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- Lentil flour formulations to develop new snack-type products by extrusion processing: phytochemicals and antioxidant capacityPublication . Morales, Patricia; Cebadera-Miranda, Laura; Cámara Hurtado, Montaña; Reis, Filipa S.; Barros, Lillian; Berrios, José de J.; Ferreira, Isabel C.F.R.; Cebadera-Miranda, LauraExtrusion process has been generally applied to the production of cereal-based snacks. However, in recent years, extrusion process has been also used for the development of pulses-based snacks, with few studies focussing on the incorporation of these food ingredients to obtain bioactive compounds enriched snacks with acceptable quality. The effects of the extrusion processing on fiber (soluble and insoluble), total available carbohydrates, tocopherols, organic acids, total phenolics, hydroxycinnamic acids, hydroxybenzoic acids and flavonols, as well as on the antioxidant capacity of different lentil-based, fiber-enriched flours were evaluated. Total dietary fiber (TDF) in the extruded flours ranged between 7.49 and 10.22 g/100 g, with a decrease from 6 to 37% which correlated with a significant increase in the fiber soluble fraction. α-, γ- and δ-Tocopherols were characterized in all the analyzed samples, being γ-tocopherol the major isoform, before and after extrusion. Additionally, a marked decrease of 83 to 94% in total tocopherol content after extrusion processing was observed and the decrease was more significant on quinic acid content. Conversely, an increase in most polyphenolic fractions was observed, probably due to the effect of extrusion process to induce hydrolysis of polyphenols bound fiber and/or proteins moieties, changing from non-extractable to extractable polyphenols. Similar increase in antioxidant activity was also observed in most formulated lentil-based flours, measured by four different antioxidant assays. Only flavonols presented an extensive content decrease of about 62 to 82%, after extrusion treatment.
- Incorporation of an extract rich in rosmarinic acid into cupcakes: Influence on the sugars and total carbohydrates compositionPublication . Caleja, Cristina; Reis, Filipa S.; Barros, Lillian; Prieto Lage, Miguel A.; Barreiro, M.F.; Oliveira, Beatriz; Ferreira, Isabel C.F.R.Currently, the food industry is focused on replacing synthetic additives with natural ingredients. Natural extracts obtained from plants have emerged as possible alternatives to synthetic preservatives, namely antioxidants [1]. Some dairy, meat and bakery products have been developed incorporating extracts of aromatic plants, spices or fruits, which have antioxidant properties [2-4]. In this work, we intend to prove the efficacy of an extract rich in rosmarinic acid as a natural preservative in cupcakes and compare these results to a synthetic additive (potassium sorbate, E202), which is normally used in pastry products. The extract of Melissa officina/is L. (lemon balm) was obtained by performing an ultrasound-assisted extraction with ethanol. After evaluation of its antioxidant (free radical scavenging activity, EC50 = 79 ± 2 J.Jg I ml; reducing power EC50 = 49 ± 1 J.Jg I ml) and antimicrobial (against 8 bacteria and 8 fungi contaminants) properties, and confirmation of toxicity absence (in a non-tumor cell line), the extract enriched in rosmarinic acid was incorporated into the cupcakes. Its effect was compared to E202 and to a control sample (cupcakes without additives) immediately after incorporation and in the 3n;l and 5th day of storage, protected from light at room temperature. The results showed that both natural (extract enriched in rosmarinic acid) and synthetic (E202) additives, did not cause significant changes in the nutritional composition of cupcakes (fat, protein and carbohydrate contents), when compared with the control sample. Free sugars were also determined by high performance liquid chromatography coupled to a refractive index detector (HPLC-RI), which indicated sucrose and glucose as the most abundant sugars in all samples. These molecules did not undergo any changes with the addition of the additives as compared to the control sample. The results obtained demonstrate that the presence of rosmarinic acid does not influence sugars and total carbohydrates content of cupcakes and may be used in the development of new pastry and bakery products, replacing synthetic preservatives and improving the functional properties of the food.
- Bioactive molecules profile of two Lactarius species from SerbiaPublication . Reis, Filipa S.; Stojković, Dejan; Soković, Marina; Ćirić, Ana; Glamočlija, Jasmina; Barros, Lillian; Ferreira, Isabel C.F.R.Mushrooms have become attractive as functional foods and as a source of many biologically active compounds. Different wild mushroom species were reported to have an excellent nutritional value and bioactivity that includes antioxidant and antitumor properties [1-3]. Hence, in recent times, the consumption of mushrooms has risen greatly, comprising a large number of species. This work presents the profile of organic acids, tocopherols and phenolic compounds of two species of wild mushrooms from Serbia: Lactarius piperatus and Lactarius rufus. Organic acids were determined by UFLCPDA; tocopherols were analysed by HPLC-fluorescence and the phenolic compounds characterization was made by HPLC-DAD/MS. It was possible to quantify oxalic, quinic, malic, citric and fumaric acids in both species, being malic acid the majority organic acid found in these samples. However, L. rufus revealed a higher content in total organic acids comparing with L. piperatus (12.43 g/100 g dw and 9.40 g/100 g dw, respectively). Concerning tocopherols, the four isoforms of vitamin E (α-, β-, γ- and δ-tocopherol) were detected in both species. Herein, L. piperatus was the species that holds the highest concentration of tocopherols (80.38 μg/100 g dw), due to the contribution of β-tocopherol (47.43 μg/100 g dw). About phenolic compounds, it was only possible to quantify p-hidroxibenzoic acid in L. rufus. Nevertheless, it was detected the related compound cinnamic acid in both species. This work results from a Project in cooperation with the University of Belgrade, and the main purpose is the enhancement of the mycological flora as functional food and as a source of compounds of interest, allowing comparison between Portuguese and Serbian species.
- Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative studyPublication . Reis, Filipa S.; Barros, Lillian; Martins, Anabela; Ferreira, Isabel C.F.R.Herein, it was reported and compared the chemical composition and nutritional value of the most consumed species as fresh cultivated mushrooms: Agaricus bisporus (White and Brown mushrooms), Pleurotus ostreatus (Oyster mushroom), Pleurotus eryngii (King oyster mushroom), Lentinula edodes (Shiitake) and Flammulina velutipes (Golden needle mushroom). Shiitake revealed the highest levels of macronutrients, unless proteins, as also the highest sugars, tocopherols and PUFA levels, and the lowest SFA content. White and brown mushrooms showed similar macronutrients composition, as also similar values of total sugars, MUFA, PUFA and total tocopherols. Oyster and king oyster mushrooms gave the highest MUFA contents with similar contents in PUFA, MUFA and SFA in both samples. They also revealed similar moisture, ash, carbohydrates and energy values. This study contributes to the elaboration of nutritional databases of the most consumed fungi species worldwide, allowing comparison between them. Moreover it was reported that cultivated and the wild samples of the same species have different chemical composition, including sugars, fatty acids and tocopherols profiles.
- Biomolecule profiles in inedible wild mushrooms with antioxidant valuePublication . Reis, Filipa S.; Pereira, Eliana; Barros, Lillian; Sousa, Maria João; Martins, Anabela; Ferreira, Isabel C.F.R.The use of natural products isolated from mushrooms, included inedible species, against infection, cancer diseases and other oxidative-stress related diseases is one of the cornerstones of modern medicine. In the present work, the antioxidant molecule profiles of inedible mushroom species were evaluated and compared with those of edible species. The order of antioxidant abundance found in inedible wild mushrooms was: phenolics > flavonoids > ascorbic acid > tocopherols > carotenoids, similar to that of edible species. Furthermore the same energetic biomolecules were found including the disaccharide trehalose, the monosaccharide alcohol derivative mannitol and the fatty acids palmitic, oleic and linoleic acids. Fomitopsis pinicola revealed a very high phenolics concentration (388 mg GAE/g extract) and powerful antioxidant properties, mainly reducing power (EC50 value 60 g/mL similar to the standard Trolox®). It could find applications in the prevention of free radical-related diseases as a source of bioactive compounds.
- DecodE – an integrated approach to improve awareness and aid transition to natural additives in collaboration with the food industryPublication . Carocho, Márcio; Barreiro, M.F.; Reis, Filipa S.; Barros, Lillian; Ferreira, Isabel C.F.R.; Rodrigues, AlírioRecent studies indicate that consumers have a higher awareness of what they eat and are paying more attention to labels to consume healthier food. For food additives, there is still a high percentage of the population that does not understand their different classes, or origin (natural or artificial), along with many misconceptions. In line with consumer’s volition to reduce the consumption of processed foods and their tendency to prefer them non- or minimally processed, the DecodE project aims to help clarify the mystification of food additives, helping consumers and the general public know the differences between them, their implications on food and especially, their impact on consumer health. Furthermore, DecodE also aims at helping the gradual transition from artificial to natural preservatives and colourants, with research being carried out on both topics. This is performed in close collaboration with pastries and bakeries to help substitute the artificial additives used in these industries, and create sustainable methods of obtaining safe natural counterparts to be gradually introduced into the market, so, reducing their drawbacks, namely stability, high obtainment cost and low efficacy. All this is carried out in an integrated framework based on the cumulative knowledge of the research centre where the project is being executed, the Mountain Research Centre in Bragança, Portugal (www.cimo.ipb.pt), supported by various researchers with expertise in different topics. One of the main focuses of the project is to be open to society, hence the open data strategy, to reduce the gap between scientific data and the public’s knowledge of important aspects regarding their food.
- Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulatesPublication . Reis, Filipa S.; Barros, Lillian; Martins, Anabela; Vasconcelos, M. Helena; Morales, Patricia; Ferreira, Isabel C.F.R.In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.
- A irradiação como tecnologia pós-colheita viável para conservação de cogumelos, vegetais e plantas aromáticasPublication . Pinela, José; Reis, Filipa S.; Pereira, Eliana; Fernandes, Ângela; Oliveira, Beatriz; Antonio, Amilcar L.; Cabo Verde, Sandra; Ferreira, Isabel C.F.R.A irradiação é um tratamento pós-colheita utilizado para processar produtos destinados à indústria alimentar e farmacêutica. Esta tecnologia é frequentemente combinada com outros processos de conservação, tais como a secagem e o embalamento, para tirar partido de possíveis efeitos sinérgicos. De facto, a elevada perecibilidade de alguns alimentos e matérias-primas torna imprescindível aplicar tratamentos de conservação capazes de aumentar o tempo de vida útil, mas com um impacto mínimo em atributos de qualidade. Este estudo pretendeu avaliar a adequabilidade da irradiação para conservar parâmetros nutricionais de cogumelos, vegetais e plantas aromáticas durante o tempo de vida útil. O cogumelo e as plantas aromáticas foram irradiados com feixe de eletrões a uma dose máxima 10 kGy e analisadas após irradiação e passados 6 e 12 meses ou 12 e 18 meses, respetivamente. Os vegetais frescos foram irradiados numa câmara experimental de 60Co a uma dose máxima de 6 kGy. Estes foram analisados antes de irradiar e após 7 ou 14 dias de armazenamento a 4 °C. O tratamento de irradiação permitiu manter mais eficazmente o perfil nutricional do cogumelos e dos vegetais durante o armazenamento, comparativamente com amostras não irradiadas. Além disso, o tempo de armazenamento teve um maior impacto no perfil nutricional do cogumelo do que a irradiação (com exceção dos ácidos gordos). No caso das plantas aromáticas, apesar de a irradiação ter atenuado perdas de qualidade durante o armazenamento, não foi possível identificar uma tendência geral pois os fatores testados tiveram efeitos dissimilares nas diferentes espécies.
- A study on the medicinal mushroom Cordyceps militaris (L.) link: chemical characterization, antioxidant, antimicrobial and antiproliferative propertiesPublication . Reis, Filipa S.; Stojković, Dejan; Glamočlija, Jasmina; Ćirić, Ana; Van Griensven, Leo J.L.D.; Soković, Marina; Ferreira, Isabel C.F.R.The Cordyceps genus is well known for its medicinal properties. A number of bioactive constituents from Cordyceps species have been reported such as antibacterial, antifungal, immunopotentiating or antitumor agents [1]. The anti-inflammatory and anti-angiogenic properties of Cordyceps militaris (L.) Link have been reported as also the antioxidant activity of its cultured mycelium [2]. In this work, the chemical characterization of C. militaris was performed, includingbioactive compounds (free sugars, unsaturated fatty acids, tocopherols, organic acidsand phenolic compounds). The antioxidant potential of its methanolic extract was evaluated (reducing power, scavenging activity and lipid peroxidationinhibition) as also the antimicrobial activity (tested towards Gram positive and Gram negative bacteria and eight microfungi) and antiproliferative properties (tested in different human tumor cell lines). Mannitol and trehalose were the main free sugars found in this species (2.01 and 24.71 g/100 g dw, respectively). The fatty acid quantified in higher amounts was linoleic acid (68.00% of total fatty acids). a-Tocopherol was the only isoform of vitam in E detected (55.86 j.lg/1 00 g dw). The organic acids found in this mushroom were oxalic (0.33 g/1 00 g dw), citric (7.97 g/1 00 g dw) and fumaric (0 .1 3 g/100 g dw) acids. p-Hydroxybenzoic acid was the only phenolic acid quantified in C. militaris (0.02 mg/1 00 g dw), but cinnamic acid was also found (0. 11 mg/1 00 g dw). The lowest EC50 values for the antioxidant potential were presented in lipid peroxidation inhibition assays, namely in 13-carotene/l inoleate and TBARS assays (1.05 mg/ml and 0. 77 mg/ml, respectively). The methanolic extract also revealed strong antibacterial and antifungal activities, and it was able to inhibit the proliferation of MCF-7 (breast}, NCI-H460 (non-smalllung),HCT-15 (colon) and Hela (cervical) human carcinoma cell lines.
- Potencial antitumoral de cogumelos silvestres: um caso de estudo com espécies Leccinum.Publication . Reis, Filipa S.; Sousa, Diana; Martins, Anabela; Morales, Patricia; Ferreira, Isabel C.F.R.; Vasconcelos, M. HelenaAlém de constituírem uma excelente opção como alimento devido ao seu valor nutricional, os cogumelos (silvestres e/ou comerciais) têm sido descritos como matrizes naturais com inúmeras propriedades bioativas, incluindo potencial antitumoral [1,2]. No presente trabalho foi estudado um extrato fenólico, obtido da espécie Leccinum vulpinum Watling, que revelou ser rico essencialmente em ácidos hidroxibenzóicos (nomeadamente ácido gálico, ácido protocatéquico e ácido p-hidroxibenzóico). Foi efetuado um screening inicial, testando o referido extrato contra um painel de linhas celulares tumorais humanas (MCF-7: adenocarcinoma de mama; NCI-H460: carcinoma de pulmão; HCT-15: adenocarcinoma colorectal; e AGS: adenocarcinoma gástrico), de forma a verificar o seu efeito no crescimento celular das linhas em estudo. Uma vez que a proliferação celular foi inibida em todas as linhas testadas, e dada a evidência de uma relação inversa entre o consumo de cogumelos e a redução do risco de incidência do cancro da mama [3], foi efetuado um estudo detalhado na linha celular MCF-7. Para tal, foram realizados alguns ensaios funcionais, de modo a verificar os efeitos do extrato na proliferação celular, no perfil de ciclo celular e apoptose. O potencial do extrato como indutor de danos ao nível do ADN foi também avaliado. De uma forma geral, o extrato fenólico da espécie L. vulpinum diminuiu a proliferação celular, sendo que o tratamento celular com uma concentração de extrato correspondente ao GI75 reduziu significativamente a percentagem de células na fase S do ciclo celular. Adicionalmente, o extrato induziu apoptose, sendo que o tratamento celular com uma concentração de extrato correspondente ao GI50 e ao GI75 aumentou a percentagem de células em apoptose para 13,4% e para 27%, respectivamente, enquanto os valores controlo de apoptose foram de 6 - 9%. Os resultados obtidos sugerem também que o extrato em estudo causou danos ao nível do ADN celular, uma vez que proteínas envolvidas na reparação de danos no ADN, nomeadamente PARP, ficaram sobre-expressas nas células tratadas com o extrato. Isto foi confirmado pelo Ensaio Cometa (Alkaline Comet Assay), tendo sido verificado um aumento significativo da percentagem de ADN na “cauda” do Cometa, após tratamento celular com 250 mg/mL de extrato.