Characterization of an oncofetal chondroitin sulfate epitope in cancer

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)Plasmodium falciparum is a malaria parasite that can infect humans. When the parasite replicates inside the erythrocytes, they become susceptible to elimination by the immune system. However, to avoid this elimination, the parasite expresses adhesion proteins on the surface of infected erythrocytes (iEs). The adhesion proteins anchor erythrocytes to specific receptors in the host. Carbohydrate called chondroitin sulfate (CS) is present in different tissues in the human body. However, in the human placenta is present a CS with very specific characteristics called oncofetal-CS (of-CS). Erythrocytes infected by the malaria parasite express to the surface the VAR2CSA protein that specifically binds to of-CS. The link between VAR2CSA-CS causes placental malaria (PM), a disease that can be deadly. The placenta cells and cancer cells have some similar characteristics, such as: rapid cell growth, migration, cell invasion and specific CS. PM affects a large part of the world's population and so it is very important to find a possible vaccine. The characterization of the CS present specifically in the placenta and the characterization of its binding to the VAR2CSA protein is fundamental, however it is very difficult to characterize CS through the common methods. Cancer is also a deadly disease that affects a large number of the world's population. The characterization of CS in cancer cells would be very important in pathogenesis, treatment and diagnosis. In the present work a human placenta sample was used from which CS was extracted through various methods of tissue purification. Posteriorly, the CS of the sample was analyzed by analytical methods, such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS). The CS chain is a combination of many characteristics that makes it specific and difficult to characterize. However, through the work performed it has been found that 4-O sulphation in the CS chain is required for its binding to the VAR2CSA protein. This search also demonstrated that the CS binding fragment to the VAR2CSA protein shows little sulfation. This work aims to contribute to new studies of therapeutic compounds and to the diagnosis of cancer, as well as to possible anti-malaria vaccine.Plasmodium falciparum é um parasita da malária que pode infetar seres humanos. Quando o parasita replica dentro dos eritrócitos, eles tornam-se suscetíveis à eliminação pelo sistema imunológico. No entanto, para evitar esta eliminação, o parasita expressa proteínas de adesão na superfície de eritrócitos infetados. As proteínas de adesão ancoram os eritrócitos a receptores específicos no hospedeiro. Carboidratos designados de chondroitin sulfate (CS) estão presentes em diferentes tecidos no corpo humano. No entanto, na placenta humana está presente um CS com características muito específicas chamado oncofetal-CS (of-CS). Os eritrócitos infetados pelo parasita da malária expressam à superfície a proteína VAR2CSA que se liga especificamente a CS. A ligação entre VAR2CSA-CS provoca malária placentária, uma doença que pode ser mortal. As células da placenta e as células de cancro têm algumas características semelhantes, tais como: crescimento rápido de células, migração, invasão celular e CS com características específicas. A malária placental afeta uma grande parte da população mundial e por isso é muito importante encontrar uma possível vacina. A caracterização do CS presente especificamente na placenta e a caracterização da sua ligação à proteína VAR2CSA é fundamental, no entanto é muito difícil caracterizar as cadeias de CS através dos métodos comuns. O cancro também é uma doença mortal que afeta um grande número da população mundial. A caracterização de CS em células de cancro seria muito importante na patogênese, tratamento e diagnóstico. No presente trabalho foi utilizada uma amostra de placenta humana a partir da qual o CS foi extraído através de vários métodos de purificação de tecidos. Posteriormente, a amostra de CS foi analisada por métodos analíticos, tais como High Performance Liquid Chromatography (HPLC) e por Liquid Chromatography-Mass Spectrometry (LC-MS). A cadeia CS é uma combinação de muitas características o que a torna muito específica e difícil de caracterizar. No entanto, através do trabalho realizado verificou-se que a sulfatação de 4-O na cadeia CS é necessária para a sua ligação à proteína VAR2CSA. Esta pesquisa demonstrou que o fragmento de ligação de CS à proteína VAR2CSA apresenta pouca sulfatação. Este trabalho tem como objetivo contribuir para novos estudos de compostos terapêuticos e para o diagnóstico de câncer, bem como a possível vacina anti-malária

Extraction of chlorophylls from natural sources

The growing consumers’ concern for possible long-term adverse effects of artificial molecules commonly used in food industry has led to an increased interest in natural products. At the same time, there is a demand for a more eco-sustainable use of natural matrices, which justifies the search for byproducts that have no other application to be explored in the development of novel food products [1,2]. In this context, the present study was designed to exploit natural pigments, more specifically chlorophylls, from bioresidues (aerial parts of carrot and tomato) for the development of food colorants. These are the most abundant pigments in plants and present, beyond their great coloring capacity, several bioactive properties, which corroborates the importance of their application in foodstuff. In this work, different extraction methodologies and techniques (maceration, ME, and ultrasound-assisted, USE) were applied to the lyophilized aerial parts of carrot and tomato to maximize the chlorophyll extraction yield. For the extraction, green solvents were prioritized, namely water, ethanol (90%), and hexane. The parameters affecting the pigments recovery were varied for each technique, namely the time, power, and solvent for USE, and the time and solvent for ME. The extractions were performed protecting the samples from light and the results were monitored through the implementation of a new chromatographic method, HPLC coupled to a diode array detector (DAD) and mass spectrometry (MS), to determine the concentration of chlorophylls and the best procedure to be performed. Both aerial parts presented chlorophylls and derivatives in significant concentrations and extraction yields up to 88% for the ethanolic extracts. The applied chromatographic method revealed to be appropriate for the analysis of this class of pigments, allowing a good peak resolution and separation, but also characteristic TIC spectrum for the tentative identification of the compounds. Therefore, the results of the present study can be explored for the development of chlorophyl-based colorants from these bioresidues, but also from similar byproducts.To the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); National funding by FCT, P.I., through the individual scientific employment program-contract for C. Pereira, M.I. Dias, and L. Barros contracts and A.K. Molina PhD grant (2020.06231.BD). To FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E and TRANSCoLAB 0612_TRANS_CO_LAB_2_P; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural® and Project GreenHealth - Norte-01-0145-FEDER-000042info:eu-repo/semantics/publishedVersio

Extraction of anthocyanins from red raspberry for natural food colorants development: Processes optimization and in vitro bioactivity

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Heat (HAE)-and ultrasound (UAE)-assisted extraction methods were implemented to recover anthocyanins from red raspberry. Processing time, ethanol concentration, and temperature or ultrasonic power were the independent variables combined in five-level rotatable central composite designs coupled with response surface methodology (RSM) for processes optimization. The extraction yield and levels of cyanidin-3-O-sophoroside (C3S) and cyanidin-3-O-glucoside (C3G) were monitored by gravimetric and HPLC-DAD-ESI/MSn methods, respectively, and used as response criteria. The constructed theoretical models were successfully fitted to the experimental data and used to determine the optimal extraction conditions. When maximizing all responses simultaneously, HAE originated slightly higher response values (61% extract weight and 8.7 mg anthocyanins/g extract) but needed 76 min processing at 38â—¦ C, with 21% ethanol (v/v), while the UAE process required 16 min sonication at 466 W, using 38% ethanol (v/v). The predictive models were experimentally validated, and the purple-red extracts obtained under optimal condition showed antioxidant activity through lipid peroxidation and oxidative hemolysis inhibition, and antibacterial effects against food-related microorganisms, such as Escherichia coli and Enterococcus faecalis. These results highlight the potential of red raspberry extracts as natural food colorants with bioactive effects and could be exploited by industries interested in the production of anthocyanin-based products.info:eu-repo/semantics/publishedVersio

A História da Alimentação: balizas historiográficas

Os M. pretenderam traçar um quadro da História da Alimentação, não como um novo ramo epistemológico da disciplina, mas como um campo em desenvolvimento de práticas e atividades especializadas, incluindo pesquisa, formação, publicações, associações, encontros acadêmicos, etc. Um breve relato das condições em que tal campo se assentou faz-se preceder de um panorama dos estudos de alimentação e temas correia tos, em geral, segundo cinco abardagens Ia biológica, a econômica, a social, a cultural e a filosófica!, assim como da identificação das contribuições mais relevantes da Antropologia, Arqueologia, Sociologia e Geografia. A fim de comentar a multiforme e volumosa bibliografia histórica, foi ela organizada segundo critérios morfológicos. A seguir, alguns tópicos importantes mereceram tratamento à parte: a fome, o alimento e o domínio religioso, as descobertas européias e a difusão mundial de alimentos, gosto e gastronomia. O artigo se encerra com um rápido balanço crítico da historiografia brasileira sobre o tema

Enfoques integrados para el impulso socioeconómico de la producción y consumo sostenible de hongos de Montesinho

El Parque Natural de Montesinho representa un área de montaña con un patrimonio micológico único. Entre las aproximadamente doscientas setas comestibles que se encuentran en este espacio protegido, la mayoría se consumen desde la antigüedad por su exquisito sabor y valor nutritivo [1]. Sin embargo, la disponibilidad de estos productos de montaña está limitada por su estacionalidad y condiciones climáticas, cuestión que se intensifica con el cambio climático. Por otro lado, las prácticas insostenibles de recolección de hongos y el comercio ilegal de especies de alto valor también han sido prácticas comunes con impactos negativos en el ecosistema y la economía regional y nacional. Todas estas cuestiones, junto con la incapacidad de los recolectores locales para garantizar la autenticidad/inocuidad de las setas recolectadas, han llevado a muchos restaurantes a evitar su inclusión en sus menús, salvaguardando su negocio y la salud de los consumidores. Así, se propone la producción de apreciados hongos comestibles en ambiente controlado ex-situ. Se está realizando una extensa caracterización nutricional, química y biológica para asegurar la alta calidad de las especies producidas y la preservación de sus características originales. El desarrollo de un sello de calidad y seguridad, “Safe2Taste”, que garantiza la trazabilidad de toda la cadena de producción, tiene como objetivo aumentar la confianza/fidelidad de los consumidores en los productosA la Fundação para a Ciência e Tecnologia (FCT; Portugal) por el apoyo financiero FCT/MCTES a CIMO (UIDB/00690/2020 e UIDP/00690/2020), SusTEC (LA/P/0007/2020), proyecto Safe2Taste (MTS/BRB/ 0056/2020) y beca de doctorado A. Saldanha (2021.08346.BD); la financiación nacional para F.C.T. y P. I. y a través del contrato del programa de empleo científico institucional para J.P, L.B., M.I.D y C.P.info:eu-repo/semantics/publishedVersio

Blueberry juice as a nutritious and bioactive beverage to be included in novel food products

Blueberry ( Vaccinium myrtillus L.) is a very popular fruit, native from the northern hemisphere and consumed worldwide. It has been widely studied for being a rich source of bioactive compounds with recognized beneficial properties for Human health [1]. Therefore, several industrialized products, such as juices and derivatives, have been developed from blueberry fruit, aiming at most practical forms of consumption. In this sense, the present work aimed to analyse the blueberry juice in terms of nutritional value, following AOAC procedures, and chemical compositions, namely in free sugars, by HPLC-RI, organic acids, by UFLC-PDA, and phenolic compounds, by HPLC-DAD/ESI-MS. Moreover, the antioxidant capacity, by TBARS and OxHLIA assays, was also assessed to validate its bioactive properties. Regarding the nutritional value, carbohydrates were the major macronutrients found in blueberry juice, followed by ash and protein. In terms of free sugars, fructose and glucose were detected, with a higher concentration of fructose. Four organic acids were also identified in the juice, namely oxalic, quinic, malic, and shikimic acids, being quinic acid the most abundant one. In what concerns the phenolic composition, four phenolic acids (quinic acid, caffeic acid hexoside, cis 5- O- caffeoylquinic acid, and trans 5- O -caffeoylquinic acid) and six anthocyanins (cyanidin-3- O -glucoside, cyanidin-3- O -pentoside, peonidin-3- O glucoside, peonidin-3- O -pentoside, malvidin-3- O -glucoside, and malvidin-3- O -pentoside) were detected. As expected, considering its chemical composition, the juice also presented a strong antioxidant capacity, being able of inhibiting the lipid peroxidation and the oxidative hemolysis. The results obtained in the present study validate the nutritional and bioactive quality of the juice obtained from Vaccinium myrtillus L., justifying its application in the development of novel foodstuff.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for M.I. Dias and L. Barros contracts and A.K. Molina for her PhD grant (2020.06231.BD). To FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P; to ERDF through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Norte-01-0145-FEDER-000042.info:eu-repo/semantics/publishedVersio

Attalea speciosa mesocarp flour in-depth characterization and its application for the development of new bakery products

Non-conventional food plants (NFPs) present themselves as a viable and efficient alternative for replacing the food products we consume today. Presenting a huge nutritional, chemical, physical, and biological potential, these plants are usually abundant and not competitors with other vegetable matrices used for human consumption.1 Attalea speciosa (Mart. Ex Spreng - Babassu) is a palm tree from the botanical family Arecaceae found in Brazil, and its mesocarp (BM) represents about 20.4% of the fruit and most of its application it’s in the manufacture of animal feed.2 Thus, the present work aimed to deepen the study the nutritional characterization of BM by AOAC methods; the determination, by chromatographic methods, of free sugars (HPLC-RI), fatty acids (GC-FID), and organic acids content (UPLC-DAD); and the study of the phenolic profile (HPLC-DAD/ESI(MS) and bioactivities (antioxidant, antimicrobial, anti-inflammatory and cytotoxicity activities) of the hydroethanolic extracts. Furthermore, given the possible industrial application, bakery products (bread) were developed with 12, 18, and 24% substitutions of wheat flour by BM flour. BM flour presented low moisture, fat, ash levels, being carbohydrates the main macronutrients, and palmitic (C16:0) and stearic (C18:0) acids were the main fatty acids found in the sample. Regarding phenolic composition, nine phenolic compounds were tentatively identified, six flavan-3-ols (catechin and epicatechin derivatives) and three Oglycosylated flavonoids (quercetin derivatives). Specifically, the flavan-3-ols group represented 99% of total amount of phenolic compounds mainly due to the presence of β-type (Epi)catechin dimer. The hydroethanolic extract of BM showed a high antioxidant capacity to inhibit lipid peroxidation and high anti-hemolytic capacity, showing an IC50 99% and 75% more effective than the positive control used (Trolox) in these assays, respectively. Moreover, it was able to inhibit all the tumor cell lines tested (MCF-7, NCIH460, CaCo, and AGS), however, it showed some toxicity towards healthy cells of PLP2 and VERO lines. The results obtained for the antibacterial activity of the BM extract were lower when compared to the positive controls used (E211 and E224). Comparing the results obtained with 100% wheat flour products, it was noted that the formulations with substitutions by BM presented remarkable results, providing a reasonable increase in the PUFAs content (despite decreasing protein content), maintaining the antioxidant capacity with lower IC50 values than the positive control used, and not presenting hepatotoxic activity (first validation of this flour for the incorporation in food matrices). In general, the formulation B24 was the one that presented greater similarity to the control bread (B0), relatively to the nutritional aspects, however, concerning the physical parameters it was the one that presented bigger discrepancy, especially regarding the specific volume, texture, and the color parameter L* (luminosity), tending for a darker and opaque coloration. All the formulations presented higher percentages of loss of rheological characteristics in the first three days of elaboration, however, formulation B24 was the one that presented the lowest percentage. This study presented innovative results regarding the nutritional, chemical and bioactive characterization of both the babassu mesocarp itself and the bread made from it, showing great potential to be applied in the food industry, however, there is still much to explore, especially in preparations that do not yet use non-conventional ingredients.The authors thanks to the Fundação para a Ciência e Tecnologia (FCT; Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020), the national funding for F.C.T. and P.I. and through the institutional scientific employment program contract for C.P., M.I.D, and L.B. To ERDF through the Regional Operational Program North 2020 within the scope of the project GreenHealth-Norte-01-0145-FEDER-000042. To Serbian Ministry of Education, Science and Technological Development (Contract No. 451-03-9/2021-14/200007).info:eu-repo/semantics/publishedVersio

Caracterización detallada de la harina del mesocarpio de Attalea speciosa y su aplicación para el desarrollo de nuevos productos de panadería

Las plantas alimenticias no convencionales se presentan como una alternativa viable y eficiente para reemplazar los productos alimenticios que consumimos hoy. Presentando un enorme potencial nutricional, químico, físico y biológico, estas plantas suelen ser abundantes y no competidoras de otras matrices vegetales [1]. Attalea speciosa (Mart. Ex Spreng - Babassu) es una palmera que se encuentra en Brasil, y su mesocarpio (MB) representa cerca del 20,4% del fruto, presentando aplicabilidad en la fabricación de alimentos para animales [2]. Así, el presente trabajo tuvo como objetivo profundizar el estudio de la caracterización nutricional de MB por métodos de la AOAC; la determinación del contenido de azúcares libres (HPLC-RI), ácidos grasos (GC-FID), ácidos orgánicos (UPLC-DAD) y compuestos fenólicos (HPLCDAD/ ESI(MS); y propiedades bioactivas (antioxidante, antimicrobiana, antiinflamatoria, citotóxica y nefrotoxicidad) de los extractos hidroetanólicos. Además, se desarrollaron productos de panadería (pan) con 12, 18 y 24% de sustitución de harina de trigo por harina MB. La harina presentó bajos niveles de humedad, grasa y cenizas, siendo los carbohidratos los principales macronutrientes, y los ácidos palmítico y esteárico los principales ácidos grasos. Se identificó tentativamente nueve compuestos fenólicos, seis flavan-3-oles (99% de la cantidad total, sobre todo por la presencia del dímero de (epi)catequina tipo β) y tres flavonoides O-glicosilados. El extracto hidroetanólico mostró una alta capacidad antioxidante y antihemolítica, mostrando una IC50 de 99% y 75% más efectiva que el control positivo (Trolox), respectivamente. Además, fue capaz de inhibir todas las líneas celulares tumorales, per mostró cierta toxicidad hacia las células sanas. Para la actividad antibacteriana fueron obtenidas concentraciones mínimas inhibitorias inferiores a la de los controles positivos (E211 y E224). Finalmente, se observó que las formulaciones de pan con sustituciones presentaron un aumento razonable en el contenido de PUFAs, manteniendo la capacidad antioxidante con valores de IC50 más bajos que los del control positivo, y no presentando actividad hepatotóxica (primera validación de esta harina para la incorporación en matrices alimentarias). En general, la formulación B24 fue la que presentó mayor similitud con el pan control, en relación a los aspectos nutricionales, sin embargo, en cuanto a los parámetros físicos fue la que presentó mayor discrepancia (en volumen específico, textura, y luminosidad). Todas las formulaciones presentaron mayores porcentajes de pérdida de características reológicas en los primeros tres días de elaboración. Este estudio presentó resultados innovadores en cuanto a la caracterización nutricional, química y bioactiva tanto del propio mesocarpio de babasú como del pan elaborado a partir de él, mostrando un gran potencial para ser aplicado en la industria alimentaria, sin embargo, aún queda mucho por explorar, especialmente en preparaciones que todavía no use ingredientes no convencionalesA Fundação para a Ciência e Tecnologia (FCT; Portugal) por el apoyo financiero FCT/MCTES a CIMO CIMO (UIDB/00690/2020 e UIDP/00690/2020), SusTEC (LA/P/0007/2020) y beca de doctorado A. Saldanha (2021.08346.BD); la financiación nacional para F.C.T. y P. I. y a través del contrato del programa de empleo científico institucional para C.P., M.I.D y L.B. A FEDER a través del Programa Operativo Regional Norte 2020 (GreenHealth-Norte-01-0145-FEDER-000042. Al Ministerio de Educación, Ciencia y Desarrollo Tecnológico de Serbia (Contrato No. 451-03-9/2021-14/200007).info:eu-repo/semantics/publishedVersio

Integrated approaches for socio-economic boosting of the sustainable production and consumption of Montesinho mushrooms

Montesinho Natural Park (MNP) represents a mountain area with a unique mycological heritage. Among the approximately two hundred edible mushrooms found in this protected area, most of them have been consumed since ancient times for their exquisite taste and nutritional value. 1 Nevertheless, the availability of these mountain products is limited by their seasonality and weather conditions, an issue intensified by climate change. On the other hand, unsustainable mushroom collection practices and illegal trade of high-value species have also been common practices with negative impacts in the ecosystem and regional and national economy. All these issues, together with the incapacity of local collectors to guarantee the authenticity/safety of the collected mushrooms, have led many restaurants to avoid their inclusion in their menus, safeguarding their business and consumer’s health. Thus, it is proposed the production of appreciated edible mushrooms in controlled ex-situ environment. An extensive nutritional/chemical/biological characterization are being performed to ensure the high quality of the produced species and the preservation of their original characteristics. The development of a quality and safety seal, “Safe2Taste”, that guarantee the traceability of the entire production chain, aims to increase consumers’ confidence/loyalty on the products.To the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020) LAQV-REQUIMTE (UIDB/50006/2020), and Safe2Taste project (MTS/BRB/0056/2020), A. Saldanha PhD grant (2021.08346.BD) and national funding by FCT, P.I., through the institutional scientific employment program-contract for L. Barros, M.I. Dias, J. Pinela, and C. Pereirainfo:eu-repo/semantics/publishedVersio

Red raspberry waste as a source of anthocyanin-rich food colorants: extraction process optimization and functionality assessment

Food colorants are increasingly used in the food industry to preserve, improve or change the food color. While the quite controversial artificial colorants are widely used in this sector, the natural counterparts have been less selected in part due to the limited availability of options and stability issues [1]. Within this class, anthocyanins are naturally occurring colorants that can be found in different plant matrices, including berries such as red raspberry (​Rubus idaeus ​L.). These water-soluble pigments show attractive colors ranging from red to purple and present health-promoting effects [2,3]. Therefore, this work aimed to develop a novel anthocyanin-rich food colorant from red raspberry waste through the optimization of a sustainable extraction methodology and to characterize this ingredient for its functionality. Heat (HAE)- and ultrasound (UAE)- assisted extraction methods were implemented to recover the anthocyanins from red raspberry. Processing time, ethanol concentration, and temperature or ultrasonic power were the independent variables analyzed in a central composite design coupled with response surface methodology for processes optimization. The extraction yield and levels of anthocyanins (cyanidin-3-​O​-sophoroside and cyanidin-3-​O​-glucoside) were monitored gravimetrically and by HPLC-DAD-ESI/MSn​ ​, respectively, and used as response criteria. The constructed theoretical models were successfully fitted to the experimental data and used to determine the optimal extraction conditions. Overall, HAE originated slightly higher response values (61% extract weight and 8.7 mg anthocyanins/g extract) but needed 76 min processing at 38 °C, with 21% ethanol, while the UAE process required 16 min sonication at 466 W, using 38% ethanol, to obtain 58% extract weightand 8.3mganthocyanins/gextract.Then,thepredictivemodelswereexperimentallyvalidatedand the purple red extracts obtained under optimal condition showed antioxidant activity through lipid peroxidation and oxidative hemolysis inhibition, and antibacterial effects against food-related bacteria, such as ​Escherichia coli ​and ​Enterococcus faecalis ​[4]. These results highlight the potential of red raspberry extracts as natural food colorants with bioactive effects. In future studies, it will be interesting to investigate the stability of the developed anthocyanin-rich extracts when exposed to different stability factors and in real food matrices.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020). This research was funded by FEDER-Interreg España-Portugal programme through the projects 0377_Iberphenol_6_E and TRANSCoLAB 0612_TRANS_CO_LAB_2_P, and by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte 01-0247-FEDER-024479: ValorNatural®. M. Añibarro-Ortega thanks FCT for his PhD grant (2020.06297.BD). National funding by FCT, P.I., through the individual and institutional scientific employment program-contract for the J. Pinela (CEECIND/01011/2018) and L. Barros contracts, respectively. The authors thank Ponto Agrícola Unipessoal, Lda for the samples.info:eu-repo/semantics/publishedVersio