Desenvolvimento de um corante alimentar natural à base de antocianinas obtido a partir do epicarpo de frutos de Prunus spinosa L.: otimização da extração e estudo de aplicação

Dupla diplomação coma UTFPR - Universidade Tecnológica Federal do ParanáEntre as propriedades organoléticas, a cor exerce um papel muito importante na perceção global de um alimento, uma vez que a visão é normalmente o primeiro contato que o consumidor tem com o produto. Neste sentido, os corantes têm sido amplamente utilizados como aditivos alimentares, para suprir ou intensificar os aspetos de coloração dos alimentos, tornando-os mais atrativos e agradáveis. No entanto, devido à preocupação que os consumidores têm com a saúde e o registo de efeitos adversos de alguns corantes artificiais, existe um grande interesse na sua substituição por alternativas naturais mais seguras. As antocianinas são um grupo de pigmentos de coloração vermelha, azul e violeta, presentes em muitas flores e frutos, podendo ser consideradas alternativas aos corantes artificiais. Prunus spinosa L. é um arbusto selvagem que dá origem a frutos de sabor amargo e adstringente pouco explorados comercialmente, mas que apresentam níveis elevados de antocianinas, mais especificamente cianidina 3-rutinósido e peonidina 3-rutinósido, concentradas no epicarpo. Assim, o presente trabalho teve como objetivo desenvolver um corante alimentar natural a partir da extração de antocianinas dos frutos de P. spinosa, aliando os crescentes interesses na substituição dos corantes artificiais por alternativas naturais mais seguras à valorização de produtos agrícolas de fontes subexploradas. Foram otimizadas as condições que maximizam a extração de antocianinas do epicarpo de frutos de P. spinosa, comparando-se duas técnicas de extração, sendo uma convencional assistida por calor (HAE) e outra alternativa assistida por ultrassons (UAE), e tendo como variáveis relevantes tempo, temperatura (HAE) ou potência (UAE) e concentração de solvente. Para isso, foi aplicada uma metodologia de superfície de resposta, utilizando um desenho composto central circunscrito de três variáveis com cinco níveis, e os compostos antociânicos foram identificados e quantificados por HPLC-DAD-ESI/MS. A extração assistida por ultrassons foi o método mais eficaz, nas condições ótimas de 5,00 ± 0,15 min, 400,00 ± 32,00 W e 47,98 ± 2,88% de etanol (v/v), onde o rendimento de extração foi de 68,60 ± 2,06%, com um teor total de antocianinas de 18,17 ± 1,82 mg/g de extrato e 11,76 ± 0,82 mg/g de epicarpo seco, originando um extrato de cor roxo-avermelhado. Além disso, estes valores de resposta foram ligeiramente melhorados quando se testou o efeito da relação sólido-líquido nas condições ótimas num formato dose-resposta, mostrando um padrão decrescente constante na faixa de 5 a 250 g/L. Para além de estudar o potencial corante deste ingrediente natural, também se pretendeu avaliar o seu potencial funcional, para isso realizou-se uma avalição das propriedades bioativas e efectuou-se um estudo de aplicação num produto alimentar do extrato obtido nas condições otimizadas. A avaliação das bioatividades foi realizada através da análise da capacidade antioxidante, anti-inflamatória, antimicrobiana e citotóxica. O extrato apresentou uma atividade antioxidade considerável, com valores de EC50 de 204 ± 2 μg/mL para o método de TBARS e EC50 de 296 ± 4 e 509 ± 3 μg/mL para o método OxHLIA aos 60 e 120 min de reação, respetivamente. A capacidade antimicrobiana foi testada em 9 estirpes de bactérias de interesse na área da saúde, nas quais o extrato não apresentou efeito bactericida, mas teve a capacidade de inibir o crescimento bacteriano em concetrações de 2,5 a 20 mg/mL. A avaliação da atividade anti-inflamatória foi realizada em células de macrófagos de rato RAW 264.7 e a citotoxicidade foi testada em 4 linhas celulares tumorais humanas e numa cultura primária de células hepáticas não tumorais, para as quais o extrato não apresentou atividade, nem toxicidade. O extrato corante foi incorporado em “beijinhos”, um produto de confeitaria tipico brasileiro que tem como ingredientes básicos leite condensado, natas e manteiga, para avaliação da sua aplicabilidade numa matriz alimentar como corante alimentar. Foram determinados os parâmetros de cor, pH e textura, a composição nutricional e o valor energético das amostras de “beijinhos” logo após a sua confeção e depois de um dia de armazenamento. Em geral, o extrato otimizado incorporado revelou uma boa capacidade corante sem alterar as caraterísticas nutricionais do produto alimentar. Com isto, foi possível evidenciar o potencial da exploração do epicarpo dos frutos de P. spinosa como fonte de antocianinas a serem utilizadas no setor alimentar, tendo como valor agregado ao poder corante, a sua capacidade bioativa.Among organoleptic properties, colour plays a very important role in the overall perception of a foodstuff, since sight is usually the first contact that consumers have with the product. In this context, colorants have been widely used as food additives, to supply or intensify food colouring aspects, making them more attractive and pleasant. However, due to consumers' concerns about the health and adverse effects of some artificial colorants, there is a strong interest in replacing them with safer natural alternatives. Anthocyanins are a group of pigments with red, blue and violet coloration, being present in many flowers and fruits and increasingly considered as potential alternatives to artificial colorants. Prunus spinosa L. is a wild shrub with bitter and astringent fruits that are poorly commercially exploited but have high levels of anthocyanins, more specifically cyanidin 3-rutinoside and peonidin 3-rutinoside, concentrated in the epicarp. The objective of this study was to develop a natural food colorant, from the extraction of anthocyanins from the fruit epicarps of P. spinosa, combining the growing interest of substituting artificial colorants for safer natural alternatives and the valorization of agricultural products from underexploited sources. The conditions that maximize the extraction of anthocyanins from the fruit epicarp of P. spinosa were optimized, comparing two extractive techniques, one conventional heat-assisted (HAE) and another alternative ultrasound assisted extraction (UAE), testing as relevant variables time, temperature (HAE) or power (UAE), and solvent concentration. For that purpose, a response surface methodology was applied, using a circumscribed central composite design with three variables and five levels, and the anthocyanin compounds were identified and quantified by HPLC-DAD-ESI/MS. Ultrasound assisted extraction was the most efficient method, under optimum conditions of 5.00 ± 0.15 min, 400.00 ± 32.00 W and 47.98 ± 2.88% ethanol, where the extraction yield was 68.60 ± 2.06% (v/v), with a total anthocyanin content of 18.17 ± 1.82 mg/g of dry extract and 11.76 ± 0.82 mg/g of dry epicarp, wich gives rise to a purple-reddish extract. Additionally, these response values were slightly improved when the solid-to-liquid ratio effect at the optimal conditions in a dose-response format was tested, showing a steady decreasing pattern in the range 5 to 250 g/L. In addition to studying the colorant potential of this natural ingredient, it was also intended to evaluate its functional potential, for which an evaluation of the bioactive properties was determined, and an application study was carried out using a food product incorporated with the extract obtained under optimized conditions The evaluation of the bioativities was carried out through analyzes of the antioxidant, anti-inflammatory, antimicrobial and cytotoxic capacity. The extract presented a considerable antioxidant activity, with EC50 values of 204 ± 2 μg/mL for the TBARS assay and EC50 of 296 ± 4 and 509 ± 3 μg/mL for OxHLIA method at 60 and 120 min of reaction, respectively. The antimicrobial capacity was tested in 9 strains of bacteria of interest in the health area, in which the extract did not present bactericidal effect, but all had their growth inhibited with an extract concentration between of 2.5 to 20 mg/mL. The anti-inflammatory activity was assessed on RAW 264.7 mouse macrophages and cytotoxicity was tested using 4 human tumor cell lines and on a primary culture of non-tumor liver cells, for which the extract showed no activity and no toxicity. The dye extract was incorporated into "beijinhos", a typical brazilian confectionery product, that has as basic ingredients condensed milk, cream and butter, to evaluate its applicability as a natural colorant ingredient for a food matrix. The color, pH, and texture parameters, nutritional composition, and energetic value of "beijinhos" samples were determined after cooking and after one day of storage. In general, the incorporated optimized extract revealed a good coloring ability without altering the nutritional characteristics of the food product. With this, it was possible to evidence the potential exploitation of P. spinosa fruit epicarp as a source of anthocyanins to be used in the food industry, having as added value to the dye power, its bioactive capacity.Este trabalho é financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289 (DeCodE) e Norte-01-0247-FEDER-024479 (projeto Mobilizador ValorNatural), e pelo programa FEDER-Interreg España-Portugal, no âmbito do projeto 0377_Iberphenol_6_E

Ultrasound as a apid and Low-cost extraction procedure to obtain Anthocyanin-based colorants from Prunus spinosa L. Fruit epicarp: comparative study with conventional heat-based extraction

An ultrasound rapid and low-cost procedure for anthocyanin-based colorants from Prunus spinosa L. fruit epicarp was developed, and the advantages were compared with conventional heat-based extraction. To obtain the conditions that maximize anthocyanins’ extraction, a response surface methodology was applied using the variables of time, temperature, and ethanol content, in the case of heat extraction, whereas for ultrasound assisted extraction, temperature was replaced by ultrasound power. Two anthocyanin compounds were identified by HPLC-DAD-ESI/MS—namely, cyanidin 3-rutinoside and peonidin 3-rutinoside. The responses used were the extraction yield and the content of the identified anthocyanins. Ultrasound extraction was the most effective method at 5.00 0.15 min, 400.00 32.00 W, and 47.98% 2.88% of ethanol obtaining 68.60% 2.06% of extracted residue, with an anthocyanin content of 18.17 mg/g (extract-basis) and 11.76 mg/g (epicarp-basis). Overall, a viable green process was achieved that could be used to support pilot-scale studies for industrial production of anthocyanin-based colorants from P. spinosa fruit epicarp.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 and C. Pereira research contract; to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E.; to 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®. This work was also financially supported by the following: Project POCI-01-0145-FEDER-006984, Associate Laboratory LSRE-LCM funded by FEDER through COMPETE2020, Programa Operacional Competitividade e Internacionalização (POCI), and national funds through FCT. The authors thank the GAIN (Xunta de Galicia) for financial support (P.P. 0000 421S 140.08) to M.A. Prieto by a post-doctoral (modality B) grant.info:eu-repo/semantics/publishedVersio

Variability in chemical profile and bioactivities of the flesh of greek pumpkin landraces

The aim of this study was to evaluate the chemical profile of the flesh and yield parameters of Greek pumpkin genotypes, including nine local landraces and two commercially available cultivars, focusing on valorizing the genetic pool of Cucurbita sp. with high added value products. Yield parameters (mean fruit weight and total fruit yield) recorded high variability with genotypes V8 and V2 showing the highest fruit yield. Moreover, genotype V11 was the most abundant in glucose and total sugars and scored the highest sweetness index suggesting good taste and promising marketing attributes. The highest antioxidant activity (OxHLIA assay) was assessed in the V8 genotype, while the V2 genotype showed the highest -, - and total tocopherols content. Oxalic acid was the main organic acid, followed by malic and citric acids, while organic acid composition varied among the tested genotypes. Moreover, the flesh extracts showed varied antimicrobial activity against several bacteria and fungi, while no toxicity against non-tumor cells was recorded. In conclusion, our results make evident the presence of high innate variability in terms of crop performance, chemical composition and bioactive properties not only between the different genotypes but also at the intrapopulational level. This finding is of high importance for the valorization of the local genetic pool of Cucurbita species through the selection of elite genotypes with high yield and quality of fruit, contributing to the conservation of valuable genetic material and limitation of the risk of genetic erosion due to neglect of local landraces.This research was funded by the Foundation for Science and Technology (FCT), P.I. (FCT, Portugal) through national funds FCT/MCTES to CIMO (UIDB/00690/2020 and UIDP/00690/2020), SusTEC (LA/P/0007/2021) and UIDB/50006/2020. National funding by FCT, P.I., through the institutional scientific employment program contract for M.I.D., R.C.C., C.P. and L.B. contracts, and M.G. and A.K.M. PhD grants (2020.06706.BD and 2020.06231.BD, respectively); FCT, P.I., within the scope of the Project PRIMA Section 2—Multi-topic 2019: PulpIng (PRIMA/0007/2019); the MICINN for the Juan de la Cierva Formación contract for T.C.S.P.P. (FJC20120-045405-I); and the General Secretariat for Research and Technology of the Ministry of Development and Investments under the PRIMA Program. PRIMA is an Art.185 initiative supported and co-funded under Horizon 2020, the European Union’s Program for Research and Plants 2022, 11, 800 16 of 18 Innovation (Prima2019-08).info:eu-repo/semantics/publishedVersio

Food preservative extracts from pumpkin by-products

Pumpkin is a vegetable widely consumed around the world, popularly known for its nutritional and pharmacological properties. Some reports in the literature reveal the rich profile of nutrients and bioactive compounds of pumpkin varieties [1,2]. However, the seeds, peels, and fibers resulting from pumpkin processing are still poorly explored by food industry. Considering the alarming scenario of losses and waste during all the food supply chain worldwide [3] and seeking to promote a more sustainable food system, the present study purposed to investigate the by-products of different varieties of pumpkin as a source of extracts with preservative capacity for food application. For that purpose, hydroethanolic extracts from the varieties “Butternut squash”, “Common pumpkin”, and “Kabocha squash” from Portugal, and “Butternut squash”, “Common pumpkin”, and “Musquée de provence” from Algeria, were evaluated in terms of their bioactivities, more specifically the antioxidant, antimicrobial, and cytotoxic capacities. All the samples presented great antioxidant capacity through two based-cell assays, namely the lipid peroxidation inhibition capacity (TBARS) and the anti-hemolytic activity (OxHLIA). Interestingly, the seeds of the common pumpkin from Algeria did not present antihemolytic properties, despite showing the strongest lipid peroxidation inhibition capacity. In fact, the seeds stood out for all the pumpkin varieties in the TBARS assay, while in the OxHLIA assay, the results were quite similar between the type of bioresidues and between the varieties. Moreover, the samples from Portugal showed greater antioxidant capacity than the Algerian ones. Regarding cytotoxicity, the effect of inhibiting non-tumor cell growth was not observed, even at the highest tested concentration of 400 μg/mL. This first validation is of great importance to prevent food safety issues. These preliminary results are the basis for future studies aiming at the valorization of these bioresidues from food industry as a great source of preservative compounds toward the replacement of synthetic additives with natural alternatives allied to health benefits, as also the promotion of a circular economyThe authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FCT, P.I., within the scope of the Project PRIMA Section 2 - Multi-topic 2019: PulpIng (PRIMA/0007/2019).info:eu-repo/semantics/publishedVersio

Biological activity of pumpkin byproducts: antimicrobial and antioxidant properties

Pumpkin fruits are widely appreciated and consumed worldwide. In addition to their balanced nutritional profile, pumpkin species also present valuable bioactive compounds that confer biological and pharmacological properties to them. However, the seeds, peels, and fibrous strands resulting from pumpkin processing are still poorly explored by the food industry. The current study used those fruit components from the genotypes of pumpkin that are economically significant in Portugal and Algeria to produce bioactive extracts. In order to support their usage as preservatives, their phenolic content (HPLC-DAD-ESI/MS) and antioxidant (OxHLIA and TBARS) and antimicrobial properties (against eight bacterial and two fungal strains) were assessed. In terms of phenolic profile, the peel of the Portuguese ‘Common Pumpkin’ showed the most diversified profile and also the highest concentration of total phenolic compounds, with considerable concentrations of (-)-epicatechin. Regarding the antioxidant capacity, the seeds of ‘Butternut Squash’ from both countries stood out, while the fibrous strands of Portuguese ‘Butternut Squash’ and the seeds of Algerian ‘Gold Nugget Pumpkin’ revealed the strongest antimicrobial activity. The bioactive compounds identified in the pumpkin byproducts may validate their enormous potential as a source of bio-based preservatives that may enhance consumers’ health and promote a circular economy.info:eu-repo/semantics/publishedVersio

Valorização de cascas, sementes e fibras de abóbora no desenvolvimento de extratos bioativos para aplicação alimentar

Na Europa, quase metade da produção de frutas e hortaliças é perdida ou desperdiçada, principalmente nas etapas de processamento, através da geração de grandes quantidades de cascas.info:eu-repo/semantics/publishedVersio

Natural pigments with high antioxidant capacity, an alternative solution for food industry

Natural matrices that are rich in anthocyanin compounds are increasingly explored by the food industry due to their coloring properties. As examples, Prunus spinosa L. and Lonicera careulea L. fruits are excellent sources of anthocyanins and are, therefore, increasingly explored for their coloring properties to be applied as food colorants, in addition to providing beneficial properties to the consumer [1-2]. This study aimed to evaluate the anthocyanin profile (HPLCDAD/ ESI-MS) and the antioxidant (TBARS and OxHLIA) and antimicrobial properties of the hydroethanolic extract of P. spinosa fruit epicarp and L. careulea fruit juice.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FEDER-Interreg España-Portugal programme for financial support through TRANSCoLAB 0612_TRANS_CO_LAB_2_P project; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479:ValorNatural®.info:eu-repo/semantics/publishedVersio

Study of Prunus spinosa L. fruit epicarp and Lonicera careulea L. fruit: alternative natural colorants with

Natural matrices that are rich in anthocyanin compounds are increasingly explored by the food industry due to their colouring properties. As examples, Prunus spinosa L. and Lonicera careulea L. fruits are excellent sources of anthocyanins and are, therefore, increasingly explored for their colouring properties to be applied as food colorants, in addition to providing beneficial properties to the consumer [1,2]. Therefore, the aim of this study was to evaluate the anthocyanin profile (HPLC-DAD/ESI-MS) and the antioxidant (TBARS and OxHLIA) and antimicrobial properties of the hydroethanolic extract of P. spinosa fruit epicarp and L. careulea fruit juice. A high content of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside was found in the juice of L. caerulea berries and in the hydroethanolic extract of the epicarp of P. spinosa, respectively. As for the antioxidant activity, in the TBARS assay, the L. caerulea berries showed a higher capacity (IC50 of 29.9±0.3 μg/mL) than the positive control, trolox (IC50 of 139±5 μg/mL). Similarly, the epicarp of P. spinosa showed the ability to inhibit lipid peroxidation, revealing an EC50 value of 204±2 μg/mL. Regarding the ability to retard oxidative haemolysis, both extracts showed activity not only at 60 min, but also at 120 min, allowing EC50 values of 145±5 μg/mL and 938±49 μg/mL, respectively, for L. caerulea, and 296±4 and 509±3 μg/mL, respectively, for P. spinosa. On the other hand, both colouring extracts revealed great antimicrobial properties. Through this work, it was possible to conclude that L. caerulea and P. spinosa berries have a high colouring capacity and bioactive potential, being suitable for the development of new products for food industry.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FEDER-Interreg España-Portugal programme for financial support through TRANSCoLAB 0612_TRANS_CO_LAB_2_P project; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®.info:eu-repo/semantics/publishedVersio

Food industry by-products applied as pumpkin-based foodstuff preservatives

Pumpkin is traditionally cultivated and consumed worldwide, being the fruit and its by-products recognized by their nutritional and health benefits. In the current pandemic scenario and towards a circular economy, the development of a healthy and green technological food product was proposed using pumpkin pulp enriched with bioactive compounds obtained from pumpkin by-products.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). The project is funded by the General Secretariat for Research and Technology of the Ministry of Development and Investments and FCT, P.I. under the PRIMA Programme. PRIMA is an Art.185 initiative supported and co-funded under Horizon 2020, the European Union’s Programme for Research and Innovation (PRIMA Section 2 - Multi-topic 2019: PulpIng (PRIMA/0007/2019 and Prima2019-08).info:eu-repo/semantics/publishedVersio

Haskap and blackthorn berries anthocyanin profile

Anthocyanin compounds are pigments that have a red, blue, and/or violet coloration widely found in nature, mainly in fruits [1]. Given the current need to replace synthetic colorants with healthier natural alternatives, this study investigated the potential of haskap (Prunus spinosa L.) and blackthorn (Lonicera careulea L.) fruits as sources of anthocyanins. For this purpose, the characterization of these fruits (the hydroethanolic extract of blachthorn epicarp and the haskap juice) was carried out by high performance liquid chromatography coupled to a diode array detector and a mass spectrometer (HPLC-DAD/ESI-MS). The identification was carried out using standards, when available, comparing their retention times, UV-Vis spectra, and mass spectra. In the absence of standards, the identification was carried out by the fragmentation profile and by comparison with the information available in the literature. The quantification was performed from the peak areas recorded at a wavelength of 520 nm, compared to the standard calibration curves. The blackthorn epicarp showed two anthocyanin compounds, being the molecules present in the highest concentrations cyanidin-3-O-rutinoside ([H]+ at m/z 595) and peonidin-3-O-rutinoside ([H]+ at m/z 609). In haskap berries, six anthocyanins were identified, namely cyanidin-O-hexoside-O-hexoside ([H]+ at m/z 611), cyanidin-O-rhamnoside-O-hexoside ([H]+ at m/z 595), pelargonidin-3-O-glucoside ([H]+ at m/z 433), peonidin-3-O-glucoside ([H]+ at m/z 463), peonidin-O-rhamnoside-O-hexoside ([H]+ at m/z 609), and cyanidin-3-O-glucoside ([H]+ at m/z 449), with the latest as the most abundant one. These results demonstrate that the blackthorn fruit epicarp and haskap fruits can be considered great sources of pigments in the red-purple colour range, having applicability both in the food and pharmaceutical industries.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FEDER-Interreg España-Portugal programme for financial support through TRANSCoLAB 0612_TRANS_CO_LAB_2_P project; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®info:eu-repo/semantics/publishedVersio