Desarrollo de un aditivo colorante natural a base de cianidina obtenido a partir de frutos de Arbutus unedo L.: optimización de la extracción y estudio de su aplicación en gofres

Mestrado em cooperação com a Universidade de SalamancaEl primer paso crítico de este estudio fue obtener las condiciones óptimas que maximizan la extracción de antocianinas a partir de frutos de Arbutus unedo L., mediante la comparación de dos técnicas extractivas asistidas por diferentes energías físicas, el calor y los ultrasonidos. Para ello, se aplicó una metodología de superficie de respuesta, utilizando el diseño compuesto central circunscrito de tres variables con cinco niveles. Se identificaron tres compuestos de antocianina por HPLC-DAD-ESI/MS, siendo cianidina-3-O-glucósido la molécula principal. La extracción asistida por calor resultó ser la técnica más efectiva, que produjo un rendimiento del 60,9 %, con un contenido total de antocianinas de 500,9 μg/g de fruto seco y 800,6 μg/g de extracto siguiendo unas condiciones de 5 min, 90 ºC y 80 % de etanol acidificado. Además, estos valores se mejoraron ligeramente al estudiar el efecto de la relación sólido/líquido en un formato de dosis-respuesta (de 5 a 150 g/L), en las condiciones óptimas de extracción, manifestando valores de extracción prácticamente constantes entre los 5 y los 40 g/L. Una vez conseguida la optimización de la extracción de antocianinas (principalmente cianidina-3-O-glucósido) a partir de frutos de Arbutus unedo L., se procedió a estudiar su potencial como aditivo natural colorante, con la finalidad de ser utilizado en la industria alimentaria como substituto de colorantes sintéticos, cuya demanda va en decadencia progresiva. En primer lugar, se analizaron las propiedades bioactivas del extracto, analizando sus capacidades antioxidantes, antimicrobianas y citotóxicas. Los resultados mostraron que el extracto optimizado presentaba importantes propiedades antioxidantes, así como actividades citotóxicas (en líneas celulares tumorales) y antimicrobianas significativas. Complementariamente, se evaluó su posible toxicidad, corroborándose efectos nulos. Una vez analizadas minuciosamente las características del extracto, se procedió a su estabilización. Para ello se sometió el extracto rico en antocianinas a las condiciones más adversas de conservación (solución acuosa) y se estudió su comportamiento teniendo en cuenta las principales variables que afectan a la misma (tiempo, temperatura y pH). Los valores utilizados como respuestas fueron las concentraciones de antocianinas de cada muestra, determinadas por HPLC-DAD. Se usaron ecuaciones matemáticas fenomenológicas para describir las respuestas, y se proporcionaron patrones para la estabilización del extracto rico en antocianinas en solución acuosa. Finalmente, el extracto se incorporó en gofres como caso práctico de estudio en productos de pastelería, con el objeto de evaluar su posible aplicación en matrices alimentarias. La estabilidad completa del perfil nutricional, los azúcares libres, los ácidos grasos y la actividad antioxidante se controlaron justo después de que los gofres se hornearan y tras 3 y 6 días de almacenamiento. Por lo tanto, los resultados obtenidos proporcionan información para: i) la posible aplicación industrial del extracto de frutos de A. unedo como fuente alternativa de antocianinas para producir un aditivo natural colorante con propiedades bioactivas; ii) predicciones de vida útil en condiciones específicas de temperatura y pH; y iii) la incorporación del extracto de A. unedo rico en antocianinas no solo no causó cambios significativos en el valor nutricional de gofres en comparación con las muestras control, sino que dicha incorporación añadió propiedades colorantes y bioactivas al producto original, lo que respalda su posible aplicación en la preparación de productos de pastelería y panadería.The first critical step of this study was to obtain the optimal conditions that maximize the anthocyanins extraction from fruits of Arbutus unedo L., by comparing two extractive techniques assisted by different physical energies, heat and ultrasounds. For this, a response surface methodology was applied, using the three-variable circumscribed central composite design with five levels. Three anthocyanin compounds were identified by HPLC-DAD-ESI/MS, being cyanidin-3-O-glucoside the major molecule. Heat assisted extraction proved to be the most effective technique, yielding a 60.9 %, with a total anthocyanin content of 500.9 μg/g of dry fruit and 800.9 μg/g of extract following conditions of 5 min, 90 °C and 80 % acidified ethanol. In addition, these values were slightly improved by studying the effect of the solid/liquid ratio under optimum conditions, in the dose-response format, showing constant extraction values between 5 and 40 g/L. Once the extraction optimization of anthocyanins (mainly cyanidin-3-O-glucoside) from fruits of Arbutus unedo L. was achieved, its potential as a natural coloring additive was studied to replace, in the food industry, artificial additives, whose demand goes in progressive decay. First, the bioactive capacities of the extract were analyzed by testing its antioxidant, antimicrobial and cytotoxicity properties, revealing high antioxidant properties, as well as significant cytotoxic (in tumor cell lines) and antimicrobial activities. Complementarily, the toxicity of the extract was evaluated, validating its non-toxic effects. Afterwards, a stability study of the extract was carried out, considering the main variables that could affect the extract (time, temperature and pH) under the most unfavorable conservation conditions (aqueous solution). Responses were measured by anthocyanin contents determined by HPLC-DAD. Mechanical and phenomenological equations were used to describe the responses, and patterns were provided for the stabilization of the rich extract in anthocyanins in aqueous solution. Finally, the extract was incorporated in waffles in order to study its possible application in food matrices, in this case, in a pastry product. A complete stability of the nutritional profile, free sugars, fatty acids and antioxidant activity were monitored right after the waffles were baked and subsequently with 3 and 6 days of storage. Therefore, the results obtained provide information for: i) the possible industrial application of A. unedo fruit extract as an alternative source of anthocyanins to produce a natural coloring additive with bioactive properties; ii) predictions of shelf life under specific conditions of temperature and pH; and iii) the incorporation of A. unedo extract rich in anthocyanins, did not only cause significant changes in the nutritional value of waffles in comparison with the control samples, but also added color and bioactive properties to the original product, supporting its potential application in the preparation of pastry and bakery products

Optimization and comparison of heat and ultrasound assisted extraction techniques to obtain anthocyanin compounds from Arbutus unedo L. Fruits

The goal of this study was to compare anthocyanin extraction from Arbutus unedo L. fruits, by using two techniques assisted by different energy sources, heat and ultrasound. In order to obtain the conditions that maximize anthocyanin extraction, a response surface methodology was applied using the circumscribed central composite design of three variables with five levels. Three anthocyanin compounds were identified by HPLC-DAD-ESI/MS, being cyanidin-3-glucoside the main molecule. Heat assisted extraction proved to be the most effective method at 5 min, 90 °C and 80% of ethanol, yielding 51.2% of extract, with a total anthocyanin content of 382.4 μg/g dried fruit, and 744.6 μg/g extract. These response values were slightly improved by studying the solid/liquid effect at the optimal conditions in dose-response format, showing steady extraction values from 5 to 40 g/L. Results show the possibility of using A. unedo fruits as a source of anthocyanin compounds for industrial applications.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), LA LSRE-LCM (POCI-01-0145-FEDER-006984), L. Barros contract and C. Caleja (SFRH/BD/93007/2013) grant. This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project NORTE- 01-0145-FEDER-023289: DeCodE and project Mobilizador Norte-01- 0247-FEDER-024479: ValorNatural®. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. To Xunta de Galicia for financial support for the post-doctoral researcher of M.A. Prieto.info:eu-repo/semantics/publishedVersio

Stability of a cyanidin-3-O-glucoside extract obtained from Arbutus unedo L. and incorporation into wafers for colouring purposes

An extract from Arbutus unedo fruits, rich in anthocyanins, was studied as a natural colorant with bioactive properties (antioxidant, antimicrobial and cytotoxic). The aqueous stability of the extract was monitored using the anthocyanins’ content as response (determined by HPLC-DAD) in function of time, temperature and pH. Aided by mechanistic/phenomenological models, the conditions that favours the stabilization of the extract were provided, highlighting the suitability of the colorant for pastry/bakery products. As a case study, the extract was incorporated into wafers and the changes on the nutritional profile, free sugars, fatty acids and antioxidant properties were monitored during 6 days of storage. The results provide information for: i) potential application of the rich extract in anthocyanins for producing a natural colorant with bioactive properties; and ii) shelf-life predictions. The extract incorporation did not cause changes in the nutritional components of wafers but added colorant and antioxidant properties.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros contract and C. Caleja (SFRH/BD/93007/2013) grant. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project NORTE- 01-0145-FEDER-023289: DeCodE and project Mobilizador Norte-01- 0247-FEDER-024479: ValorNatural®. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. To Xunta de Galicia for financial support for the post-doctoral researcher of M.A. Prieto.info:eu-repo/semantics/publishedVersio

Scientific basis for the industrialization of traditionally used plants of the Rosaceae family

© 2020 Elsevier Ltd Plants have been traditionally used for the treatment of different types of illness, due to biomolecules with recognised benefits. Rosaceae family is used in traditional Galician medicine. The following plants Agrimonia eupatoria, Crataegus monogyna, Filipendula ulmaria, Geum urbanum, Potentilla erecta and Rosa canina are usually found in treatments. The aim of this study is to perform an ethnobotanical review about the bioactive compounds of these plants and their different bioactivities, both studied in vitro and in vivo. The nature of the bioactive compounds is varied, highlighting the presence of different phenolic compounds, such as phenolic acids, flavonoids or tannins. Understanding the beneficial effects of the administration of the whole plant or target tissues from A. eupatoria, C. monogyna, F. ulmaria, G. urbanum, P. erecta and R. canina as well as those from their individual compounds could lead to the development of new drugs based on the use of natural ingredients.info:eu-repo/semantics/publishedVersio

Main bioactive phenolic compounds in marine algae and their mechanisms of action supporting potential health benefits

Given the growing tendency of consumers to choose products with natural ingredients, food industries have directed scientific research in this direction. In this regard, algae are an attractive option for the research, since they can synthesize a group of secondary metabolites, called phenolic compounds, associated with really promising properties and bioactivities. The objective of this work was to classify the major phenolic compounds, compare the effectiveness of the different extractive techniques used for their extraction, from traditional systems (like heat assisted extraction) to the most advance ones (such as ultrasound, microwave or supercritical fluid extraction); the available methods for identification and quantification; the stability of the enriched extract in phenolic compounds and the main bioactivities described for these secondary metabolites, to offer an overview of the situation to consider if it is possible and/or convenient an orientation of phenolic compounds from algae towards an industrial application.The research leading to these results was funded by FEDER under the program Interreg V Spain-Portugal by POPTEC, IBERPHENOL Project (ref. 0377-Iberphenol-6-E) and by NANOEATERS Project (ref. 0181-NANOEATERS-01-E) that supports the pre-doctoral work of C. Jimenez-Lopez; by MICINN supporting the Ramón&Cajal grant for M.A. Prieto (RYC-2017-22891); by Xunta de Galicia and University of Vigo supporting the post-doctoral grant for M. Fraga-Corral (ED481B-2019/ 096), and the pre-doctoral grants for A.G. Pereira (ED481A-2019/0228) and P. García-Oliveira (ED481A-2019/295); by the company AlgaMar (www.algamar.com) and Axudas Conecta Peme (Xunta de Galicia) supporting the IN852A 2018/58 NeuroFood Project that financed the pre-doctoral grant for C. Lourenço-Lopes; Ibero-American Program on Science and Technology (CYTED - AQUA-CIBUS, P317RT0003) and UP4HEALTH Project (H2020-BBI-JTI-2019) for financial support. Lucía Cassani thanks the Secretaría General Iberoamericana (SEGIB) - Fundación Carolina for a grant.info:eu-repo/semantics/publishedVersio

Evolution of flavors in extra virgin olive oil shelf-life

Extra virgin olive oil (EVOO) is one of the most distinctive ingredients of the Mediterranean diet. There are many properties related to this golden ingredient, from supreme organoleptic characteristics to benefits for human health. EVOO contains in its composition molecules capable of exerting bioactivities such as cardio protection, antioxidant, anti-inflammatory, antidiabetic, and anticancer activity, among others, mainly caused by unsaturated fatty acids and certain minor compounds such as tocopherols or phenolic compounds. EVOO is considered the highest quality vegetable oil, which also implies a high sensory quality. The organoleptic properties related to the flavor of this valued product are also due to the presence of a series of compounds in its composition, mainly some carbonyl compounds found in the volatile fraction, although some minor compounds such as phenolic compounds also contribute. However, these properties are greatly affected by the incidence of certain factors, both intrinsic, such as the olive variety, and extrinsic, such as the growing conditions, so that each EVOO has a particular flavor. Furthermore, these flavors are susceptible to change under the influence of other factors throughout the oil's shelf-life, such as oxidation or temperature. This work offers a description of some of the most remarkable compounds responsible for EVOO’s unique flavor and aroma, the factors affecting them, the mechanism that lead to the degradation of EVOO, and how flavors can be altered during the shelf-life of the oil, as well as several strategies suggested for the preservation of this flavor, on which the quality of the product also depends.The research leading to these results was funded by Xunta de Galicia supporting the Axudas Conecta Peme, the IN852A 2018/58 NeuroFood Project and the program EXCELENCIAED431F 2020/12; to Ibero-American Program on Science and Technology (CYTED—AQUA-CIBUS, P317RT0003) and to the Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBI-JTI-2019). The JU receives support from the European Union’s Horizon 2020 research and innovation program and the Bio Based Industries Consortium. The project SYSTEMIC Knowledge hub on Nutrition and Food Security, has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT), and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched in 2019 under the ERA-NET ERA-HDHL (nº 696295).info:eu-repo/semantics/publishedVersio

Optimization and stabilization ofa cyanidin-3-O-glucoside rich extract obtained from fruits of Arbutus unedo L..

Currently, there is a tendency for consumers to choose more natural foods [l]. Anthocyanins are secondary metabolites ofplants that have aroused industry attenüon for its coloring potential combined with confirmation ofvarious beneficiai efFects to human health [2]. However, due to their very sensitive chemical structure to several factors such as temperature, pH, oxygen and light, studies that allow its stabilization are indispensable [3]. This work aimed to study the stability of an extract obtained from Arbutus unedo L. fruits, considering 3 variables (time, temperature and pH) in aqueous solution by analyzing the content of anthocyanins. The extract was obtained by a heat-assisted extraction technique, with the optimal condiüons of 5 minutes, 90°C, 80% ethanol and 15 g/L solid/liquid ratio, obtained after an optimization process by applying a response surface methodology. The identification of three anthocyanin compounds (cyanidin- 3-0-glucoside as the major molecide) was performed by HPLC-DAD-ESI/MS, and revealed the foUowing responses, 60.9 %, with a total anthocyanin content of 500.9 ^ig/g of dry fruit and 800.9 ^ig/g of extract. Moreover, the extract was analyzed in terms of its antioxidant, antimicrobial and cytotoxic activity revealing a high bioactive potential. Finally, the anthocyanin-rich extract was stabilized by subjecting it to adverse storage conditions. Time, temperature and pH were the variables considered to evaluate the behavior of the extract and the concentration of anthocyanins from each sample wére the response factors used. In order to describe the responses, mechanical and phenomenological equations were used with a global performing fitting analysis from a multivariable perspective. The results showed that the three analyzed variables exert a direct influence on the responses obtained. It hás been found that increasing pH and T values cause an exponential increase in anthocyanin degradation. In the overall analysis of ali variables, it was concluded that the optimum conditions ofanthocyanins stability were verified at pH <3.5 and T <30 °C for a period of at least 72 h. At the industrial levei this type of studies is indispensable, in order to guarantee the production of functional extracts in the production of high-quality new foods. Therefore, it is essential to find the optimum conditions of its stability and processing.FCT, Portugal and FEDER under Programme PT2020 for financiai support to CIMO (UID/AGR/00690/2013), C. Caleja (SFRH/BD/93007/2013) and L. Barras contract; FEDER-Interreg Espana-Portugal programme for financiai support through the project 0377_Iberphenol_6_E; European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope ofProject NORTE-01-0145-FEDER-023289: DeCodE. Junta de Galicia for the financiai support to M.A. Prieto.info:eu-repo/semantics/publishedVersio

Extraction of phenolic compounds by high hydrostatic pressure from eight edible algae species from the North-West coast of Spain: process modelling and optimization

Two thirds of the word are covered by oceans and a vast majority of that upper layer are inhabited by photoautotrophic organisms such as algae. Algae are not only of high ecological, but also of great economic importance. The industrial exploitation of algae has suffered a boom in the last two decades, revealing a source of compounds relevant to a diverse range of sectors (agriculture, energy, food science, cosmeceutical, pharmacology, etc). In consequence, many possible applications have emerged, such as production of biofuels for energy production, as nutraceutical ingredients in fish meals, as fertilizer in landfill applications, anti-biofilm activity in food science, incorporation in cosmeceutical and pharmacological products due to the rich content in bioactive compounds, among others [1]. In this work, high hydrostatic pressure (HHP) was applied to the extraction of phenolic compounds from eight edible algae species (Table 1). The process was optimized by response surface methodology using a five-level central composite design combining the independent variables of processing time (t, 5-90 min), pressure (P, 10-600 MPa) and solvent (S, 0-100 % of ethanol, v/v) [2]. The individual and grouped phenolic compounds were analysed, and the extraction yield were used as response variables. The theoretical models were fitted to the experimental data, statistically validated, and used in the prediction and optimization steps. In general, the optimum extraction conditions for phenolic acids for all eight species analysed were found at shorter values of t, high values of P and high values of S. The identified phenolic compounds were also clustered according to the conditions that maximize their extraction. HHP was highlighted as a promising emerging technology to extract phenolic compounds from edible algae species using a green solvent and reduced extraction times. The analysis presented provides important data that allows the comparison between different extraction conditions, in terms of efficiency, and consequent related decision making. In an industrial level, these methodologies reduce costs related to energy, solvent consumption, equipment investment, etc. Achieving the optimal conditions and maximizing the responses is an important step to guide the choice of a suitable and sustainable process. In conclusion, the present study contributes in the valorisation of edible algae species, common in the North-West region of Spain, by the obtainment of rich extracts in phenolic compounds that potentially can be applied as ingredients in different industrial fields.To AlgaMar for the algae material provided (www.algamar.com). To MICINN for the financial support for the Ramón&Cajal researcher of M.A. Prieto. To University of Vigo for the financial support for the pre-doctoral researchers of A.G. Pereira and C. Jiménez López. The authors are also grateful to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio

Aplicação em waffles de um corante natural obtido de frutos de Arbutus unedo L.

Os corantes são um dos aditivos mais importantes em termos de marketing, uma vez que a cor pode influenciar diretamente as escolhas e preferências dos consumidores [1]. No entanto, têm sido descritos alguns efeitos indesejáveis de determinados corantes artificiais. Contrariamente, os corantes naturais (e.g. ricos em compostos antociânicos) são genericamente considerados inofensivos, para além de poderem proporcionar efeitos bioativos [2]. Neste estudo, um extrato rico em antocianinas, obtido a partir de frutos de Arbutus unedo L. (medronho), foi aplicado como corante natural num produto de pastelaria. Para a preparação do extrato, otimizou-se o processo de extração utilizando duas metodologias: extração assistida por calor e extração assistida por ultrassons, de forma a obter as condições que maximizam a concentração de antocianinas. Foi aplicanda uma metodologia de superfície de resposta, seguindo um delineamento experimental de 5 níveis e as respostas foram obtidas através de HPLC-DAD-ESI/MS, onde foram identificados três compostos antociânicos; a cianidina-3-glucósido foi a molécula maioritária. A técnica de extração assistida por calor demostrou ser o método mais eficaz para a extração de antocianinas, produzindo 51,2% de extrato, com um teor total de antocianinas de 382,4 μg/g fruto seco e 744,6 μg/g de extrato, nas condições ótimas de 5 minutos, 90ºC, 80% de etanol e 15 g/L de razão sólido: líquido. Posteriormente, o extrato foi incorporado em waffles, tendo-se avaliado a sua composição em macronutrientes, bem como em açúcares livres e ácidos gordos, ao longo de 6 dias de armazenamento. Os resultados obtidos demonstraram que a incorporação do extrato não causou alterações nos componentes nutricionais. Validam também a possibilidade de utilizar frutos de A. unedo como fonte de compostos antociânicos muito interessantes para utilização como corantes alimentares naturaisFCT, Portugal e ao FEDER no âmbito do programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013), bolsa de C. Caleja (SFRH/BD/93007/2013) e contrato de L. Barros; FEDER através do Programa Operacional Regional Norte 2020, no âmbito do Projeto Norte-01-0247-FEDER-024479: ValorNatural®; FEDER-Interreg España-Portugal no âmbito do Projeto 0377_Iberphenol_6_E. Xunta de Galicia pelo apoio financeiro a M.A. Prieto.info:eu-repo/semantics/publishedVersio

Aplicação em waffles de um corante natural obtido de frutos de Arbutus unedo L.

Os corantes são um dos aditivos mais importantes em termos de marketing, uma vez que a cor pode influenciar diretamente as escolhas e preferências dos consumidores [1]. No entanto, têm sido descritos alguns efeitos indesejáveis de determinados corantes artificiais. Contrariamente, os corantes naturais (e.g. ricos em compostos antociânicos) são genericamente considerados inofensivos, para além de poderem proporcionar efeitos bioativos [2]. Neste estudo, um extrato rico em antocianinas, obtido a partir de frutos de Arbutus unedo L. (medronho), foi aplicado como corante natural num produto de pastelaria. Para a preparação do extrato, otimizou-se o processo de extração utilizando duas metodologias: extração assistida por calor e extração assistida por ultrassons, de forma a obter as condições que maximizam a concentração de antocianinas. Foi aplicanda uma metodologia de superfície de resposta, seguindo um delineamento experimental de 5 níveis e as respostas foram obtidas através de HPLC-DAD-ESI/MS, onde foram identificados três compostos antociânicos; a cianidina-3-glucósido foi a molécula maioritária. A técnica de extração assistida por calor demostrou ser o método mais eficaz para a extração de antocianinas, produzindo 51,2% de extrato, com um teor total de antocianinas de 382,4 μg/g fruto seco e 744,6 μg/g de extrato, nas condições ótimas de 5 minutos, 90ºC, 80% de etanol e 15 g/L de razão sólido: líquido. Posteriormente, o extrato foi incorporado em waffles, tendo-se avaliado a sua composição em macronutrientes, bem como em açúcares livres e ácidos gordos, ao longo de 6 dias de armazenamento. Os resultados obtidos demonstraram que a incorporação do extrato não causou alterações nos componentes nutricionais. Validam também a possibilidade de utilizar frutos de A. unedo como fonte de compostos antociânicos muito interessantes para utilização como corantes alimentares naturais.FCT, Portugal e ao FEDER no âmbito do programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013), bolsa de C. Caleja (SFRH/BD/93007/2013) e contrato de L. Barros; FEDER através do Programa Operacional Regional Norte 2020, no âmbito do Projeto Norte-01-0247-FEDER-024479: ValorNatural®; FEDER-Interreg España-Portugal no âmbito do Projeto 0377_Iberphenol_6_E. Xunta de Galicia pelo apoio financeiro a M.A. Prietoinfo:eu-repo/semantics/publishedVersio