fractions from Saccharina and Alaria residues. While the higher cellulose purity films presented more desirable characteristics in terms of mechanical properties (with elastic moduli of ca. 5–7 GPa and elongation values of ca. 3–5%) and visual appearance, the presence of non-cellulosic components in the films from less purified fractions reduced their water sensitivity and promoted greater water barrier (with water permeability values of ca. 4–6 kg·m/s·m2·Pa). These results point towards the potential of a simple alkaline extraction to generate cellulose-based films from seaweed residuals with the best compromise between functional properties and economical and environmental efficiency

Production of hybrid protein-polysaccharide extracts from Ulva spp. seaweed with potential as food ingredients

This work explores the production of hybrid protein-polysaccharide extracts from the green seaweed Ulva spp. with potential for food-related applications by means of a pH-shifting method combined by a pre-treatment step. The optimum solubilization (pH = 12) and precipitation (pH = 3) conditions were first determined to maximize protein extraction; however, the extraction yield (21%) was still quite low. The application of a short (5 min) ultrasound pre-treatment prior to the solubilization step was seen to effectively disrupt the cell walls and facilitate protein release, hence increased up to 2-fold the protein extraction yields, and achieving a maximum of 47%. Interestingly, preservation of the seaweed biomass by freeze-drying caused a collapse in the cell wall structure, making protein extraction more difficult and giving rise to lower extraction yields (3-fold lower as compared to the frozen seaweed). The obtained extracts were composed mostly of proteins (21–40%) and carbohydrates (29–51%). The most abundant amino acids (AA) were alanine, glycine, aspartic acid and glutamic acid and the essential AA content accounted for 37–41%, superior to other proteins of non-animal origin. On the other hand, ulvans were the major polysaccharides found in the extracts, which may confer them interesting rheological and bioactive properties. As an important point for their application in the food industry, the low levels of metals and inorganic compounds in the extracts suggested low risk for human consumption. These results evidence the potential of Ulva spp. as a source of proteins with high interest for the production of novel food products based on alternative proteins.This research work was financially supported by the grant TED2021-129711B–I00, funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. Irene Molina-Gilarranz was recipient of a pre-doctoral grant JAE Intro CSIC (JAEINT_22_00353). Cynthia Fontes-Candia was supported by a Margarita Salas Postdoctoral Fellowship from Universitat Politectica de Valencia, funded by Ministerio de Universidades and the Eropean Union-Next Generation EU.Peer reviewe