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The 17 goals of sustainable development address several topics, such as: (2) Zero hunger and sustainable agriculture; (9) Industry, Innovation, and Infrastructure; (12) Responsible consumption and production; that are essential for the promotion of the circular economy, product development and conscious production [1]. Fig is a food matrix, cultivated in Portugal and valued by the Portuguese people for consumption in natura, being also used in wines, liqueurs, and jams. As this fruit is very appreciated and consumed, it´s cultivation leads to the production of tons of leaves, usually discarded [2]. Therefore, in the present work, the leaves of five fig varieties (Figure 1), namely Dauphine (Da), Longue d'Aout (La), Pasteliere (Pa), Marseille (Ma) and Bourjassote Noire (Bn), were nutritionally and chemically characterized to detect possible bioactive molecules. The antioxidant and antimicrobial, activities were also analyzed, to provide the food industry with natural additives in alternative to the artificial ones; and at the same time, promote the circular economy. Regarding the nutritional profile of the five leaves, La sample exhibited the highest amount in proteins (18.0±0.6 g/100g dw), while Pa revealed the highest content in fats (2.2±0.1 g/100g dw). The highest moisture content was presented by Da leaves (17.3±0.1 g/100g fw), and for the ashes, La sample was the one that presented the highest value (14.18±0.06 g/100g fw). Concerning the organic acids, these molecules were most abundant in Ma leaves, where it was possible to identify oxalic, malic and citric acids with a total of 139.6±0.4 mg/g dw. For the soluble sugars profiling, in all samples it was possible to identify five sugars, namely, fructose, glucose, sucrose, trehalose, and raffinose in different concentrations; however, Da leaves revealed the higher amount (17±1 g/100g dw). Tocopherols where also analyzed, and in all samples, three of the four isoforms where detected, being Pa sample standing for the predominance of these compounds (4.14±0.05 mg/100 g dw). For the bioactive analysis, different assays were performed, and Pa sample showed the strongest antioxidant potential for the TBARS assay, with an EC50 value of 105±5 mg/mL. For the antimicrobial activity assay, Da leaf extract was the one displaying the best results, by presenting Minimum Inhibitory Concentrations (MIC) ranging from 1.25 to 10 mg/mL against the tested bacterial strains. On the other hand, for the antifungal activity, the samples present very similar profiles, with the exception of the Pa sample, that present the lowest MIC of 5 mg/mL for Aspergillus fumigatus. In general, these leaf extracts can be used in the food industry namely in pastry and bakery products as promising sources of bioactive compounds, and at the same time, this reuse of biowaste promotes circular economy, and reduces the impact of biowaste resulting from the fig industry, thus meeting some of the goals of sustainable development. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). S.Heleno and M. Carocho thank FCT for their individual employment program–contract (CEEC-IND/00831/2018, CEECIND/03040/2017), and L. Barros also thanks to the national funding by FCT through the institutional scientific employment program–contract for her contract. the European Regional Development Fund (ERDF) through the Competitiveness and Internationalization Operational Program for financial support to the project 100% Figo (POCI-01-0247-FEDER-064977) and for C. Shirashi PhD grant. info:eu-repo/semantics/publishedVersion

Wild plants have received high importance at different locations and times of the human history given their ability to provide nutrients and protection[1]. In the Northeastern region of Portugal, a mountainous land with a vast biodiversity and cultural heritage, a large number of edible wild plants have become underutilized over time. This abandonment was mainly due to altered lifestyles of the modern society and massive utilization of a restrict number of crops. However, some wild species are now emerging in gardens and kitchens around Europe and increasingly found in farmers' markets, gourmet food shops, and restaurants[1]. Following this trend, this study aimed to characterize five species traditionally consumed as vegetable (Montia fontana L., Nasturtium officinale R. Br., and Rumex induratus Boiss. & Reut.) or as condiment/infusion (Pterospartum tridentatum (L.) Willk and Thymus pulegioides L.) in terms of nutrients and bioactive phytochemicals. Wild specimens were studied for their nutritional value following AOAC procedures; free sugars, fatty acids, tocopherols, and ascorbic acid were analysed by chromatographic techniques; and phenolic compounds were screened by colorimetric methods and characterized by HPLC-DAD-ESI/MS[2–5]. All plants revealed low protein contents. The highest levels of carbohydrates were found in the flowering parts of the two spice plants. Fructose and glucose predominated in all samples except in T. pulegioides in which sucrose prevailed. These low-fat foods had healthy fatty acid profiles mainly composed by α-linolenic acid, a precursor of n-3 polyunsaturated fatty acids. M. fontana and R. induratus were sources of ascorbic acid; a 100 g portion of R. induratus contain more than 50% of the recommended dietary allowances (RDA) of vitamin C for adults. R. induratus, T. pulegioides, and P. tridentatum had high α-tocopherol levels, with 100 g servings contributing > 30% of the RDA. Regarding bioactive non-nutrients, while P. tridentatum was found particularly rich in total phenolics (particularly flavonoids and dihydroflavonois), T. pulegioides showed the highest total flavonoids content. This work demonstrates that the selected wild edible plants have a healthy nutritional profile and can be considered as interesting contemporary gourmet foods. To FCT (Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); for the grants of M. Añibarro-Ortega (2020.06297.BD), A.K. Molina (2020.06231.BD) and C.L. Roriz (SFRH/ BD/117995/2016), the contracts of J. Pinela (CEECIND/01011/2018), C. Pereira, Â. Fernandes, and L. Barros through the scientific employment program-contract; to FEDER-Interreg España-Portugal programme for financial support through Project TRANSCoLAB 0612_TRANS_CO_LAB_2_P and to FEDER through the Regional Operational Program North 2020, under the Project GreenHealth, Norte-01-0145-FEDER-000042. info:eu-repo/semantics/publishedVersion

Nos últimos anos, consumidores e indústria têm procurado corantes naturais que sejam seguros e, simultaneamente, associados a efeitos benéficos para a saúde. Existem alguns corantes naturais autorizados pela EFSA, no entanto, não conseguem satisfazer todas as necessidades da indústria pela sua instabilidade, nomeadamente a alterações de temperatura, pH, luminosidade entre outros fatores [1]. Assim, a comunidade científica tem-se debruçado no estudo de matrizes naturais para obtenção de agentes com diferentes funções, nomeadamente função corante [2]. Entre as moléculas naturais com capacidade corante encontram-se as betacianinas, compostos pigmentados, com elevado poder corante, obtidas preferencialmente a partir de beterraba (tonalidades na gama dos vermelhos). Neste sentido, o presente trabalho visa a utilização de flores de Gomphrena globosa L. (perpétua roxa), como fonte de betacianinas para potencial aplicação na indústria alimentar [3]. As flores de G. globosa foram sujeitas a uma extração assistida por ultrassons previamente otimizada (t = 22 min, P = 300 W, Et = 0% e S/L = 5 g/L), obtendo-se 47 ± 5 mg gonfrenina/g de extrato. Os extratos obtidos foram liofilizados e incorporados em biscoitos para avaliação das suas características físicas nomeadamente: a) cor, com recurso a um colorímetro para análise das coordenadas L* = Luminosidade, a* = vermelho/verde e b* = amarelo/azul; b) textura, através de um texturómetro, analisando os parâmetros de dureza, adesividade, elasticidade, coesão, mastigabilidade e resiliência. Estes parâmetros foram analisados ao longo de um tempo de prateleira de 30 dias e comparados com: i) biscoitos sem corante, ii) biscoitos com extrato corante liofilizado; iii) biscoitos com extrato de corante comercial (E162). De acordo com os resultados obtidos nos parâmetros de avaliação de cor, verificou-se que o extrato de G. globosa liofilizado apresentou resultados mais promissores em termos de estabilidade (L *56; a *25.5; b *4.3), mantendo uma coloração rosa após a cozedura e durante os 30 dias de análise, enquanto que, o corante comercial E162 (L *60.7; a *23,2; b *18,1) sofreu alterações na capacidade corante, provocadas pela cozedura, no entanto, apresentou também grande estabilidade ao longo dos 30 dias de análise. Nos parâmetros de textura avaliados, as mudanças gerais na textura foram resultado de uma interação significativa entre o tipo de corante e a passagem do tempo. A dureza dos biscoitos diminuiu devido à retrogradação, enquanto os parâmetros de adesividade, elasticidade, coesão e resiliência aumentaram, mas não de uma forma significativa. Desta forma, com o decorrer deste trabalho foi possível concluir que o extrato obtido a partir de flores de G. globosa, é uma fonte de betacianinas com potencial para aplicação na indústria alimentar como corante natural. De realçar que este extrato apresenta uma tonalidade rosa diferente do corante comercial (avermelhado), pelo que pode ser considerado como possível candidato corante de tonalidade rosa para a indústria alimentar. Estão em curso mais estudos de estabilidade, de forma a garantir as melhores condições de armazenamento e aplicação deste extrato. Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portugal) e ao Fundo Europeu de Desenvolvimento Regional (FEDER), no âmbito do Programa PT2020, pelo apoio financeiro ao CIMO (UID/AGR/00690/2019); bolsa de doutoramento de C. L. Roriz (SFRH/BD/117995/2016), L. Barros e S. Heleno agradecem ao financiamento nacional pela FCT, I.P., no âmbito da celebração do contrato-programa de emprego científico institucional e individual, respetivamente. Este trabalho também foi financiado pelo FEDER, através do Programa Operacional Regional Norte 2020, no âmbito do projeto Mobilizador Norte-01-0247-FEDER-024479 (ValorNatural). M. Carocho agradece também ao ValorNatural pelo seu contrato. Os autores também agradecem ao programa FEDER-Interreg España-Portugal pelo apoio financeiro através do projeto TRANSCoLAB (0612_TRANS_CO_LAB_2_P). info:eu-repo/semantics/publishedVersion

Numerous plants have been used all over the world and in addition to the food properties, their consumption seems to be associated with different beneficial properties for consumers’ health. In this way, several scientific studies have been carried out with the objective of confirming these properties besides the assessment of their nutritional quality1. Hibiscus sabdariffa L., is an annual or perennial plant with red stems and calyces, belonging to the Malvaceae family. It is cultivated mainly in the tropical and subtropical areas of both hemispheres2. The present study aimed to perform a targeted metabolites’ analysis (free sugars, organic acids, tocopherols and fatty acids) of H. sabdariffa dried calyces (Guinea-Bissauan origin), as well as the individual phenolic composition of its infusion and hydroethanolic extracts. Free sugars were identified through an HPLC-RI system, the organic acids by UFLC-PDA, tocopherols by HPLC-fluorescence, fatty acids by GC-FID and the individual phenolic compounds were analysed through HPLC-DAD-ESI/MS. Glucose and quinic acid showed the major concentration for sugars and organic acids, respectively. Palmitic acid and α-tocopherol were the most abundant lipophilic compounds. In the individual phenolic profile, thirteen compounds were identified, five phenolic acids and flavonols, and three anthocyanins. The hydromethanolic extract presented all the identified compounds, while the infusion revealed the presence of twelve molecules, with the absence of caffeic acid. In general, the hydroethanolic extraction seems to have favoured the extraction of non-anthocyanin phenolic compounds, while the infusion was the ideal methodology for the anthocyanins’ extraction. Nevertheless, 3-O-caffeoylquinic acid was the major non-anthocyanin compound, while, delphinidin-3-O-sambubioside was the most abundant anthocyanin, in both extracts. This study demonstrates the high potential of this species highlighting its usage as a functional food or beverage, and as a source of a possible nutraceuticals and natural pigments, giving an added value for future applications in several industrial sectors. 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/00690/2019). C. L. Roriz thanks to FCT for the PhD grant (SFRH/BD/117995/2016).L. Barros thanks FCT, P.I., through the institutional scientific employment program-contract for her contract. The authors are also grateful to the Interreg España-Portugal for financial support through the projects 0377_Iberphenol_6_E and TRANSCoLAB 0612_TRANS_CO_LAB_2_P; European Regional Development Fund (ERDF) throug the Regional Operational Program North 2020, within the scope of project Mobilizador Norte- 01-0247-FEDER-024479: ValorNatural®, and to the Ministry of Education, Sciences and Technological Development of Republic of Serbia, grant No. 173032. info:eu-repo/semantics/publishedVersion

Betalains are a group of secondary metabolites named chromoalkaloids that are synthesized from tyrosine. These compounds have gained some attention in the last few years mainly due to their interesting bioactive potential, namely antioxidant, antimicrobial, and other bioactive properties [1]. Their strong and vibrant colours are also one of the characteristics by which these compounds have gained visibility in the food and pharmaceutical industries [2]. Betalains can be divided in two groups regarding the colour range: betaxanthins in the orange to yellow range, and betacyanins in the purple to pink range. Thereby, these compounds can be used as natural colouring agents, providing alternatives to the massively used artificial counterparts [3]. Although there are already some natural options in the market, these are not enough to meet the needs of the food industry, due to the growing concern of consumers regarding what they eat. Thus, the objectives of this work were to: i) obtain bioactive extracts and with strong colouring capacity from the epicarp of two pitaya varieties, white-fleshed pitaya Hylocereus undatus (Haworth) Britton & Rose (WFP) and red-fleshed pitaya Hylocereus costaricensis (F.A.C.Weber) Britton & Rose (RFP); ii) chemically characterize the betalains’ content through HPLC-DAD-ESI/MS; iii) evaluate the bioactive properties of the extracts, namely antimicrobial activity. The extracts were obtained through a dynamic maceration assisted by heat, and the betacyanins’ profile was characterized by HPLC-DAD-ESI/MS. The antibacterial capacity was determined against a panel of Gram-negative and Gram-positive bacteria using the colorimetric method of rapid detection with p-iodonitrotetrazolium chloride (INT), and the responses obtained were expressed as minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. By the chromatographic analysis of the extracts it was possible to identify six compounds in both samples. For the WFP the major compounds identified were 6'-O-malonylbetanin (phylocactin), followed by 4'-malonyl-betanin, while for the RFP the major compound found was phylocactin. In the evaluation of the antibacterial activity, both extracts showed MIC and MBC values that ranged from 10 to 20 mg/mL. Gram-positive bacteria showed to be more susceptible to both extracts then the negative strains. Further studies need to be conducted to better understand the correlation between the bioactive potential and the betacyanins’ composition. Nevertheless, these natural matrixes proved to be viable alternatives for obtaining colouring extracts with antimicrobial properties. To FCT and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019); C. Lobo Roriz (SFRH/BD/117995/2016) and T.C.S. Pires (SFRH/BD/129551/2017) grants and L. Barros contract. This work is funded by 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® info:eu-repo/semantics/publishedVersion

Betalains are a group of secondary metabolites named chromoalkaloids that are synthesized from tyrosine. These compounds have gained some attention in the last few years mainly due to their interesting bioactive potential, namely antioxidant, antimicrobial, and other bioactive properties [1]. Their strong and vibrant colours are also one of the characteristics by which these compounds have gained visibility in the food and pharmaceutical industries [2]. Betalains can be divided in two groups regarding the colour range: betaxanthins in the orange to yellow range, and betacyanins in the purple to pink range. Thereby, these compounds can be used as natural colouring agents, providing alternatives to the massively used artificial counterparts [3]. Although there are already some natural options in the market, these are not enough to meet the needs of the food industry, due to the growing concern of consumers regarding what they eat. To FCT and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019); C. Lobo Roriz (SFRH/BD/117995/2016) and T.C.S. Pires (SFRH/BD/129551/2017) grants and National funding by FCT- Foundation for Science and Technology, P.I., through the institutional scientific employment program-contract for L. Barros contract. This work is funded 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®. info:eu-repo/semantics/publishedVersion

A seletividade do consumidor na escolha de produtos alimentares contendo aditivos tem desafiado a indústria alimentar e os centros de investigação a encontrar novas fontes e métodos de extração eficientes para a produção sustentável de ingredientes de base natural com capacidade corante [1]. Nesta vertente, as plantas surgem como uma fonte promissora de pigmentos naturais bioativos com potencial aplicação no setor industrial. Hibiscus sabdariffa L. é um bom exemplo, pois trata-se de uma espécie cujo cálice detém uma elevada concentração de antocianinas [2]. Visando maximizar a recuperação destes compostos antociânicos a partir do cálice desta espécie para posterior utilização como corantes alimentares, este estudo foi delineado com o objetivo de otimizar dois métodos de extração, assistidos por calor (HE) e ultrassons (UE), utilizando a metodologia de superfície de resposta (RSM). Para otimização, foi implementado um desenho compostos central de cinco níveis combinando três variáveis independentes, nomeadamente tempo (t), percentagem de etanol (S) e temperatura (T) ou potência (P) do ultrassons. Os teores das antocianinas delfinidina-3-O-sambubiósido e cianidina-3-O-sambubiósido foram quantificados por LC-DAD-ESI/MSn e usadas como critérios de resposta para otimização por RSM. Os modelos teóricos foram ajustados através de uma equação polinomial de segunda ordem e as respostas maximizadas pelo método Simplex [3]. Após otimizar as condições para as variáveis t, T e S ou P, procedeu-se à otimização da razão sólido/liquido (S/L) para HE e UE. Os modelos desenvolvidos foram ajustados com sucesso aos dados experimentais e utilizados para determinar as condições ideais de extração. A UE foi mais eficiente, permitindo recuperar 23,83 mg de antocianinas por cada g de material vegetal seco e obter 51,76 mg desses pigmentos por cada g de extrato ao aplicar as condições de processamento ótimas: t = 26,1 min, P = 296,6 W e S = 39,1% etanol. A quantidade de antocianinas obtida através da extração UE foi cerca de 3 vezes superior à quantidade obtida por HE. Quanto à razão S/L, o incremento desta quarta variável aumentou os rendimentos de extração de antocianinas, atingindo um máximo ao utilizar 200 g/L [3]. Os resultados demonstram que H. sabdariffa é uma fonte de antocianinas viável para produzir corantes de base natural, através de uma metodologia economicamente exequível à escala industrial. Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portugal) e ao Fundo Europeu de Desenvolvimento Regional (FEDER), no âmbito do Programa PT2020, pelo apoio financeiro ao CIMO (UID/AGR/00690/2019) e ao FEDER, através do POCI-COMPETE2020 e da FCT, pelo suporte financeiro ao LA LSRE-LCM (POCI-01-0145-FEDER-006984); pela bolsa de doutoramento de Custódio Lobo Roriz (SFRH/BD/117995/2016), e pelos contratos de J. Pinela e L. Barros ao abrigo do CEEC institucional. Trabalho financiado por FEDER, através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289 (DeCodE) e do projeto Mobilizador Norte-01-0247-FEDER-024479 (ValorNatural). Também agradecem ao programa FEDER-Interreg España-Portugal pelo apoio financeiro através do projeto 0377_Iberphenol_6_E e ao TRANSCoLAB (0612_TRANS_CO_LAB_2_P). info:eu-repo/semantics/publishedVersion

In order to respond to the industry’s need for additives of natural origin, the exploration of new natural sources for different compounds has increased. Compounds with dyeing capacity are of great interest because, throughout the industrial processing of foods, they eventually see their initial color compromised.1 Thus, it is necessary to add coloring compounds, preferably of natural origin, to meet the demands of consumers, who are aware of the harmful effects that are attributed to some food additives of artificial origin. The addition of a natural colorant to a particular food has several advantages, besides imparting color, it also enriches the food, due to its intrinsic biological benefits. Betacyanins, for instance, are pigmented compounds that have a powerful pink color, being quite abundant in Gomphrena globosa L. flowers, which have strong coloring ability, in addition to their high antioxidant and chemopreventive effects2. In the present work, betacyanins were obtained from purple-colored flowers of G. globosa by ultrasound assisted extraction (UAE) and were further submitted to stabilization processes. The obtained coloring formulations were incorporated in cookies and the influence of this incorporation in the chemical profile regarding fatty acids (GC-FID), sugars (HPLC-RI) and tocopherols (HPLC-fluorescence) of the cookies were evaluated along a shelf-life of 30 days. Furthermore, the cookies were also analyzed for their physical characteristics through the evaluation of the color using a colorimeter (D65 illuminant), analyzing the L*, a* and b* coordinates, where L* represents lightness, a* represents the redness and b* the yellowness. Texture was analyzed with a TA.XT texturometer, evaluating the effects on the hardness, adhesiveness, springness, cohesiveness, chewiness and resilience over the 30 days. Cookies with a commercial colorant and ones with no colorant were used as control samples. As expected, the natural colorants incorporation caused no significant differences among the chemical analyses of the cookies, as the definition of colorants implies that beyond the color imparting no other parameter of the food should be altered. In terms of the cookie profiles, the most abundant individual fatty acid was palmitic acid (C16:0), followed by oleic (C18:1) and linoleic acid (C18:2) contributing to the major prevalence of the saturated fatty acids (SFA), followed by the monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids. Sucrose was the only detected sugar and corresponded to  48.5  2.0 g/100 g dw. Regarding the tocopherols, only three of the four isoforms were detected, namely α-, β-, and δ-tocopherol, being α-tocopherol the most abundant ( 29.2  0.8 mg/100 g dw), followed by δ-tocopherol ( 8.0  0.8 mg/100 g dw), and β-tocopherol ( 2.0  0.2 mg/100 g dw). Regarding the physical parameters, among the prepared formulations, the one stabilized by lyophilization presented a loss of the original color. Comparing the developed formulations with the pink commercial colorant, the natural ones showed a better pink color, since this commercial colorant displays a more reddish color, highlight the potential of the developed formulations as real pink colorants. Concerning the texture profile, no significant differences were verified between the samples over the shelf-life of 30 days, although hardness decreased due to retrogradation. The results demonstrated the strong potential of G. globosa as a promising source of betacyanins with a stable pink colour with high applicability in the food industry. 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/00690/2019); C. L. Roriz PhD grant (SFRH/BD/117995/2016), L. Barros, FCT, P.I., through the institutional scientific employment program-contract for her contract. The authors are also grateful to the Interreg España-Portugal for financial support through the projects 0377_Iberphenol_6_E and TRANSCoLAB 0612_TRANS_CO_LAB_2_P; 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. Carocho also acknowledges ValorNatural® for his contract. info:eu-repo/semantics/publishedVersion

Na última década, e devido à crescente preocupação dos consumidores, várias matrizes naturais têm sido utilizadas na procura de ingredientes corantes, alternativos aos artificiais, que têm sido associados a alguns efeitos tóxicos. As betacianinas são um grupo de compostos naturais com capacidade corante e têm sido amplamente utilizados na indústria alimentar, não só por causa do seu elevado poder de coloração, mas também devido às suas reconhecidas propriedades bioativas [l]. A principal fonte deste tipo de compostos é a beterraba, mas existem outras matrizes naturais, ainda pouco exploradas, que possuem na sua composição esta classe de moléculas. Exemplos disso são as espécies Gomphrena globosa L. e Amaranthus caudatus L., duas plantas da família das Amaranthaceae que apresentam flores com uma coloração viva, entre o rosa e o violeta, sendo as betacianinas os compostos responsáveis pelo seu efeito corante [2]. Assim, este trabalho teve como objetivo a obtenção de extraías ricos em pigmentos a partir das flores de G. globosa e A. caudatus, de forma a serem utüizados como corantes alimentares, oferecendo alternativas para a indústria alimentar que, para além de conferir cor, são ainda capazes de oferecer propriedades bioativas. Os extratos foram obtidos através de uma maceração dinâmica assistida por calor, sendo posteriormente caracterizados quanto ao seu perfil em betacianmas por cromatografia líquida de alta eficiência acoplada a um detetor de díodos e espetrometria de massa (HPLC-DAD-ESI/MS). A capacidade antimicrobiana foi determinada utüizando um painel de microrganismos Gram-negativo, Gram-positivo e fungos, utilizando um método colorimétrico de deteção rápida com cloreto de p-iodonitrotetrazolio (INT) e os resultados obtidos foram expressos em concentrações mínünas mibitórias do crescimento dos microrganismos (MIC). Pela análise cromatográfica dos extratos foi possível identificar quatro compostos em cada uma das amostras. Em G. globosa os compostos maioritários identificados foram a gonfrenina III (17.9 ± 0.8 mg/g extrato) e a isogonfrenina III (13.5 ± 0.7 mg/g extrato), e os minoritários a gonfrenina II (8.6 ± 0.5 mg/g extrato) e a isogonfrenina II (6.9 ± 0.5 mg/g extraio). O extrato de A. caudatus apresentou como compostos maioritários a amarantina (171 ± l mg/g extrato) e a isoamarantma (38 ± l mg/g), enquanto que a betanina (1.6 ± 0.1 mg/g extrato) e a isobetanina (1.3 ± 0. 1 mg/g extrato) foram encontrados em menor concentração. Na avaliação da atividade antimicrobiana o extrato de G. globosa apresentou MICs na ordem dos 0, 15-0,35 mg/mL, enquanto que o extrato de A. caudatus revelou MICs na ordem dos 10-20 mg/mL. Apesar de apresentarem diferenças no que diz respeito aos compostos que as constituem, estas espécies apresentam capacidade corante, pelo que ambas as plantas podem ser consideradas como fontes alternativas para obter ingredientes corantes de origem natural. FCT e FEDER sob o Programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013), bolsa de C.L. Roriz (SFRH/BD/117995/2016) e contrato de L. Barras; Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289: DeCodE e do Projeto Mobilizador Norte-01-0247- FEDER-024479: ValorNatural*. info:eu-repo/semantics/publishedVersion

Amaranthus caudatus L. is a plant belonging to the Amaranthaceae family, and is a very interesting crop, since it resists drought, heat, and pests, and adapts readily to new environments. The visual appearance of this plant is a bright red-violet, being betacyanins the compounds responsible for the color, and several bioactivities. Therefore, and despite being considered a good source of nutrients with high quality proteins, vitamins, minerals and bioactive molecules, such as phenolic compounds, A. caudatus can also be commercially exploited as a source of natural colorants. In the last decade, and due to the growing demand of consumers, natural matrices have been used to search natural colorant alternatives, in order to replace the artificial counterparts, which have been revealed some toxic effects. Betacyanins are natural colorants and have been used in the food industry, not only because of the high colouring power, but also due to recognized bioactive properties, such as antioxidant and antimicrobial, among others. Thus, the objective of this work was to obtain an extract rich in betacyanins to be used as food coloring agents, offering an alternative source of these coloring compounds, but at the same time conferring bioactive properties. A dynamic maceration extraction was carried out, being the extracts characterized by HPLC-DAD-ESI/MS regarding betacyanins’ composition. The antibacterial activity was evaluated against a panel of four Gram-positive and five Gram-negative bacteria, and the absence of cytotoxicity was confirmed in a porcine liver primary culture. By the chromatographic analysis it was possible to identify four compounds, being amaranthine (171 ± 1 mg/g extract) and isomaranthine (38 ± 1 mg/g) the two major compounds, and betanin (1.6 ± 0.1 mg/g) and isobetanin (1.3 ± 0.1 mg/g) the minor compounds. The extract presented minimum growth inhibitory concentrations (MICs) ranging from 20-10 mg/mL, with no cytotoxicity. After such promising results, this plant can be a viable alternative to obtain natural colorant ingredients. To the Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013); C. Lobo Roriz (SFRH/BD/117995/2016) grant and L. Barros contract. 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®. info:eu-repo/semantics/publishedVersion

Os efeitos nocivos de alguns aditivos sintéticos preocupam cada vez mais os consumidores, impulsionando uma tendência crescente para a sua substituição por compostos/extratos de origem natural. No presente trabalho, foram desenvolvidos dois protocolos de incorporação de extratos naturais em alimentos, visando obter efeitos de conservação e de funcionalização do produto final. O 1º protocolo consistiu na incorporação de um extrato obtido a partir da decocção de Foeniculum vulgare Mill. (funcho) e Matricaria recutita L. (camomila), na sua forma livre, em iogurtes, não se tendo verificado alterações significativas do seu valor nutricional e propriedades organolépticas, quando comparadas com amostras aditivadas com um conservante sintético (sorbato de potássio, E202). No entanto, verificou-se que os iogurtes incorporados com os extratos de funcho e camomila apresentaram atividade antioxidante superior. No 2º protocolo fez-se a incorporação de microesferas de alginato enriquecidas com o extrato obtido a partir da infusão de Fragaria vesca L. silvestre (morangueiro) em gelatinas. As microesferas produzidas apresentaram forma esférica, uma eficiência de encapsulação de 95% e capacidade de reidratação adequada. A dissolução da k-carragenina a temperaturas elevadas não afetou a integridade das microesferas. A atividade antioxidante dos extratos protegidos foi, assim, preservada comparativamente à sua forma livre. Este trabalho mostrou que os extratos aquosos de funcho e camomila apresentam potencial para o desenvolvimento de conservantes naturais, enquanto que os extratos de morangueiro silvestre são alternativas de interesse para a formulação de novos produtos nutracêuticos. The harmful effects of some synthetic additives are increasingly concerning some consumers, motivating a growing trend for their replacement by natural compounds/extracts. In the present work, two protocols were developed to incorporate natural extracts in food matrices to confer conservation and bioactive effects to the final product. The first protocol was the addition of an extract obtained from the decoction of Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (Chamomile), in the free form, in yogurts. No significant changes of their nutritional value and organoleptic properties were observed when compared with samples with a synthetic preservative (potassium sorbate E202). However, it was possible to confirm that yogurts incorporated with fennel and chamomile extracts exhibited higher antioxidant activity. The second protocol was the incorporation of alginate microspheres enriched with an extract obtained from the infusion of wild Fragaria vesca L. (strawberry) in gelatine. The microspheres produced presented spherical shape, encapsulation efficiency of 95% and proper rehydration. The dissolution of the k-carrageenan at high temperatures did not affect the integrity of the microspheres. The antioxidant activity of the protected extract was preserved when compared with the free form. Overall, this work showed the potential of aqueous extracts of fennel and chamomile to be used in the development of natural preservatives, while wild strawberry extracts are an interest alternative for formulating new nutraceuticals products. Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portugal) e ao FEDER no âmbito do Programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013), REQUIMTE (UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265), bolsa de C. Caleja (SFRH/BD/93007/2013), bolsa de C. Roriz (SFRH/BD/117995/2016) e contrato de L. Barros. Ao POCI-01-0145-FEDER-006984 - Laboratório Associado LSRE-LCM financiado pelo FEDER através do COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) - e por fundos nacionais através da FCT - Fundação para a Ciência e Tecnologia. info:eu-repo/semantics/publishedVersion

Devido à grande preocupação por parte dos consumidores em relação aos aditivos artificiais presentes nos alimentos, a procura de alternativas de origem natural tem vindo a ganhar uma grande importância. Este trabalho, focou-se na otimização de três técnicas de extração, extração assistida por calor (EAC), por micro-ondas (EAM) e por ultrassons (EAU) de flores de Gomphrena globosa L., aplicando uma metodologia de superfície de resposta (MSR), tendo-se estudado os efeitos das variáveis, tempo de processamento (t), temperatura (T) ou potência (P), concentração de etanol (%Et) e razão sólido/líquido (S/L). Como respostas foram utilizados o rendimento de extração e a concentração total de betacianinas determinadas por LC-DAD-ESI/MS. A técnica de extração que apresentou melhores resultados foi a EAU: t = 22 min, P = 500 W; %Et = 0%, e S/L = 5 g/L, conduzindo a um valor de betacianinas totais de 46,9 ± 4,8 mg/g [1,2]. Após ter sido determinada a melhor técnica de extração foi aplicada novamente a MSR no sentido de obter um extrato rico em betacianinas com elevada atividade antimicrobiana. As respostas foram avaliadas em termos das concentrações de extrato necessárias para obter os melhores valores de MIC (concentração mínima inibitória do crescimento) e MBC (concentração mínima bactericida). Os resultados indicaram as seguintes condições ótimas: t = 10,8 min, P = 410,5 W; %Et = 57,8%, e razão S/L = 5 g/L, fornecendo as seguintes respostas de MIC ~0,15 a 0,35 g/L e MBC ~0,30 a 0,65 g/L [3]. Este extrato rico em betacianinas e com elevada atividade antimicrobiana, foi incorporado num gelado, tendo-se verificado ao longo do tempo de prateleira (60 dias) a coloração desejada e sem alteração do seu perfil nutricional. Desta forma, foi possível com o extrato aquoso de flores de G. globosa atribuir a cor pretendida ao gelado e, simultaneamente, conferi-lhe capacidade antimicrobiana. FCT e FEDER sob o Programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013); bolsa de C.L. Roriz (SFRH/BD/117995/2016) e contrato de L. Barros; Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289: DeCodE e do Projeto Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®. info:eu-repo/semantics/publishedVersion

Ice cream is a complex food colloid formed of air bubbles, fat globules, ice crystals and a serum unfrozen phase [1]. Although presenting a complex composition, including milk, sweeteners, emulsifiers, stabilizers, colourants and flavouring agents, ice cream is very poor in some essential components such as natural antioxidants, dietary fibres, minerals and vitamins, all representing features desired by consumers [2]. Nevertheless, the addition of a natural food colourant to ice cream, besides fulfilling its main colouring function, might be an effective way to functionalize it, due to the antioxidant activity of most natural colourants (e.g., anthocyanins, carotenoids or betacyanins). Gomphrena globosa L. is a good example of an alternative source of colouring compounds of natural origin, namely betacyanins, which present a threefold higher colouring capacity than its commercial analogue (anthocyanins), additionally possessing antioxidant, antiviral, and antimicrobial properties [3]. Therefore, betacyanins extracted from the purple flowers of G. globosa were evaluated as colouring/functionalizing agents in icecream formulations, aiming to verify potential improvements on fatty acids profile, analysed by gas chromatography (GC) coupled to a flame ionization detector (FID). In order to acquire comprehensive conclusions, other ice-cream formulations (IF) were prepared and analysed in 5 storage times (ST). Besides the ice cream containing G. globosa extract, three formulations were prepared: control ice cream (without colourants), ice cream with commercial betalain and ice cream with Beta vulgaris extract (E-162). The profiles in fatty acids were especially sensitive to ST, which induced a significant effect in all cases except C18:1 (p=0.271) and MUFA (p=0.108), in contrast with IF, which was only significant in the cases of C14:0, C16:1, C18:1 and MUFA. In general, the new colouring approach allowed maintain the most relevant fatty acids unchanged. The authors are grateful to the Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), C. Lobo Roriz (SFRH/BD/117995/2016) grant and L. Barros and J.C.M. Barreira contracts. Authors also thank the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of NORTE-01-0145-FEDER-023289: DeCodE and ValorNatural® projects. info:eu-repo/semantics/publishedVersion

A aplicação de corantes em alimentos visa tornar os produtos mais atrativos para o consumidor. A curcumina, um diaril-heptanoide extraído dos rizomas de Curcuma longa Linn., apresenta coloração laranja-amarelada e elevado potencial para substituir corantes artificiais, compostos associados muitas vezes a efeitos colaterais adversos. A encapsulação da curcumina configura uma alternativa para ultrapassar a baixa solubilidade em água e controlar a coloração (dependente do pH). Neste estudo, utilizaram-se diferentes formulações de curcumina: i) forma pura (PC); ii) nanoencapsulada em poli(vinil pirrolidona) (PVP) pela técnica de dispersão sólida (NC); iii) forma dispersável em água, comercialmente disponível (DC). Estas formulações foram posteriormente utilizadas como agentes funcionais/corantes numa matriz de iogurte natural que foi posteriormente comparada quanto ao teor de ácidos gordos (GCFID) e açúcares livres (HPLC-RI) (controlo: formulação sem curcumina) para três tempo de armazenamento a 4ºC (TA: 0, 7 e 15 dias). No que concerne aos diferentes tipos de iogurte (TI: PC, NC e DC), e considerando os resultados obtidos para os três tempos, verificou-se que as diferenças foram apenas significativas para os teores de lactose (máxima para TI=PC), C10:0 (máximo para TI=DC), C12:0 (máximo para TI=DC) e MUFA (máximos nos iogurtes com PC). Igualmente, o efeito induzido por TA nos diferentes TI foi apenas significativo nos casos da lactose, C10:0 (máxima para T=15 dias), C12:0 (máxima para T=15 dias) e MUFA (máxima para T=0 dias). Não se registou qualquer diferença significativa, quer em TI quer em TA, para os teores de glucose, C6:0, C14:0, C16:0, C18:0, C18:1n9c, C18:2n6c, SFA e PUFA. A manutenção destes parâmetros valida o uso eficaz das nanocápsulas de PVP/ curcumina na funcionalização de iogurte, cujo perfil em açúcares livres e ácidos gordos, com especial destaque para o C18:1n9c e C18:2n6c, se mantém. FEEI através do NORTE 2020 (Projetos DeCodE (NORTE-01-0145-FEDER-023289), AIProcMat@N2020 (NORTE-01-0145-FEDER-000006) e Mobilizador ValorNatural®); FEDER através do POCI-COMPETE2020 e FCT (POCI-01-0145-FEDER-006984 (LSRE-LCM) e UID/AGR/00690/2013 (CIMO)). info:eu-repo/semantics/publishedVersion

A curcumina tem reconhecidas propriedades bioativas e corantes. Porém, a sua aplicação em produtos alimentares é dificultada pela sua baixa solubilidade em meios hidrofílicos. De forma a ultrapassar esta limitação, foram preparadas nanopartículas de poli(vinil-pirrolidona) (PVP, 40.000g/mol)/curcumina com base na técnica de dispersão sólida, utilizando Tween 80 como estabilizante e etanol como solvente. As nanopartículas foram caracterizadas quanto à morfologia (microscopia eletrónica de transmissão) indicando forma esférica e tamanho (dispersão dinâmica de luz) com índice de polidispersão = 0,050±0,009 e diâmetro médio em intensidade = 377±15 nm. As propriedades térmicas (análise termogravimétrica, TG), interações químicas (Espectroscopia de Infravermelho com Transformada de Fourier, FTIR). As análises por TG revelaram que as nanopartículas não apresentaram perda de humidade (não se observou nenhuma transição antes dos 100 ºC), justificada pelo facto de os pontos de ligação da PVP à água terem dado origem a interações com a curcumina na sequência da encapsulação [1]. A análise por FTIR comprovou este facto dado a ausência da banda característica a 3510 cm-1 na zona do estiramento dos OHs. As propriedades bioativas (atividade antioxidante, atividade anti-inflamatória e citotoxicidade) das nanopartículas de PVP/curcumina (NC) foram comparadas às da curcumina pura utilizada no procedimento de nanoencapsulação (PC) (Sigma Aldrich) e às de uma amostra comercial dispersável (DC) (CHR Hansen, Vegex Tumeric WS5). Os valores de EC50 (μg/mL) para a atividade antioxidante (DPPH, poder redutor, β-caroteno e TBARS) evidenciaram o seguinte perfil de atividade: PC>NC>DC, similarmente à resposta anti-inflamatória (concentração responsável por 50% da inibição da produção de óxido nítrico em comparação com o controlo negativo). Quanto à citotoxicidade, a PC revelou uma atividade superior em todas as linhas celulares, tendo os valores de GI50 (μg/mL) indicado atividade NC>DC apenas nos casos das linhas HeLa, HepG2 e PLP2 e indistinta nas MCF-7 e NCI-H460. Na etapa seguinte, as formulações PC, NC e DC foram aplicadas em iogurtes, para os quais se avaliou o perfil nutricional (humidade, cinzas, gordura, proteínas, hidratos de carbono e energia) e os parâmetros de cor (L*, a* e b*) ao longo do tempo de armazenamento (TA = 0, 7 e 15 dias, 4 ºC). Independentemente de TA, os iogurtes com DC apresentaram os teores de cinza, gordura, energia e L* superiores, enquanto os valores a* e b* foram superiores para os com PC. O teor máximo de proteína foi registado para os iogurtes com NC e o teor máximo de hidratos de carbono para as amostras controlo. Quanto a TA, as amostras armazenadas durante 7 dias caracterizaram-se por teores superiores de proteína, cinza (tal como as amostras armazenadas durante 15 dias), hidratos de carbono e energia, enquanto as amostras analisadas no dia de preparação registaram os valores mais elevados para gordura e a*. O efeito do tempo de armazenamento não foi significativo nos parâmetros L* e b*. Em síntese, e apesar de se terem observado algumas diferenças significativas nos indicadores nutricionais e parâmetros de cor, pode concluir-se que as nanopartículas de PVP/curcumina são uma boa estratégia para coloração do iogurte, não se tendo registado sem alterações relevantes do seu valor nutricional e aparência. Adicionalmente, e dado a bioatividade evidenciada, a sua utilização resultará em benefícios adicionais não conseguidos com a PC (baixa solubilidade em meio hidrofílico). FEEI através do NORTE 2020 (Projetos DeCodE (NORTE-01-0145-FEDER-023289), AIProcMat@N2020 (NORTE- 01-0145-FEDER-000006) e Mobilizador ValorNatural®); FEDER através do POCI-COMPETE2020 e FCT (POCI-01- 0145-FEDER-006984 (LSRE-LCM) e UID/AGR/00690/2013 (CIMO)). info:eu-repo/semantics/publishedVersion

There is a huge diversity of pigments from natural origin that can be exploited as colorants in the food industry. Nonetheless, they can additionally provide several bioactive properties, which represent an added-value for food products. Carotenoids, chlorophylls, anthocyanins and betalains are some of the most studied plant-based pigments. Betalains, can be subdivided according to their chemical structure into betacyanins and betaxanthins [1]. Although less exploited than Beta vulgaris L., Gomphrena globosa L. is an important source of betacyanins [2], with antimicrobial and antifungal activity [3]. Thus, the aim of this study was to obtain a pigmented extract from G. globosa with a high antimicrobial and antifungal activity. This objective was implemented by the application of the response surface methodology (RSM), a robust optimization technique that allows to study jointly the effects of several variables and responses, namely in extraction procedures (in this case ultrasound assisted extraction (UAE)). To evaluate the antimicrobial and antifungal properties, the responses were the minimum inhibitory (MIC), minimum bactericidal (MBC) and minimum fungicidal (MFC) concentrations. The optimized UAE conditions were: 10.8 min, 410.5 W, 57.8% ethanol content (ethanol-water mixtures were used) and 5 g/L as solid-liquid ratio, providing the following responses: 1) antibacterial activity: MIC of ~0.15-0.35 g/L) and MBC of ~0.30-0.65 g/L; and 2) antifungal activity: MIC of ~0.20-0.30 g/L and MFC of ~0.40-0.65 g/L. In conclusion, the obtained results evidenced the potential application of the extracts from G. globosa as sources of betacyanins (natural colorants), but also as important antimicrobial and antifungal agents. The Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013); Project POCI-01-0145-FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER through COMPETE 2020; C. Lobo Roriz (SFRH/BD/117995/2016) grant and L. Barros contract. 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 ValorNatural®. info:eu-repo/semantics/publishedVersion

Os efeitos nocivos de alguns aditivos sintéticos preocupam cada vez mais os consumidores, impulsionando uma tendência crescente para a sua substituição por compostos/extratos de origem natural. No presente trabalho, foram desenvolvidos dois protocolos de incorporação de extratos naturais em alimentos, visando obter efeitos de conservação e de funcionalização do produto final. O 1º protocolo consistiu na incorporação de um extrato obtido a partir da decocção de Foeniculum vulgare Mill. (funcho) e Matricaria recutita L. (camomila), na sua forma livre, em iogurtes, não se tendo verificado alterações significativas do seu valor nutricional e propriedades organolépticas, quando comparadas com amostras aditivadas com um conservante sintético (sorbato de potássio, E202). No entanto, verificou-se que os iogurtes incorporados com os extratos de funcho e camomila apresentaram atividade antioxidante superior. No 2º protocolo fez-se a incorporação de microesferas de alginato enriquecidas com o extrato obtido a partir da infusão de Fragaria vesca L. silvestre (morangueiro) em gelatinas. As microesferas produzidas apresentaram forma esférica, uma eficiência de encapsulação de 95% e capacidade de reidratação adequada. A dissolução da k-carragenina a temperaturas elevadas não afetou a integridade das microesferas. A atividade antioxidante dos extratos protegidos foi, assim, preservada comparativamente à sua forma livre. Este trabalho mostrou que os extratos aquosos de funcho e camomila apresentam potencial para o desenvolvimento de conservantes naturais, enquanto que os extratos de morangueiro silvestre são alternativas de interesse para a formulação de novos produtos nutracêuticos. The harmful effects of some synthetic additives are increasingly concerning some consumers, motivating a growing trend for their replacement by natural compounds/extracts. In the present work, two protocols were developed to incorporate natural extracts in food matrices to confer conservation and bioactive effects to the final product. The first protocol was the addition of an extract obtained from the decoction of Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (Chamomile), in the free form, in yogurts. No significant changes of their nutritional value and organoleptic properties were observed when compared with samples with a synthetic preservative (potassium sorbate E202). However, it was possible to confirm that yogurts incorporated with fennel and chamomile extracts exhibited higher antioxidant activity. The second protocol was the incorporation of alginate microspheres enriched with an extract obtained from the infusion of wild Fragaria vesca L. (strawberry) in gelatine. The microspheres produced presented spherical shape, encapsulation efficiency of 95% and proper rehydration. The dissolution of the k-carrageenan at high temperatures did not affect the integrity of the microspheres. The antioxidant activity of the protected extract was preserved when compared with the free form. Overall, this work showed the potential of aqueous extracts of fennel and chamomile to be used in the development of natural preservatives, while wild strawberry extracts are an interest alternative for formulating new nutraceuticals products. N/A

Plant-derived colorants have an extensive history of use around the world for enhancing food and clothing. However, artificial colorants are nowadays massively used in the food industry due to their abundance, cheaper prices, and bright colors. These additives are recognized for their potentially toxic and allergic effects to humans, and contribution to environmental pollution. This leads the current, more conscientious consumer to opt for foods containing natural coloring agents, which may also have health-promoting effects. A current challenge is to find natural matrices rich in molecules with coloring capacity and optimize their recovery and stabilization. A chemical prospecting study allowed us to select globe amaranth (Gomphrena globosa L.) as a rich source of betalains, a group of chromoalkaloids that can be divided in betacyanins (red-violet pigments) and betaxanthins (yellow pigments). Given the powerful colorant capacity of these molecules and the great interest to use natural colorants in the food industry to replace the artificial counterparts, this study aimed to optimize the production of a natural colorant from globe amaranth using efficient and more sustainable processes. The non-conventional methods of microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) were used for samples processing. For production of the coloring ingredients, a full factorial design was implemented combining the independent variables of processing time (t), temperature (T) or power (P), ethanol concentration (Et) and solid/liquid ratio (S/L) and response surface methodology was used for optimization. The extraction yield and coloring capacity of the extracts was maximized based on different responses: extracted residue, determined by gravimetric analysis; betacyanins levels, monitored by high-performance liquid chromatography coupled with mass spectrometry; and color intensity, measured by UV-Visible spectroscopy and colorimetry. The optimum processing conditions for MAE (t= 8 min, T= 60 ºC, Et= 0%, and S/L= 5 g/L) originated 39.6±1.8 mg/g, while for UAE (t= 22 min, P= 500%; Et= 0%, and S/L= 5 g/L) led to 46.9±4.8 mg/g, which supports the use of UAE for recovery of betacyanins from the studied plant. This study highlighted globe amaranth as an alternative source of red-violet colorants with high potential to be used as natural food additives. info:eu-repo/semantics/publishedVersion

The production of ice cream consists of two essential steps: firstly, the mixture is prepared and aerated, and afterwards it is frozen. This complex frozen dessert consists of several components, such as air bubbles (to give volume), water (in the form of ice crystals to confer weight), a partially frozen serum phase, and small amounts of emulsifiers, stabilizers and colourants [1]. Probably, ice cream is not the best product to answer to the growing health concerns, mainly due to the increased rate of obesity, metabolic syndrome and diabetes. This has been bothering consumers, leading them to seek healthier foods [2]. Nevertheless, ice cream is a very appreciated desert all over the world, representing a suitable food matrix to be submitted to any improvement process, such as the addition of natural additives with acknowledged bioactive properties. Currently, there is a trend to replace the artificial colourants used in ice cream preparation. The flowers of Gomphrena globosa L., contain high levels of betacyanins, which have a strong colouring capacity, in addition to their high antioxidant activity and chemopreventive effects [3]. Accordingly, aqueous extracts from G. globosa flowers were incorporated in ice cream, benefiting from their dual colouring/functionalizing effects. To validate their suitability, the prepared formulations were compared to other ice cream (i) free of any colourant, (ii) added with commercial betalain, or (iii) added with Beta vulgaris extract (E-162). Among other parameters, the individual sugar profiles were evaluated by high-performance liquid chromatography (HPLC) coupled to a refractive index detector (RI) throughout storage time (up to 60 days). Overall, the tested formulations did not induce significant changes in individual sugars profile, except for the lower sucrose contents detected in ice creams prepared with B. vulgaris extract. Therefore, the extracts of G. globosa might be considered as an effective colouring solution, without compromising individual sugars profile, one of the most important parameters to assess ice cream quality. The authors are grateful to the Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), C. Lobo Roriz (SFRH/BD/117995/2016) grant and L. Barros and J.C.M. Barreira contracts. Authors also thank the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of NORTE-01-0145-FEDER-023289: DeCodE and ValorNatural® projects. info:eu-repo/semantics/publishedVersion

Food appearance is as important as its taste. Colour preservation, for instance, is one of the top concerns throughout food processing [1] . Nowadays, the main trend is developing visually appealing foods, which simultaneously should contain enhanced functional properties. Products typically containing limited levels of bioactive compounds, such as the widely consumed ice cream, present better possibilities for improvement [2]. Ice creams are complex colloidal systems combining milk, sweeteners, emulsifiers, stabilizers, colouring and flavouring agents. These ingredients are often artificial, but they could be replaced by natural alternatives, fulfilling the current concern about cleaner labels. Colouring agents, in particular, are highly valued by consumers, who prefer natural compounds instead artificial ones, often perceived as being harmful [3]. In fact, some natural dyes are already available on the market, but other alternative sources could be exploited. Betalains, which can be divided in red-violet betacyanins and yellow-orange betaxanthins are a good example of compounds deserving higher attention. Betacyanins are usually derived from beetroot, but they can be obtained from different sources such as the purple flowers of Gomphrena g/abosa L. (Amaranthaceae) [4]. Accordingly, betacyanins from G. globosa were used as colouring agents in ice creams and further compared with other ice cream formulations (containing commercial betalain or free of any colouring agents). Besides the colouring strength, the nutritional parameters, individual sugars and fatty acids profiles were also compared. To obtain a thorough characterization, all parameters were evaluated in five different times: preparation day and after 15, 30, 45 and 60 days of storage. Nutritional parameters, except protein along storage time (ST) and ash among ice cream formulations (IF) showed significant differences either comparing different ST or IF. Nevertheless, while colour parameters remained unchanged along ST (except L * in fresh samples), they varied significantly in each IF. On the other hand, ST proved to be a stronger effector on fatty acids than IF, which caused no significant changes in most cases. Overall, both natural extracts proved to be suitable alternatives to the commercial dye (betalain), according to the maintenance of nutritional and fatty acids profiles and improvement in the appearance of the tested food product. The authors are grateful to the Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), C. Lobo Roriz (SFRH/BD/11799S/2016) grant and L. Barros and J.C.M. Barreira contracts. Authors also thank the European Structural and Investment Funds (FEEl) through the Regional Operational Program North 2020, within the scope of NORTE-01-0145-FEDER-023289: DeCodE and ValorNatural® projects. info:eu-repo/semantics/publishedVersion

Betacyanins are betalain pigments that display a red-violet colour which have been reported to be three times stronger than the red-violet dye produced by anthocyanins [1]. The applications of betacyanins cover a wide range of matrices, mainly as additives or ingredients in the food industry, cosmetics, pharmaceuticals and livestock feed. Although, being less commonly used than anthocyanins and carotenoids, betacyanins are stable between pH 3 to 7 and suitable for colouring in low acid matrices. In addition, betacyanins have been reported to display interesting medicinal character as powerful antioxidant and chemopreventive compounds either in vitro or in vivo models [2]. Betacyanins are obtained mainly from the red beet of Beta vulgaris plant (between I 0 to 20 mg per I 00 g pulp) but alternative primary sources are needed [3]. In addition, independently of the source used, the effect of the variables that affect the extraction of betacyanins have not been properly described and quantified. Therefore, the aim of this study was to identifY and optimize the conditions that maximize betacyanins extraction using the tepals of Gomphrena globosa L. flowers as an alternative source. Assisted by the statistical technique of response surface methodology, an experimental design was developed for testing the significant explanatory variables of the extraction (time, temperature, solid-liquid ratio and ethanolwater ratio). The identification was performed using high-performance liquid chromatography coupled with a photodiode array detector and mass spectrometry with electron spray ionization (HPLC-PDAMS/ ESI) and the response was measured by the quantification of these compounds using HPLC-PDA. Afterwards, a response surface analysis was performed to evaluate the results. The major betacyanin compounds identified were gomphrenin 11 and Ill and isogomphrenin IJ and Ill. The highest total betacyanins content was obtained by using the following conditions: 45 min of extraction. time, 35•c, 35 g/L of solid-liquid ratio and 25% of ethanol. These values would not be found without optimizing the conditions of the betacyanins extraction, which moreover showed contrary trends to what it has been described in the scientific bibliography. More specifically, concerning the time and temperature variables, an increase of both values (from the common ones used in the bibliography) showed a considerable improvement on the betacyanins extraction yield without displaying any type of degradation patterns.

A perceção, as opiniões e os desejos dos consumidores têm um enorme impacto na indústria alimentar. Na perceção visual, a cor torna-se um fator fundamental e, neste campo, os corantes alimentares assumem uma extrema importância. A cor pode ser considerada um dos atributos mais impressionantes dos géneros alimentícios, que influencia diretamente a preferência e a seleção dos consumidores[1]. Existem muitos corantes naturais utilizados na indústria alimentar, tais como carotenóides, antocianinas e betalaínas. As betalaínas incluem compostos com cores que vão do vermelho-violeta (betacianidinas) ao amarelo-laranja (betaxantinas). As betalaínas não têm sido tão extensamente estudadas como as antocianinas, mas possuem uma capacidade corante três-vezes maior. A única betalaína autorizada como corante natural deriva da beterraba(E-162)[2], mas existem outras fontes alternativas de betacianidinas ,como a que se apresenta neste trabalho: Gomphrenaglobosa L., vulgarmente designada por perpétua roxa.

A current worldwide trend of research is the search for alternative natural sources of colorants to avoid the unsafe effects caused by the synthetic counterparts. Betacyanins, which are betalain pigments displaying a red‐violet colour, have been reported to be three fold stronger than the yellow‐orange dye produced by anthocyanins. Although being less commonly used than anthocyanins and carotenoids, betacyanin applications cover a wide range of matrices, where they are mainly used as additives or supplements in the food, cosmetics, pharmaceuticals and livestock feed industries. Betacyanins are obtained mainly from the red beet, Beta vulgaris L. (between 10 to 20 mg per 100 g of fresh pulp), but alternative primary sources are needed. The floral parts of Gomphrena globosa L. represent a potential source of betacyanins. In this context, the present study aims to evaluate: 1) a process for the selection and separation of pigmented floral bracts and bracteoles (~4 % of the dried plant material); 2) the identification of the major betacyanin compounds (gomphrenin II and III, and isogomphrenin II and III); 3) the most appropriate extraction procedures (from maceration, microwave and ultrasound assisted techniques) together with the optimal conditions that maximize betacyanin extraction (time, temperature, solidliquid ratio and ethanol‐water ratio); and 4) shelf life and colour stability of the obtained natural agent. The responses were assessed by the quantification of betacyanins by high‐performance liquid chromatography coupled with a photodiode array detector and mass spectrometry with electron spray ionization. In addition, results clarified some contradictory trends described in the literature concerning the time and temperature variables, finding a considerable improvement on the betacyanins yield (higher than that typically found for Beta vulgaris), without displaying any type of degradation patterns. info:eu-repo/semantics/publishedVersion

Os corantes são utilizados há muito pela indústria alimentar, sendo utilizados por diversas razões: compensar a perda de cor devido às condições de manufatura e armazenamento; realçar a sua cor natural; dar cor a um alimento sem cor; mas também permitir aos consumidores identificar produtos visualmente. Hoje em dia muitos dos consumidores preferem alimentos com aditivos naturais em detrimento dos sintéticos, que têm sido associados com alguns efeitos tóxicos. Existem vários corantes naturais utilizados pela indústria alimentar, em particular carotenoides, antocianinas e betalaínas. As betalaínas contêm compostos que possuem cores que vão do vermelhovioleta (betacianinas) ao amarelo-laranja (betaxantinas) [1]. De entre as várias fontes de betalaínas, a fonte mais explorada é a beterraba, devido a sua elevada concentração nestes pigmentos [2]. No entanto, fontes alternativas menos exploradas, como as flores de Gomphrena globosa L., uma amaranthaceae, nativa da américa latina e comumente designada por perpétua roxa, possuindo na sua composição uma variedade de compostos com atividade biológica, sendo as betacianidinas um deles, tornando esta planta uma ótima candidata como alternativa na obtenção destes pigmentos [3]. Desta modo e de forma a obter um maior rendimento destes compostos, o objetivo deste estudo foi a otimização do sistema de extração (maceração, uma metodologia tradicional), levada a cabo com o auxílio de uma metodologia de resposta de superfície, na qual várias variáveis foram tidas em conta (tempo (t), temperatura (T), relação sólido-liquido (S/L) e relação água/etanol (Et)). As respostas de extração foram avaliadas tendo em conta o rendimento de extração (peso seco), monitorização das betacianinas por HPLC acoplado a diferentes detetores (DAD e MS) e a sua capacidade corante medida por espectroscopia UV/Vis e colorimetria (Spectra Magic Nx). Estes resultados permitiram estimar as condições ótimas de trabalho, maximizando o potencial corante, diminuindo custos, tempos de produção e aumentando a sua eficiência. Após análise dos resultados concluímos que os valores ótimos de extração para as diferentes variáveis são, t=165 min, T=25°C, Et=0 % e S/L=5 g/L obtendo assim uma concentração ótima de compostos corantes de betacianidinas de 45.1±1.3 mg/g de Gomphrena globosa seca com um rendimento de extração de 27.3±1.3 %. Os autores agradecem à FCT e FEDER no âmbito do programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2013) e L. Barros (SFRH/BPD/107855/2015). Agradecer ao POCI-01-0145-FEDER-006984 (LA LSRE-LCM), financiados pelo FEDER, através do POCI-COMPETE2020 e FCT. À Junta de Galícia pelo financiamento da bolsa de pós-doutoramento de M.A. Prieto. info:eu-repo/semantics/publishedVersion

Betalains are plant derived natural pigments that are presently gaining popularity for use as natural colorants in food industry. Although being betalains from red beetroot already used as food colorant (E- 162), these compounds are not as well studied as compared to other natural pigments such as anthocyanins, carotenoids or chlorophylls (I]. Since food additives are on the focus of public interest, it is becoming increasingly important to meet consumers' expectations for natural and healthy products. Hence, the search for new plant-derived colorants for the food industry is still necessary [2]. Betalains were originally called 'nitrogenous anthocyanins', which incorrectly implied structural similarities between the two pigment classes. There are two structurally different types of betalains: the yellow/orange betaxanthins which are the condensation products of betalamic acid and assorted amino compounds, and the red betacyanins which are formed by glycosylation and acylation of cyclo-DOPA [3]. Looking at the chemical structure of the pigment, the addition of an acid to the extraction solvent will increase the affinity of the pigment with the solvent. The aim of this study was to use Gomphrena globosa L. flowers, as an alternative plant source to obtain these pigments and to evaluate the best acid to be used within the extraction procedure. For that purpose three different acids (acetic, hydrochloric and phosphoric acids, all ofthem allowed by the food industry), adjusted at the same pH, were tested during a maceration extraction procedure. After the extraction a purification through C18 column was performed in order to obtain a more concentrate extract in betacyanins. The results were analysed by HPLC-PDA-MSIESI. The betacyanin profile allowed the identification of gomphrenin IIJIII and isogomphrenin IIIIII and the best results were achieved by performing the extraction procedure using hydrochloric acid (6.6 mg/g extract), while phosphoric acid only presented trace amounts of these compounds. When acetic acid was used, the pigment extracted was 6.8 times less (0.97 mg/g extract) when compared to HCI. In conclusion hydrochloric acid can be considered the most suitable acid to be applied in the extraction procedure of these pigments.

Mestrado de dupla diplomação com a Universidad de Salamanca Pterospartum tridentatum (L.) Willk., Gomphrena globosa L. and Cymbopogon citratus (DC) Stapf. são plantas medicinais que requerem uma caracterização química mais detalhada, devido à importância do seu consumo em infusões. Assim, o perfil individual em tocoferóis, açúcares livres e ácidos orgânicos foi efetuado por cromatografia líquida de alta eficiência (HPLC) acoplada a diferentes detetores (florescência, índice de refração e detetor de díodos, respetivamente). A composição fenólica (e.g., ácidos fenólicos e flavonoides) foi também analisada por técnicas de cromatografia e espetrometria de massa. A atividade antioxidante dos extratos metanólicos e das infusões foi avaliada através da atividade captadora de radicais livres, poder redutor, inibição da descoloração do β-caroteno e inibição da peroxidação lipídica em homogeneizados cerebrais. Nos últimos anos, têm surgido estudos de avaliação dos efeitos sinergistas resultantes da combinação de plantas, destacando o maior potencial das misturas quando comparados com as plantas individuais. As três espécies mencionadas anteriormente são exemplos de plantas medicinais com propriedades antioxidantes próprias, mas que podem mostrar melhorias quando combinadas. Assim, avaliou-se também a atividade antioxidante das infusões preparadas a partir das plantas individuais e de misturas dessas plantas em diferentes proporções. C. citratus revelou o maior teor em α-tocoferol, tocoferóis totais, glucose sacarose, ácidos succínico e ascórbico, tendo ainda apresentado a mais elevada capacidade de inibição da descoloração do β-caroteno e peroxidação lipídica; a luteolina 2''-O-ramnosil-6-C-glucósido foi o composto maioritário no extrato metanólico dessa planta. P. tridentatum apresentou a maior concentração de frutose e açúcares totais, revelando também a maior atividade captadora de radicais livres e poder redutor; derivados de di-hidroflavonol e isoflavona foram os compostos mais significativos no extrato metanólico. Finalmente, G. globosa demonstrou a maior concentração em ácidos orgânicos, sendo o canferol 3-O-rutinósido o composto fenólico mais abundante no extrato metanólico; foram ainda identificadas betacianidinas nesta amostra. O perfil fenólico foi muito similar nos extratos metanólicos e nas infusões. Para as infusões, a ordem de eficácia antioxidante foi a seguinte P. tridentatum> C. citratus> G. globosa, o que pode estar relacionado com a diferente composição em compostos fenólicos, principalmente flavonoides. Quando se combinaram as plantas em estudo, observaram-se maioritariamente efeitos sinergistas na atividade antioxidante das infusões; os efeitos foram mais significativos nas proporções de 40%:60% (tanto de P. tridentatum e C. citratus, como G. globosa e C. citratus). A infusão obtida a partir de 40% de P. tridentatum e 60% de C. citratus foi a que apresentou propriedades antioxidantes mais elevadas, devido ao aumento da percentagem individual da planta com maior atividade antioxidante, comparativamente à mistura de 25%:75%. Tanto quanto sabemos, este é o primeiro trabalho que descreve a composição química de G. globosa e C. citratus. Dada a importância do consumo das três plantas mencionadas, o estudo da sua composição fitoquímica e das suas propriedades antioxidantes é de particular interesse. O presente trabalho valida ainda a comercialização das espécies combinadas em proporções específicas. Pterospartum tridentatum (L.) Willk., Gomphrena globosa L. and Cymbopogon citratus (DC) Stapf. are medicinal plants that require a more detailed chemical characterization, given the importance of their consumption as infusions. Therefore, the individual profiles in tocopherols, free sugars and organic acids were obtained by high performance liquid chromatography (HPLC) coupled to different detectors (fluorescence, refraction index and photodiode array, respectively). Phenolic composition (e.g., phenolic acids and flavonoids) was also analysed by chromatographic and mass spectrometry techniques, and the antioxidant activity was also accessed through free radicals scavenging activity, reducing power and inhibition of lipid peroxidation in brain homogenates, for methanol extracts as for infusions. In recent years, studies focused on the evaluation of synergistic effects of combined plants are emerging and seem to highlight the potential of the mixtures when compared with the individual plants. These three plants are examples of medicinal plants with antioxidant properties by their own, but that can be improved when mixed. In the present work, the antioxidant activity and phenolic compounds were determined in the infusions prepared from the individual plants, as mentioned above, and from mixtures of these plants in different proportions. C. citratus revealed the highest content of α-tocopherol, total tocopherols, glucose, sucrose, succinic and ascorbic acids, with the highest -carotene bleaching and lipid peroxidation inhibitions; luteolin 2´´-O-rhamnosyl-6-C-glucoside was the main compound in its methanolic extract. P. tridentatum presented the highest fructose and total sugars content, revealing the highest free radicals scavenging activity and reducing power; dihydroflavonol and isoflavone derivatives were mainly detected in the methanolic extract. Finnaly, G. globosa showed the highest organic acids concentration, being kaempferol 3-O-rutinoside the most abundant phenolic compound in the methanoli extract; betacyanins were also identified in this sample. The phenolic profile was very similar in the methanolic extracts and infusions. Regarding infusions, P. tridentatum > C. citratus > G. globosa was the order observed for antioxidant efficacy, which can be related to their different composition in phenolic compounds namely, flavonoids. Synergism was the main effect observed among the tested mixtures, mainly for the infusions prepared from the plants in proportion 40%:60% (either P. tridentatum and C. citratus; or G. globosa and C. citratus). The infusion obtained with 40% of P. tridentatum and 60% of C. citratus gave the highest antioxidant properties, due to the increasing percentage of the individual plant with the highest antioxidant activity, in comparison with the mixture 25%:75%. As far as we know, this is the first study reporting chemical composition of G. globosa and C. citratus. It is very interesting to study the phytochemical composition of these plants, and their properties given the importance of their consumption. The present study also validates the commercialization of the studied species combined in specific proportions.

The World Health Organization estimates that 80% of the world’s population uses medicinal plants in primary health needs. Pterospartum tridentatum (L.) Willk. (Fabaceae) and Cymbopogon citratus (DC) Stapf. (Poaceae) are examples of such medicinal plants. P. tridentatum is a small shrub, which can be found in the western part of the Iberian Peninsula and northern Morocco. Traditionally its flowers can be used to treat pneumonia, colds, bronchitis and hepatic disease [1]. C. citratus, commonly known as lemon grass, is a tall, aromatic, perennial densely tufted grass from Southeast Asia. There are many effects that can be attributed to this species, such as diuretics effects; it can also be used in hepatic disorders and to treat depression, flatulence and intestinal cramps [2]. The mixture of these two species opens a new panoply of therapeutic effects that can range from the treatment in case of stomach pain, cramping and flatulence, diarrhea, vomiting and flu. This is due to the interaction of these two species. In this study we aimed to evaluate the antioxidant activity of the single plants and of the mixture of the same plants (infusions and methanolic extracts. The antioxidant activity was accessed by four in vitro assays: scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals, reducing power, inhibition of β-carotene bleaching and inhibition of lipid peroxidation in brain cell homogenates by TBARS (thiobarbituric acid reactive substances) assay. In general, infusions revealed higher antioxidant activity than methanolic extracts, being higher for P. tridentatum. With the exception of the β-carotene bleaching inhibition assay, the mixture of the two plants revealed significant synergistic effects, allowing higher antioxidant activity. Although the interesting results obtained, the authors recommend a safe consume of these plant materials, including restricting the dose and duration of use to minimize the risks of potentially interactions with conventional medicines. Further studies will be conducted in order to identify the compounds responsible for the synergisms observed.