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Garcinia mangostana L., commonly known as mangosteen or queen of fruit, is a tropical fruit belonging to the Clusiaceae family. This shrub is native from South Asia, but can also be found in other tropical territories, such as South America [1,2,3]. The fruit comprises an inedible dark purple or red pericarp (> 60%) that encases the edible succulent pulp [2]. Nowadays, mangosteen pulp and pericarp have been used in beverages as a food supplement by virtue of the traditional knowledge about its benefits. However, a poor correlation between the medicinal use of mangosteen and its chemical composition has been described [2,3]. Aiming to contribute to the elucidation of the phytochemical composition of this fruit pericarp, the present study was carried out to characterize anthocyanins by HPLC-DAD-ESI/MS. In addition, the bioactivities of the hydroethanolic extract obtained by solid-liquid extraction were evaluated. The extract cytotoxicity to four human tumour cell lines (NCI-H460 - lung carcinoma, MCF-7 - breast carcinoma, HepG2 - hepatocellular carcinoma, and HeLa - cervical carcinoma) was tested by the sulforhodamine B assay. The anti-inflammatory activity was evaluated by the ability to inhibit the lipopolysaccharide-induced nitric oxide (NO) production by murine macrophage RAW 264.7 cells. Lastly, the antioxidant activity was measured by the extract capacity to inhibit the oxidative haemolysis (OxHLIA) and the formation of thiobarbituric acid reactive species (TBARS), using sheep erythrocytes and porcine brain cells as oxidizable substrates. The chromatographic analysis allowed the identification of two anthocyanins in the mangosteen pericarp, namely cyanidin-O-dihexoside, which was the most abundant, and delphinidin-O-pentoside. Regarding the in vitro evaluated bioactive properties, the extract showed cytotoxic activity against the four human tumour cell lines, with GI50 values lower than 75 μg/mL. It also exhibited antioxidant and anti-inflammatory activities at low concentrations, with IC50 values of 8.7 and 17.3 μg/mL for the TBARS and OxHLIA assays, and an EC50 value of 85 μg/mL for the NO production inhibition assay, respectively. The results obtained in this study allow to conclude that G. mangostana pericarp contains an interesting amount of anthocyanins and also exhibits strong bioactivities, which makes it an interesting source of bioactive ingredients. To the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UIDB/00690/2020). M.I. Dias, J. Pinela, R.C. Calhelha and L. Barros thank their research contracts (national funding by FCT, P.I., through the institutional scientific employment program-contract) and B.R. Albuquerque thanks her PhD grant (SFRH/BD/136370/2018). This work was funded by the FEDER 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®. To FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

Table tomato (Solanum lycopersicum L.) is highly consumed worldwide and its annual production generates a considerable amount of plant by-products [1]. Although tomatoes are well-known for their composition in nutrients and bioactive compounds [2,3], little information is available in literature regarding tomato leaves, probably because these by-products have no commercial value. Therefore, considering the enormous production of this biomass and the current trends of circularity and sustainable development [3], this study was carried out to evaluate the polyphenolic composition and bioactive properties of tomato leaves resulting from the regeneration of table tomato landraces conserved ex-situ in the Portuguese Genebank (BPGV), in Braga, Portugal. The tomato plant material resulting from pruning performed after the flowering season was lyophilized and ground to a fine powder, used to prepare hydroethanolic extracts [4]. These extracts were characterized for their composition in polyphenols (by HPLC-DAD-ESI/MSn) and used to evaluate the antioxidant (by DPPH• scavenging capacity and reduction power), antihemolytic (using sheep erythrocytes and AAPH as a free radical generator), and antimicrobial (tested against food-borne bacterial and fungal strains by the serial microdilution and p-iodonitrotetrazolium violet colorimetric methods) activities [4]. The chromatographic analysis allowed identifying phenolic acids and flavonoids, with prevalence of quercetin-3-O-rutinoside (rutin). The leaf extracts showed antioxidant activity, with EC50 values lower than those previously reported for the respective tomato fruits, thus translating a higher activity [2]. The extracts were also effective in some extent in protecting the erythrocytes from the oxidative hemolysis caused the thermal decomposition of the free-radical initiator AAPH. Despite their low activity against the tested microfungi, some extracts had ability to inhibit and kill some bacteria (including Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and Enterobacter cloacae) more effectively than the antibiotic ampicillin. It was concluded that table tomato crop by-products can be used to produce rutin-rich extracts with antioxidant and antibacterial activities for possible use in the agri-food sector as natural preservatives. 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); to FCT for J. Pinela (CEECIND/01011/2018) and L. Barros contracts through the individual and institutional scientific employment program-contract, respectively, and for M. Añibarro-Ortega PhD grant (2020.06297.BD); to FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P; and to the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007). info:eu-repo/semantics/publishedVersion

The use of unconventional food plants (PANC) has been an asset for the food industry, due to its abundance, the non-competing with other vegetable matrices used for human consumption, and their nutritional properties, chemical and bioactive potentiality [1,2]. The present work aimed to study the flour of three PANC seeds, namely Guizotia abyssinica (Lf, niger) Cass., Panicum miliaceum L. (millet) and Phalaris canariensis L. (birdssed), starting with the study of its granulometry and water absorption index. Furthermore, the nutritional value (AOAC methods), free sugars (HPLC-RI), fatty acids (GC-FID), organic acids (UPLC-DAD), tocopherols (HPLC - fluorescence) and phenolic compounds (HPLC-DAD / ESI (MS) composition as also assessed, followed by the antioxidant, hepatotoxic and antimicrobial potential of the hydroethanolic extracts. Finally, bread samples were prepared with partial replacement of the wheat flour (20% of the PANC’s flour), and a centroid simplex statistical method was applied to understand the effect of applying PANC flours on the final physical-chemical characteristics of the breads obtained. The flour of the three seeds presented high granulometry, associated with high water absorption index (indicating that their use would have to be complemented with other flours for bakery application). Niger seed was the one that stood out the most, presenting higher contents of total fat, PUFA, sugars, tocopherols and phenolic compounds. The hydroethanolic extracts presented relatively low IC50 and MIC values for inhibition of lipid peroxidation and for antimicrobial activity, respectively. The results obtained for antifungal activity of niger and millet were lower when compared with the positive controls used (E211 and E224). The samples didn’t present hepatotoxicity. The partial replacement of 20% wheat flour by millet and birdseed flour allowed to obtain similar breads as the control samples in terms of texture, specific volume and color. This study allowed to present innovative results in relation to the nutritional, chemical and bioactive properties of seeds that have been little studied and consumed, but with great potential to be applied in the food industry. Considering their composition in bioactive compounds, the use of these seeds is highly advisable in the context of a fortified diet, being sources of compounds of high nutritional value and with beneficial effects for the health of the consumer. To the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019); the national funding by FCT, P.I., through the institutional and individual scientific employment program-contract for L. Barros/M.I. Dias/C.Pereira; to FEDER-Interreg España-Portugal programme (TRANSCoLAB 0612_TRANS_CO_LAB_2_P). To the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Digital strategies in biological assets to improve well-being and promote green health, Norte-01-0145-FEDER-000042; to the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007). info:eu-repo/semantics/publishedVersion

Edible mushrooms have been appreciated globally for their organoleptic, nutritional and chemical properties. In the present study, fruiting body of Calocybe gambosa , wild growing in Serbia has been chemically characterized (content of macronutrients, soluble sugars, tocopherols, fatty and organic acids) and its bioactive properties (antimicrobial and antioxidant) evaluated. Obtained results suggest that this mushroom is a source of carbohydrates and proteins, with low fat content. Sugar analysis revealed presence of trehalose and mannitol. Tocopherol composition revealed presence of α-tocopherol, while fatty acid analysis revealed presence of 24 fatty acids with prevalence of polyunsaturated fatty acids. Amongst organic acids, oxalic, quinic, malic, citric and fumaric acid were detected. Comprehensive antioxidant analysis (reducing power, DPPH scavenging activity, β -caroten/linoleic acid and TBARS assay) indicate that mushroom is a perspective antioxidant, whereas its antimicrobial potential turned out to be moderate. Nevertheless, at sub-inhibitory level methanolic extract disrupted cell-to-cell communication using Pseudomonas aeruginosa PAO1 as a model system. Finally, enrichment of oatmeal cookies with C. gambosa flakes not only improved nutritional value of cookies, but was praised among participants in sensory evaluation test ( Table 1 ), indicating that along with results of chemical composition and biological activity, this mushroom has potential to be regarded as functional food. This research was funded by Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 451-03-68/2020-14/200007. info:eu-repo/semantics/publishedVersion

Tomatoes are highly consumed worldwide and their annual production generates a significant amount of plant by-products, especially aerial biomass. Although tomatoes are well-known for their nutritional composition and health-promoting effects [1], little information is available in the literature on the plant remains of this crop. Therefore, considering the substantial availability of this natural resource and the current trends of circularity and sustainable development, this study was carried out to evaluate the phenolic profile and bioactive properties of tomato plant biomass resulting from the regeneration of table tomato landraces conserved ex-situ in the Portuguese Genebank (BPGV), Braga, Portugal. The plant material resultant from the pruning performed after the flowering season was lyophilized and ground to prepare hydroethanolic extracts, which were characterized for their polyphenols composition (by HPLC-DAD-ESI/MS) and used to evaluate the antioxidant activity (through oxidative haemolysis inhibition, DPPH scavenging capacity, and reduction power assays) and antimicrobial potential against food-borne bacteria and fungi (by serial microdilution and p-INT methods) [2]. The chromatographic analysis allowed identifying phenolic acids and flavonoids, with prevalence of quercetin-3-O-rutinoside. The extracts showed antioxidant activity, with EC50 values lower than those previously reported for the respective table tomato fruits, thus translating a higher activity [2]. The extracts were also effective in some extent in protecting the erythrocyte population from the oxidative hemolysis caused the thermal decomposition of the free-radical initiator AAPH. Despite their low activity against the tested microfungi, some extracts had ability to inhibit and kill some bacteria (including Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and Enterobacter cloacae) more effectively than ampicillin. Overall, it was concluded that table tomato plant biomass can be valorized to produce phenolic-rich extracts with antioxidant and antibacterial activities for possible use in the agri- food sector as natural preservatives. The authors are grateful to Foundation for Science and Technology (FCT) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); to FCT for the J. Pinela (CEECIND/01011/2018) and L. Barros contracts through the scientific employment program-contract, and for the M. Añibarro-Ortega grant (2020.06297.BD); to FEDER-Interreg España-Portugal programme for financial support through project TRANSCoLAB 0612_TRANS_CO_LAB_2_P; to the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007). info:eu-repo/semantics/publishedVersion

The planet’s resources are increasingly scarce, a reason why their most efficient use is gaining more atten- tion. Several studies have shown the influence of genetic background, environmental conditions, and different plant tissues on the polyphenolic composition and consequently on its bioactive potential, leading to different industrial applications.1 Cynara cardunculus L. (Asteraceae), commonly known as cardoon, is a Mediterranean herbaceous plant with important economic value as a result of its diversified industrial applications (i.e. pro- duction of biomass, bioenergy, paper pulp, pharmaceutics and cosmetics).1,2 The present work aims to valorize underused parts of cardoon by evaluating the phenolic profile and bioactive properties of different plant tissues. Petioles, blades, heads, bracts, and seeds from a Greek cardoon genotype were collected at different growth stages throughout the growing season depending on the plant part, e.g. petioles and blades were col- lected throughout the growing season, heads and bracts during the flowering stage and seeds after seed for- mation and until the seed ripening stage. The phenolic profile was determined by HPLC-DAD-ESI/MS and the antioxidant activity were measured through cell-based assays, namely TBARS and OxHLIA. Cytotoxic effects were screened in four human tumor cell lines and hepatotoxicity in a non-tumor cell line (PLP2) by the sul- forhodamine B assay. Anti-inflammatory potential was tested through the inhibition of NO production. Finally, the antibacterial and antifungal activities were evaluated by the broth microdilution method. The blades of car- doon presented the highest bioactive activity and variety in phenolic compounds. Considering the maturation stage, the bracts stand out at the intermediate stage, showing a higher antioxidant potential for TBARS assay, and the petioles a higher anti-inflammatory activity. In turn, at the most advanced stage of maturity, the seeds presented lower EC50 values in the OxHLIA assay. More immature plant tissues presented a higher content in phenolic compounds, and as expected, higher antioxidant activity in the OxHLIA assay. The most advanced states of maturation also presented highest cytotoxic and anti-inflammatory activity. For the antibacterial and antifungal activities, the results obtained were not affected by different plant parts or by the maturation stages studied. The results obtained in the present work, allows us to choose the best harvesting time of cardoon, allowing achieving a greater variety of phenolic compounds, and consequently, a highest bioactive potential, and ultimately the most appropriate use of their constituents. Nevertheless, further studies are needed to better understand the compounds responsible for the observed activities. To the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019); PhD grant of Filipa Mandim (SFRH/146614/2019); and M.I. Dias, Â. Fernandes, J. Pinela, and L. Barros contracts. Also, to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E and TRANSCoLAB 0612_TRANS_CO_LAB_2_P. info:eu-repo/semantics/publishedVersion

The innovation in traditional Portuguese foods can be a differentiating and dynamic factor in the food industry. In line with the market tendencies to promote the consumption of healthier foods, plants (dried material and aqueous decoctions) were incorporated in sheep cheese. The used plants were chestnut flowers (Castanea sativaMill.), lemon balm (Melissa officinalisL.) and basil (Ocimum basilicumL.). The incorporations were carried during cheese production, being left at the dairy Queijos Casa Matias, for one month for ripening. After that time, one lot was subject to various assays, while another lot was left for six months to mature, after which, they were also subject to the same assays. The analysed parameters were the antioxidant activity, nutritional composition, fatty acids and minerals. The plants and their extracts functionalized the cheeses. Nutritionally, proteins and fat were the most abundant nutrients. In terms of individual fatty acids, palmitic (C16:0) and oleic (C18:1) acids were the most abundant. The saturated fatty acids were the most prevalent followed by the monounsaturated. Finally, in terms of minerals, calcium and sodium were the most abundant elements. Conservation wise, the incorporation allowed a faster loss of moisture along the storage time, which reduced the risk of contamination by microorganisms, while also preserving some unsaturated fatty acids. Furthermore, the functionalization of the cheeses allowed conferring beneficial properties to the consumer’s health., A inovação em alimentos tradicionais Portugueses pode ser um fator de diferenciação e dinamizador da indústria alimentar. Em linha com a tendência de mercado de impulsionar o consumo de alimentos mais saudáveis, foram incorporadas plantas (material seco e extratos aquosos obtidos por decocção) em lotes de queijo de ovelha. As plantas usadas foram flores de castanheiro (Castanea sativaMill.), cidreira (Melissa officinalisL.) e manjericão (Ocimum basilicumL.). As incorporações no queijo foram efetuadas durante o seu fabrico, maturando durante um mês na empresa Queijos Casa Matias. Ao fim desse tempo foram sujeitos a vários ensaios após chegada ao laboratório e após seis meses de modo a verificar as alterações durante a maturação. Foi analisada a atividade antioxidante, bem como a composição nutricional, ácidos gordos individuais e minerais. Ficou patente que as plantas e os seus extratos funcionalizaram os queijos, conferindo-lhe bioatividade. A nível nutricional, os nutrientes mais abundantes foram as proteínas e a gordura. Em relação aos ácidos gordos individuais, o mais abundante foi o ácido palmítico (C16:0) seguido do ácido oleico (C18:1). Os ácidos gordos saturados prevaleceram, seguidos dos monoinsaturados. Quanto aos minerais, os mais abundantes foram o cálcio e o sódio. Ao nível da conservação, a incorporação das plantas nos queijos resultou numa diminuição de humidade mais rápida, reduzindo o risco de contaminação por microrganismos, ao mesmo tempo que permitiu a conservação de alguns ácidos gordos insaturados. Por outro lado, a funcionalização dos queijos permitiu conferir algumas propriedades benéficas à saúde.

Cynara cardunculus L., commonly known as cardoon, is a perennial plant that belongs to the Asteraceae family. Native to the Mediterranean basin, it is also found in other regions of the world such as Argentina, Africa, Chile, China, United States, and Peru [1]. It has been used in folk medicine due to its diuretic and dyspeptic properties, such as liver tonic and in hepatic disease treatments [2]. Its flowers are the most commonly used part due to its milk coagulating properties (vegetable rennet in goat and sheep cheeses manufacture), but its immature heads are also widely consumed as food in several dishes. In recent years, the expansion of different industrial applications of crop residues (e.g. forage, paper pulp industry, or biodiesel and energy production) has been a key lever to the growth of Mediterranean cardoon cultivation [3]. Cardoon is considered a functional food as a result of its richness in compounds with high addedvalue and proved health benefits [1,4]. Several studies make reference to its high content in phenolic compounds, such as phenolic acids and flavonoids including anthocyanins. The polyphenolic content of different plant parts of this species is strongly influenced by environmental conditions and harvest stage. Therefore, studies regarding the influence of these factors are extremely important to better understand the benefits associated to this plant. In the present study, the phenolic compounds composition of different plant tissues of cardoon (petioles, blades, heads, bracts and seeds) was analysed. Samples of cultivated cardoon (var. altilis) were collected in Greece in June 2018. The phenolic composition was analysed in hydroethanolic extracts by HPLC-DAD-ESI/MS. Cardoon petioles, blades, heads, bracts and seeds presented differences in the phenolic composition, as also in their contents of individual compounds. Blades showed the highest number of phenolic compounds, being identified thirteen molecules. In petioles and bracts, a total of eleven phenolic acids and flavonoids were found, while in heads and seeds a total of nine and seven molecules were profiled, respectively. Blades were the plant part with the highest concentration of phenolic compounds, followed by bracts, petioles, seeds and heads. Caffeoylquinic and dicaffeoylquinic acids were the main compounds present in all the studied tissues. The results obtained demonstrated that phenolic compounds allocation varies among cardoon plant parts harvested at the same time period, with blades being the part that showed the highest abundance and variety of phenolic compounds. 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) and L. Barros contract. Also, to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

Extrusion-cooking technology is a high-temperature and short-time process, necessary to cause structural, physico-chemical and nutritional changes of raw materials, forcing the material to flow under different conditions (temperature, moisture, screw speed, and feed). Rice and legumes have a great potential in the development of healthier gluten-free products than traditional snacks [1]. Rice flour (Oryza Sativa L.) has been reported as a good raw material to obtain expanded food products [1]. Dry beans (Phaseolus vulgaris L.) are rich in proteins, dietary fibre, complex carbohydrates (leading to low glycaemic index), minerals and numerous phytochemicals endowed with useful biological activities [2]. Carob fruit (Ceratonia siliqua L.) is a tree leguminous native to the Mediterranean region. The fortification with carob fruit would provide adequate fibre content and a good amount of bioactive compounds. In previous studies, carob evidenced therapeutical properties against several diseases, such us, regulatory effect in blood glucose level, reduction in low-density-lipoprotein cholesterol in hypercholesteraemic patients, body weight benefits, and improved digestion, and lipid utilization [3]. In this study, novel gluten-free extruded foods (composed by rice: 50-80%, bean: 20-40%, and carob: 0-10%) were performed using a twin-screw extruded at CARTIF (Valladolid, Spain) and the effects of extrusion were evaluated regarding bioactive properties. Commercial extruded rice was used as external control. Raw materials, non-extruded, and extruded samples were extracted with ethanol/water (80:20, v:v) and purified using C18 SepPak® Vac 3 cc cartridge [4,5], in order to obtain an extract rich in bioactive compounds. The cytotoxicity, anti-inflammatory, and antimicrobial activity were evaluated following previously reported procedures [6, 7]. Carob, commercial extruded rice and most of the extruded samples, showed cytotoxicity in the majority of the tumour cell lines tested (HeLa - cervical carcinoma, HepG2 - hepatocellular carcinoma, MCF-7 - breast adenocarcinoma, and NCI-H460 - non-small cell lung cancer). While, bean, rice, and most of the non-extruded samples presented no toxicity (GI50 value >400 µg/mL) using a non-tumour porcine liver cell culture (PLP2). In general, it was observed that extrusion process improves the cytotoxic potential in the rice-legumes sample mixtures, revealing lower GI50 concentrations (ranging between 115 and 362 µg/mL). The anti-inflammatory activity revealed a high heterogeneity, presenting bean and carob samples, the highest activity in comparison to the extruded samples. Concerning the antimicrobial activity, which was tested using a panel of multi-resistant isolated clinical strains, a low potential was observed, with non-extruded and extruded samples revealing higher values of minimum inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC). In conclusion, rice-legumes flours, rice blends, beans, and carob fruits are a great alternative for the development of new gluten-free snacks products, in a market dominated mainly by cereals, due to the presence of different bioactive compounds, such as phenolic compounds, which can give healthier benefits to the consumers. The authors are grateful to FCT, Portugal and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros and R. Calhelha contracts. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E, to the Spanish Ministry of Economy and Competitiveness (Project RTA2012-00042-C02) and INIA for the financial support of C. Arribas. info:eu-repo/semantics/publishedVersion

Cardoon is a multi-purpose crop for several industries. In this study, cardoon seeds were separated according to the viability and characterized for their chemical composition and bioactivities. Viable seeds contained higher levels of α-tocopherol (6.7 mg/100 g), lipids (23.11 g/100 g, manly oleic and linoleic acids), and free sugars (5.4 g/100 g) than the unviable ones. The hydroethanolic extract of viable seeds presented a higher concentration of phenolic compounds, namely 5-O-caffeoylquinic (8.0 mg/g) and 3,5-O-dicaffeoylquinic (43.9 mg/ g) acids, and greater in vitro antioxidant activity. Both extracts showed antibacterial properties, but the best results were achieved with unviable seeds. The extracts had similar antifungal activity but did not reveal antiinflammatory capacity or cytotoxicity to the tested cell lines. Therefore, while viable seeds contained antioxidant phytochemicals and an energy and carbon source for germination, unavailable seeds stood out for their potential to be used in the development of bio-based antibacterial ingredients. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). National funding by FCT, through the institutional scientific employment program-contract with M.I. Dias, J. Pinela and L. Barros. To FCT for the PhD grant (SFRH/BD/ 146614/2019) of F. Mandim. The authors are also grateful to FEDERInterreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E, to the project TRANSCoLAB (0612_TRANS_CO_LAB_2_P), and to the Ministry of Education, Science and Technological Development of the Republic of Serbia for Grant No. 173032. The authors are grateful to Manuela Antunes from Cynatura for the seed recollection. info:eu-repo/semantics/publishedVersion

Background: Mushrooms are a good source of protein, dietary fibre, vitamins, minerals and phenolic compounds. However, mushrooms are a very perishable food and the implementation of preservation methods is essential to extend their shelf-life. The preservation methods for mushrooms can be classified into three categories: thermal (drying/freezing), chemical (edible coatings, films and washing solutions) and physical (packing, irradiation, pulsed electric field and ultrasound) processes. These processes can change the nutritional value and bioactive properties of this commodity. Scope and approach: The goal of this review is to critically update and discuss the existing information about the effect of postharvest preservation methods on the nutritional value and bioactive properties of edible mushrooms. Key findings and conclusions: Drying, especially when high temperatures are applied, can cause the degradation of polysaccharides, proteins and flavour compounds. Freezing is one of the best methods to extend mushrooms’ shelf life but cause the loss of vitamins. Edible coatings and films improve the total sugar, ascorbic acid and bioactive compounds preservation during the storage period. Washing solutions decrease amino acids content. Gamma and electron-beam irradiation decrease the unsaturated fatty acid content, whereas UV-B irradiation significantly increases the vitamin D content. However, there is still limited information about the impact of chemical processes, packaging, pulsed electric field and ultrasound on the nutritional composition and bioactive properties of mushrooms, opening research opportunities for future. This review presents technological and economic alternatives that may support the mushroom processing industries to obtain value-added edible mushrooms and related products. The authors are grateful to the European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating [PDR2020-101- 031472] and European Regional Development Fund (ERDF) through the Competitiveness and Internationalization Operational Program (POCI), within the scope of project MicoBioExtract [POCI-01-0247-FEDER- 033939]. We would also like to thank the National Funds from FCT - Fundação para a Ciência e a Tecnologia through project UID/Multi/50016/2019, FCT/MEC (PIDDAC) - IF/00588/2015 and FCT individual PhD grant number SFRH/BD/145301/2019. The authors are also grateful to FCT, Portugal for financial support through national funds FCT/MCTES to CIMO [UIDB/00690/2020] and national funding by FCT, P.I., through the institutional scientific employment programcontract for L. Barros. info:eu-repo/semantics/publishedVersion

N-3 fatty acids are considered essential for human health, since they are associated with beneficial effects against many diseases of modern world. The most common dietary sources of n-3 fatty acids are fishes such as salmon and sardines among others, which are also incriminated with high heavy metals content. Flax or linseed is the most widely known plant source of n-3 fatty acids and is the main ingredient of food supplements. The aim of the present study was to evaluate seeds and seed oils of purslane as alternative sources of n-3 fatty acids. For this purpose, purslane seeds, oils from seeds extracted with three methods (screw press methods with or without a cooler), and seed presscakes were evaluated in terms of fatty acids composition and cytotoxicity. For comparison purposes, three commercial linseed oils, one cucurbit oil and one luffa oil were also analyzed in terms of fatty acids composition and cytotoxicity against various human tumor cell lines. The results of the study showed that linseed oils were abundant in α-linolenic acid (65.62%-71-90%), followed by linoleic and oleic acids (12.87%-17.98% and 6.43%-7.09%, respectively), while n6/n3 and PUFA/SFA ratios were lower than 4.0 and higher than 0.45, respectively. In contrast, cucurbit and luffa oils have very low amount of α-linolenic acid, since they contained mostly linoleic, oleic, palmitic and stearic acid, which resulted in very high values of n6/n3 ratios. The main detected fatty acids in purslane seed oils were α-linolenic and linoleic in amounts that depended on the extraction method. Seeds and seed presscakes also contained high amounts of α-linolenic acid and linoleic acids; however, extraction method affected fatty acids composition and values of n6/n3 and PUFA/SFA ratios. Finally, luffa oil showed the highest cytotoxicity against HeLa and NCI-H460 tumor cell lines, followed by cucurbit and one of the tested linseed oils. In conclusion, purslane seeds and seed oils could be considered as alternative n-3 fatty acids, while they have a balanced fatty acids composition and show moderate cytotoxicity against specific tumor cell lines. info:eu-repo/semantics/publishedVersion

Phosphorus is a macronutrient regularly applied in olive groves even though no studies exist demonstrating the need for its application. In this work, results of two field trials and two pot experiments are presented where the response of olive tree to phosphorus application was studied from 2013 to 2017. One of the field trials was installed in a three-year-old olive grove where it was already possible to start evaluating olive yield. The second field trial started from just planted young cuttings to evaluate the biomass produced and phosphorus uptake. One of the pot experiments consisted on the use of four phosphorus rates and the other on the use of four different soils and two phosphorus rates. In the first trial, there was no significant response to phosphorus application in olive yield or biometric parameters of the fruit such as fruit size and pulp/ pit ratio. In the other three trials, only in the second pot experiment an increase in biomass production by the application of phosphorus was observed. This experiment included acidic soils, which may have greatly influenced the availability of phosphorus to the plants. In three of the four experiments leaf phosphorus concentration increased in response to phosphorus application. The pot experiments showed that roots accumulate appreciable amounts of phosphorus and that the application of phosphorus increased proportionally more the concentration of phosphorus in roots than in leaves or stems. In one of the experiments the root/shoot ratio increased with the application of phosphorus. These results seem to indicate that roots are important tissues for phosphorus accumulation which can buffer phosphorus in the shoots in periods of lower phosphorus availability in the soils and may contribute to explain the difficulty to find a response of the olive tree to the phosphorus application under field conditions. info:eu-repo/semantics/publishedVersion

Garlic (Allium sativum L.) is one of the most economically important species of the Allium genus throughout the world. One of the major beneficial effects of garlic is related with antioxidant properties which have been associated with many therapeutic effects, including cancer prevention, antithrombotic effects, cardiovascular protection and anti-aging effects. The aim of the present study was to determine the bioactive properties from various Greek garlics in order to determine the variability in their antimicrobial and antioxidant properties, as well as to compare them with commercial cultivars. The collected samples included: one local garlic cultivar from Evros “Nea Vissa” (samples G1 and G2); one sample of a local landrace and one sample of Chinese origin cultivated in Neapoli; (samples G3 and G4, respectively); one sample from Euboea Prefecture of Chinese origin (G5); one sample of local cultivar of “Platykampos”, one commercial sample of Chinese origin and one sample of commercial cultivar Gardos, all cultivated in Magnissia Perfecture (G6-G8, respectively), and three samples from Arcadia Prefecture of local cultivar of “Tripoli” (G9-11). The antioxidant activity was evaluated by DPPH radical-scavenging activity, reducing power, inhibition of beta-carotene bleaching and inhibition of lipid peroxidation. The antimicrobial activity was evaluated against four Gram positive bacteria, six Gram negative bacteria and two fungi, using the microdilution method, in order to obtain the minimal growth inhibititory concentrations and minimal bactericidal/funcicidal concentrations. Genotype G5 showed the lowest EC50 values for all the tested antioxidant activity assays. The antimicrobial activity was significant, especially against the bacteria P. mirabilis and antibiotic resistant E. coli. Significant variation was observed between the studied garlic genotypes, indicating the importance of both growing conditions and genotype on the bioactive properties of dry garlic. This variation could be further exploited in breeding programs in order to select elite genotypes with increased bioactive properties. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), grant to A. Fernandes (SFRH/BPD/114753/2016) and L. Barros contract. info:eu-repo/semantics/publishedVersion

Taking into account the role of potassium in plant nutrition it is poorly understood the lack of studies on olive tree response to potassium application. This paper reports the results of two field trials and two pot experiments on potassium fertilization in olive carried out from 2013 to 2017. The two field trials and one of the pot experiments were classic experiments on the plant response to the nutrient application. The second pot experiment was arranged in a factorial design with two leveis of potassium, two water regimes and two cultivars ('Cobrançosa' and 'Arbequina'). The application of potassium did not increase the growth and yield of the trees although it increased the concentration of the nutrient in the tissues. The application of potassium increased the shoot/root ratio. The potassium concentration in the roots was lower than in the aerial parts for low leveis of potassium in the soil but increased more than proportionally in the root relative to the shoot as the availability of potassium in the soil increased. These results seem to indicate that the shoots are a priority sink for K and that the roots can act as a reservoir when the soil K availability is high. lndices of plant water status and chlorophyll a fluorescence parameters were not significantly affected by K applications. Plants suffering from water stress, confirmed by índices of leaf water status and chlorophyll a fluorescence parameters, yielded less phytomass. 'Cobrançosa' appeared as a more tolerant cv. to water stress than 'Arbequina; being the former more suitable to be grown under rainfed conditions and probably more adapted to climate change. info:eu-repo/semantics/publishedVersion

Renewed interest on relationship between poor nutrition and etiology and the development of various diseases affecting mankind has led to the increased number of scientific studies dealing with the consumption of certain food as medicine. Factors that contribute to this include rapid advances in science and technology and raising awareness of consumers in obtaining good health through quality diet wherein functional foods occupy an important place. Since renowned wild growing Oyster mushroom has a long tradition of use as a food, herein, we reported a detailed chemical profile regarding nutrient and selected metabolite profile. In addition, we evaluated the ability of methanolic extract obtained from mushroom basidiocarps to reduce in vitro growth of selected pathogenic microorganisms in order to assess functional properties of the mushroom that can provide health benefits for the consumers. Obtained results indicate carbohydrates were the most abundant compounds, followed by proteins and fat. Free sugar trehalose was present in considerable amount, unlike glucose and fructose. With low energy value, fruiting bodies of wild growing Oyster mushroom represent a healthy, low-calorie food, recommended for use on a daily basis. As for selected metabolite analyses, results revealed the presence of α-tocopherol, oxalic, malic, citric, fumaric and p-hydroxybenzoic acid. Results of antibacterial activity revealed potent activity against eight species of bacteria, among which Pseudomonas aeruginosa was the most susceptible to the activity of methanolic extract. Overall, proximate composition and energy value, as well as the presence of various metabolites (phenolic and organic acids as well as tocopherols) and antibacterial activity, indicate that Oyster mushroom may be a candidate for functional food or a source of functional ingredients used for the development of new, enriched food products. info:eu-repo/semantics/publishedVersion

Ficus carica L. is one of the first domesticated trees of the Neolithic Revolution; it is native to western Asia and was later spread to the Mediterranean region. Its fruits, figs, are widely consumed in fresh or dried form, or used for spirit beverage and jam due to their sweet taste and well-known nutritional properties [1, 2]. Nevertheless, the fruit peel is often discarded in the preparation of food products that only require the pulp, and this part of the fruit is reported as a good source of phenolic compounds with several beneficial properties for human health [3], thus the importance of studying the different parts separately is justified. Therefore, the aim of this study was to explore and compare the chemical composition of green fig pulp and peel. For that purpose, the phenolic composition was assessed by HPLC-DAD-ESI/MS, organic acids by HPLC-PDA, and tocopherols by HPLC-fluorescence. Regarding phenolic compounds, the highest concentration was found in the peel, which revealed the presence of flavonoids and phenolic acids in similar concentrations, with quercetin-3-O-rutinoside, 5-O-caffeoylquinic acid, and vanilic acid malonyl-di-deoxyhexoside as the major compounds detected. On the other hand, the pulp presented a prevalence of phenolic acids, among which caffeic acid hexoside was the most abundant one. In terms of organic acids, despite the higher amounts of oxalic and succinic acids found in the pulp, the peel showed higher concentrations of quinic, malic, citric, and total organic acids. The tocopherols profile was similar for both samples, with the four isoforms being detected; α-, β-, and total tocopherols were mostly found in the peels, whereas γ- and δ-tocopherol were prevalent in the pulp. The results presented highlight the importance of this fruit in human diet, given its richness in nutritional and bioactive compounds that contribute for a balanced and functional diet. Additionally, the chemical composition of fig peel justifies the recovery of this part of the fruit for further application in food industry, in the cases where it is discarded for the preparation of fig-based products. 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), C. Pereira grant (SFRH/BPD/122650/2016), and L. Barros contract. To FEDER-Interreg España-Portugal programme for financial support through project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

In the present study, the eff ect of the number of harvests on yield and chemical composition of Cichorium spinosum L. plants was examined. Seeds of were sown in seed trays containing peat and young seedlings were transplanted in 2L pots containing peat and perlite (1:1 v/v). Plants were harvested two or three times during the growing period, while control plants (no prior harvests) were also harvested at the same time. Rosette diameter, number of leaves, SPAD index, and fresh and dry weight were recorded at harvest, while chemical composition of leaves was also assessed (fatty-acids, sugars, organic acids, and tocopherols content). Regardless of the number of harvests, total fresh weight and number of leaves were higher comparing to control plants, whereas dry weight and rosette diameter were higher in control plants. SPAD index was mostly aff ected by harvesting time (growth stage) and decreased during the growing season. Signifi cant diff erences were also observed in chemical composition of the aerial parts of plants in relation to the number of harvests, with control treatment for the third cutting having the higher content in sugars, organic acids, saturated and monounsaturated fatty acids, and 1st cutting showed the best results for tocopherols. In conclusion, successive harvests (two or three harvests) increased total yield of C. spinosum plants, comparing to control plants, whereas they had a negative eff ect on sugar, organic and fatty acids content. info:eu-repo/semantics/publishedVersion

Tomato (Solanum lycopersicum L.) is a key element of the Mediterranean diet and a source of nourishment for the world's population [1]. During the evolution and domestication of this plant, genetic modifications and severe inbreeding of limited genotypes occurred [2]. As a result, a wide range of fruits with different morphological and sensorial attributes gave rise to a number of landraces that have been cultivated for centuries. These landraces represent a reservoir of genetic diversity that can be used in fruit breeding programs. However, since information about the chemical variation present among tomato landrace populations is still limited, this study aimed to profile the bioactive compounds of Portuguese landraces. Seeds from tive accessions of tomato landraces ("coração de boi", "maçã", "tomate", "redondo" and "vermelho") from the "Santarém" region, conserved ex-situ at the Portuguese Bank of Plant Germplasm, in Braga, were grown in order to regenerate and characterize these plant materiais. The ripe fruits were hand harvested and analyzed for lycopene (by a spectrophotometric method), ascorbic acid (by HPLC coupled to a photodiode array detector) and tocopherols (by HPLC coupled to a fluorescence detector) [3]. The antioxidant activity of hydroalcoholic extracts was evaluated using the in vitro assays of DPPH' scavenging activity, reducing power and p-carotene bleaching inhibition capacity. Total phenolics and flavonoids were estimated by colorimetric methods [3]. Different leveis of vitamins and other phytochemicals were detected in the studied samples, as well as different antioxidant activities. In general, the highest leveis of lycopene and tocopherols (important lipophilic antioxidants) were found in the "coração de boi" and "vermelho" landraces. a-Tocopherol was the most abundant isoform in ali samples, followed by Y-tocopherol. Ascorbic acid was particularly abundant in the "redondo" and "coração de boi" varieties. The "vermelho" tomato also revealed an interesting levei of total phenolics and a high reducing capacity. High leveis of total flavonoids and a high DPPH' scavenging activity were attributed to the "maçã" landrace. This study highlights Portuguese tomato landraces as health-promoting superfoods (or functional foods) with high leveis of powerful antioxidants involved in protection against free radical-induced diseases. To the Foundation for Science and Technology (FCT, Portugal) and FEDER under Pmgramme PT2020 for financiai support to CIMO (UID/AGR/00690/2013) and J. Pinela grant (SFRH/BD/92994/2013). info:eu-repo/semantics/publishedVersion

The species from the Cactaceae family are important invaders in the Mediterranean. The seeds are usually discarded and proper utilisation of these waste products could lead to an important source of bioactive compounds. The seeds have been described to be rich in health-promoting polyunsaturated fatty acids and may potentially be included in animal and human diets [1]. Moreover, seeds could also be recovered for their high nutritional value, as sources of sugars, tocopherols, dietary fiber and polyphenols instead of being discarded, as it currently occurs. In this study, the composition in soluble sugars, fatty acids, organic acids, tocopherols and phenolic composition were determined in seeds of Opuntia macrorhiza (Engelm.) and Opuntia microdasys (Lehm.). The individual sugars profile was determined by HPLC-RI, fatty acids by GC-FID, organic acids by HPLC-DAD, tocopherols by HPLC-fluorescence and phenolic compounds by HPLC-DAD-ESI/MS. In both cases, the overall concentrations of soluble sugars in seeds were quite lower than those detected in other Opuntia sp. parts. Quinic (0.30 g/100 g dw) and oxalic (0.32 g/100 g dw) acids were the major organic acids detected in O. microdasys and O. macrorhiza, respectively. The fatty acid profiles detected in the seeds of both species were similar, with linoleic acid (C18:2n6) as the major fatty acid (71% in O. microdasys; 74% in O. macrorhiza). γ-Tocopherol was the most abundant tocopherol vitamer in both species (7.4 mg/100 g dw in O. microdasys; 5.8 mg/100 g dw in O. macrorhiza). Regarding the phenolic profile, eight compounds were identified: seven phenolic acid derivatives and one flavonoid, and O. macrorhiza seeds (1016 μg/g extract) revealed a higher concentration than O. microdasys (450 μg/g extract). Feruloyl di-hexoside was the most abundant phenolic acid in both species (225 μg/g extract in O. microdasys and 718 μg/g extract in O. macrorhiza). The seeds of Opuntia sp. presented several bioactive phytochemicals, which may allow considering this botanical part as highly interesting by-products. 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) and L. Barros contract; to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

Mushrooms are consumed worldwide not only as a part of the normal diet, but also as a delicacy due to their highly desirable taste and aroma. In addition to their nutritional value, mushrooms have been considered functional foods [1] and even as adjuvants in some therapies, namely chemotherapy [2]. This is because they are natural matrices rich in bioactive molecules, such as phenolic compounds, tocopherols, ascorbic acid, or carotenoids. In particular, tocopherols are powerful antioxidants that can be explored as natural food preservatives, in order to replace the widely used synthetic counterparts. With these aspects in mind, and given the evidence that the in vitro culture of mushrooms promotes the production of tocopherols [3], the present work aimed to sub-culture two different mushroom species by in vitro techniques, in order to enhance the production of tocopherols, and use the mycelium as sources of preservative ingredients for foodstuff. The species chosen for the study were two of the most consumed species worldwide, Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. and Pleurotus eryngii (DC.) Quél. Firstly, both mushroom species were cultivated in different culture media (i.e., MMN complete, MMN incomplete and PDA), in order to optimize the ideal growth conditions. Since the best growth was obtained on PDA medium, both species were cultivated in PDB liquid medium, in order to obtain the amount of biomass required for the assays. After a period of approximately five weeks, the mycelia were recovered, freeze-dried and stored until further analyses. The tocopherols content was evaluated by high performance liquid chromatography coupled with a fluorescence detector. A formulation enriched in tocopherols will be prepared by extraction from the mycelium and further incorporated in cakes. Its preservative capacity will be assessed, through the evaluation of the antioxidant and antimicrobial properties before and after incorporation in the foodstuff. The results will be compared with commercial available and used preservatives (i.e., ascorbic acid- natural antioxidant and potassium sorbate- synthetic preservative). info:eu-repo/semantics/publishedVersion

Chestnut tree is the major crop of the northern highlands of Portugal. Despite the stressful situation caused by some phytosanitary problems, good nut prices have led farmers to invest in chestnut groves. Current fertilization strategies consist in liming and NPK fertilizers rich in phosphorus, based on the fact that soils are usually acidic and poor in phosphorus. This work reports results of a study in which leaf and soil samples were collected in 80 chestnut orchards. The study showed a predominance of acid and very acid soils (73%), low levels of phosphorus (68%) and high levels of potassium (87%). The content of organic matter was low to medium (80%). However, the data obtained from leaf analysis does not seem to be in agreement with the data obtained from soil analysis. Leaf nitrogen concentration is below the lower limit of the sufficiency in 47% of the samples. To soils with very low phosphorus levels do not seem correspond trees with so big nutritional problem, since only 15% of the orchards revealed leaf phosphorus concentration below the adequate range. Although soils show high potassium levels, plant nutritional status does not seem to be so good, since 31% of the samples showed leaf potassium levels below the adequate range. The high acidity seems to be affecting leaf calcium levels, as 52% of the samples showed low leaf calcium concentration. Thus, much still seems to have to be done in the region to adjust the fertilization strategy to the actual needs of the plants. info:eu-repo/semantics/publishedVersion

The overwhelming growth of the world population is causing direct consequences on food demands, which has forced the food industry to apply synthetic additives in order to meet the needs of the consumer and ensuring the global quality of the food products including aspect, texture and flavor. However, due to the publication of some studies describing adverse effects associated with these synthetic additives, the natural ones have been gaining increasing interest. Arbutus unedo L. (strawberry tree) fruits and Ocimum basilicum L. (basil) leaves, are important sources of bioactive natural molecules, especially phenolic compounds, including flavan-3- ols and phenolic acids, respectively . The aim of this work was to demonstrate the antioxidant potential (free radical scavenging effect, reducing power and inhibition of lipid peroxidation) and the absence of toxicity (porcine liver primary cells) of ethanol extracts, obtained from the previously mentioned plants by using optimal conditions. Subsequently, the extracts were incorporated as natural ingredients in loaf bread samples, with two objectives: to functionalize loaf bread, introducing bioactive properties; and as natural ingredients with preservation capacity. Five groups of loaf bread samples were prepared: i) control (without natural or synthetic additives); ii) loaf bread incorporated with strawberry tree extract; iii) loaf bread incorporated with basil extract; iv) loaf bread incorporated with ascorbic acid- E300 (natural additive); and v) loaf bread incorporated with potassium sorbate- E202 (synthetic additive). Both extracts (strawberry tree fruits and basil leaves) presented antioxidant activity (EC50 values lower the 595 and 351 μg/mL, respectively), without toxicity up to the maximal tested dose, and the main compounds present were (+)-catechin and rosmarinic acid, respectively. The introduction of both natural and synthetic additives in loaf bread when compared to the control sample, did not cause significant changes, neither in pH values, nor in color or any of the evaluated nutritional parameters. The obtained results demonstrate that these extracts could be used in the development of new loaf bread replacing the synthetic preservatives, without interfering with the physical and nutritional characteristics. info:eu-repo/semantics/publishedVersion

Medicinal plants are recognized since ancient times as therapeutic agents with several beneficiai properties for human health. Herein, the phenolic profile of three formulations (infusions, pills and syrups) based on artichoke, milk thistle and borututu was analysed as well as their bioactivity, namely the antioxidant, hepatoprotective and antimicrobial activities. In what concems the phenolic composition, the studied plants proved to represent a good source of bioactive compounds, especially phenolic acids and flavonoids. Regarding artichoke and milk thistle, phenolic compounds were higher in the infusion preparations when compared to the other formulations, but in the case of borututu, the syrup revealed the highest contents. Luteolin-7-0- glucuronide was the major flavonoid found in artichoke infusion, protocatechuic acid was the main constituent of borututu infusion, and isorhamnetin-0-deoxyhexoside-O-hexoside was the most abundant compound in milk thistle syrup. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financiai support to CIMO (UIDMGR/00690/2013). info:eu-repo/semantics/publishedVersion

In the present study, the effect of nutrient solution composition on plant growth and quality of Cichorium spinosum L. was examined. Five fertilizer treatments were applied through irrigation water differing in the nitrate: ammonium nitrogen ratio of total nitrogen, namely (1) 100:0, (2) 75:25, (3) 50:50, (4) 25:75, 5) 0:100 NO3:NH4, while an extra treatment (6) with total nitrogen only in urea form was applied. All the treatments received the same amount of fertilizer units (20-20-20 mg L-1 of N, P and K, respectively). Plants were grown in 2 L pots containing peat (Klassman-Deilmann KTS2) and harvested three times during the growing period, and when they reached marketable size. At each harvest day, plant development was assessed (number, fresh and dry weight of leaves, and rosette diameter). The results suggest that nitrogen form has a significant effect on plant growth and yield of C. spinosum plants. In particular, fresh weight and number of leaves, and rosette diameter were significantly increased when ratio of nitrate: ammonium nitrogen was 75:25 or 0:100, especially in the 1st harvest, while in the 3rd harvest treatment 100:0 showed the best results. However, this did not affect total fresh weight and number of leaves which were higher for treatments 100:0 and 75:25, respectively. Therefore, higher ammonium nitrogen rates seem to be beneficial for plant development only during the early stages, while at later growth stages nitrate nitrogen has better results. In any case, ureic nitrogen is not suggested since it has severe effects on plant development, probably due to toxicity issues. info:eu-repo/semantics/publishedVersion

Fungi of the genus Penicillium are promising sources of bioactive substances. Fungal strains isolated from poorly characterized habitais are currently under active investigation as potential producers of biologically active compounds. Penicillium nordicum is mostly known by its ability to produce the mycotoxin ochratoxin A in several protein- and salt-rich food matrices like processed meat and cheese, but it can also produce an array of other secondary metabolites. In a previous study, strains of this species were found to produce, under specific growth conditions, one unidentified compound in apparently big amounts, without producing ochratoxin A. The aim of the present study was to evaluate the bioactive properties of this compound in terms of antioxidant, antimicrobial, cytotoxic and herbicidal properties. For that purpose, the fungus was grown on ham-based médium and the aqueous extract of the compound was used for further tests. The extract showed significant effects on seed germination as well as on root and shoot growth of tomato, lettuce, wheat and oat. The root growth inhibition ranged from 74% to 98%, ana the shoot growth was reduced in 23 to 88%, being tomato the most sensitive plant. The extract did not show any cytotoxic activity in non-tumor porcine liver primary culture cells and human tumor cell lines. The antibacterial effect on human clinicai isolates and antifungal effect on foodbome and environmental isolates was also not significant. Pestalotin was the predominant compound in the fungal extract, with questiomycin and 7- hydroxypestalotin also being found at lower concentrations, as detected by Liquid Chromatography- Mass Spectrometry (HPLC-MS/MS) based multi-metabolite method. The extract was considered as a potential bioherbicide without noticeable negative effects on human and animal cells and also on the environment. Further tests on herbicidal potential are now under development. The authors are grateful to the Foundatton for Science and Technology (FCT, Portugal) anü FEDER under ProgrammePT2020 for financiai suppoit to CIMO (UID/AGR/00690/2013) info:eu-repo/semantics/publishedVersion

Edible mushrooms are widely consumed in many countries as a food having a high commercial and culinary value, mainly due to organoleptic properties, such as texture and flavor, and it is possible to distinguish a mushroom species based on its characteristic odor or flavor (Guillamón et al., 2010a). Described as rich in minerals, water, proteins, essential and non‐essential amino acids, carbohydrates, and poor in lipids, mushrooms are highly nutritious; mushroom proteins are considered of higher nutritional quality than those of vegetables, being comparable to proteins of animal origin such as meat, eggs, and milk (Wang et al., 2014). There are available many studies showing that mushrooms have a wide variety of biomolecules with medicinal properties, with great value in the prevention and treatment of various diseases, being recognized as functional foods and as a source for the development of pharmaceuticals and nutraceuticals (Newman et al., 2000; Ferreira et al., 2009, 2010; Barreira et al., 2014; Gyawali et al., 2014). Mushrooms’ bioactive molecules are responsible for their antioxidant (Ferreira et al., 2009), antitumor (Ferreira et al., 2010), antibacterial (Alves et al., 2012), immunostimulatory, antifibrotic, anti‐inflammatory (Kohno et al., 2008), antiviral (Barreira et al., 2014), antifungal (Alves et al., 2013), anti‐atherosclerotic (Guillamón et al., 2010b; De Silva et al., 2012), hypoallergenic, antiatherogenic, hypoglycemic, hepatoprotective (Nitha et al., 2013), and hypotensive (Guillamón et al., 2010b) properties. According to the available reports on mushrooms’ bioactivities, the refined extracts obtained from the mycelium or fruiting body, that can be consumed in the form of tablets or capsules as dietary supplements and have potential therapeutic application, are considered nutraceuticals, products derived from food sources that provide nutritional value but also provide extra health benefits (Chang and Buswell, 1996). In this chapter, the chemical composition, nutritional value, and bioactive properties of mushrooms species are reviewed and will be highlighted in the following sections. info:eu-repo/semantics/publishedVersion

Extrusion cooking is a high temperature short time process, which modifies flour properties through starch gelatinization, protein denaturation, complex formation between amylose and lipids, degradation of pigments and improvement of sensory characteristics [1]. Vegetables contain numerous phytochemicals, such as tocopherols, useful for their nutritional and nutraceutical properties. Tocopherols (constituents of vitamin E) appear in several active forms, presenting α-tocopherol the highest biological activity, and being γ-tocopherol the most abundant in vegetable foods, such as sesame seed, soybean, black bean and peanut. Due to its action as a free radical scavenger, vitamin E also plays a role on body protecting against degenerative abnormalities, mainly cancer and cardiovascular diseases [2]. The aim of this study was to evaluate the changes induced by extrusion-cooking on tocopherols content in functional novel formulations of flours containing different proportions of rice (50-80%), bean (20-40%), and carob (5-10%) using the raw materials as control. Tocopherols were determined in the different flours mixtures of ricelegumes by high performance liquid chromatography coupled to a fluorescence detector (HPLC-FL) programmed for excitation at 290 nm and emission at 330 nm, following a procedure previously described by Barros et al. [3]. In general, the samples showed low levels of tocopherols and, in some cases, namely in extrusion samples, the total absence of this vitamin was verified. 𝛼-, γ- and δ- Tocopherols were the vitamers detected in several flours, highlighting bean with the highest concentration of total tocopherols (180 ± 1 μg/100 g). In the samples where tocopherols were detected, the raw materials and in all the evaluated mixtures, γ-tocopherol was the predominant vitamer, being present in greater concentration in bean with values of 172 ± 1 μg/100 g. In this study it was also observed that, after extrusion, a significant reduction occurred in the total tocopherols content, being verified the absence of these molecules in different flour mixtures. In addition, the sensitivity of vitamin E to extrusion cooking depends on the extrusion processing variables and conditions used, particularly extrusion temperatures (that promotes the decrease in α- tocopherol) and moisture during extrusion (decreasing γ-tocopherol content). This work was supported by the Spanish Ministry of Economy and Competitiveness (RTA2012-00042-C02 and RTA2015-00003-C02-01). C. Arribas was supported by a PhD contract from INIA. The authors are also grateful to FCT, Portugal and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013). info:eu-repo/semantics/publishedVersion

Allium sativum L., commonly known as garlic, has been used by populations for dietary purposes and also for its medicinal interest, since antiquity. Garlic presents an interesting nutritional profile as well as bioactive compounds related to its medicinal properties: antimicrobial, antiseptic, antiviral, antioxidant, anticancer, immunostimulatory, cardioprotective and hypoglycemic [1). This study has tested (i) three samples of fresh white garlic, from different origins: Spain (commercial variety), Tn3s-os-Montes and Algarve, Portugal (both traditional cultivated varieties); (ii) and a sample of black garlic. Black garlic is the result of a thermic treatment in which garlic bulbs are subjected to controlled temperature and humidity, resulting in organoleptic changes such as colour and flavour. Nutritional and chemical compositions also suffer changes in this process [2] . The samples were tested to determine free sugars content, using high performance liquid chromatography coupled to a refraction index detector (HPLC-RI). Four different sugars were identified: fructose, sucrose, xylose and glucose. Fructose was the most abundant sugar in black garlic (BG), while sucrose was the most abundant in commercial garlic (CG), followed by the traditional cultivated garlic from Algarve (TGA) and traditional cultivated sample from Tras-os-Montes (TGTM). Xylose was only detected in BG sample. Sucrose was found in higher concentration in TGA sample and at lower levels in BG sample. The highest content of glucose and fructose was found in BG, while the lowest content was observed in TGA. Total sugars content was significantly higher in BG sample (33.55 g/1 00 g of fresh weight), than in the other samples: CG (1.32 g/1 00 g fw), TGA ( 1 .48 g/1 00 g fw) and TGTM (0. 70 g/1 00 g fw). These results show some variability on the sugars content, less relevant between the white garlic samples when compared to the black garlic sample. This study shows the possible variations in the sugars' profile accordingly to various factors, such as variety or processing treatments. Therefore, these experiments are highly important to have a better understanding of the products used in our diet. The authors are grateful to the Foundation for Science and Technology (FCT, Porlugal) and FEDER under Program PT2020 for financial supporl to CIMO {UID/AGR/00690/2013), A.F grant (SFRH/BPD11475312016) and L. B contract. info:eu-repo/semantics/publishedVersion

Stevia [Stevia rebaudianaBertoni] is a crop expanding worldwide due to the increasing use in the food industry. The aims of this work were to check the adaptation of Stevia to the ecological conditions of the northeast of Portugal and to assess the response of the plant to the application of different rates of nitrogen, phosphorus and potassium as fertilizers and two different cutting regimes, once and twice during the growing season. The field trials were carried out in Bragança and Vilariça valley, both located in NE Portugal but in very different ecological situations, respectively in one of colder and warmer places in the region. It was assessed the winter survival of stevia plants, as well as dry matter yield, tissue elemental concentration and several vegetation indices. The results revealed that the plants did not survive the winter, with mortality exceeding 95%, meaning that Stevia can only be grown as an annual crop, which requires the installation of the new fields every year. The dry matter yield significantly increased with nitrogen rate. In the most productive treatment (100kgN/ha), 1560kg/ha of dry leaves and 4961kg/ha of total aboveground dry matter (leaves + stems) were reached. Leaf nitrogen concentration did not significantly varied with nitrogen rate, despite the increase observed in dry matter production, which may mean that difficulties can arise in monitoring the nitrogen nutritional status of plants in the field and in the establishment of adequate fertilization programs for this crop., A stévia [Stevia rebaudianaBertoni] é uma cultura em expansão em todo o mundo devido à sua crescente utilização na indústria alimentar. Este trabalho foi iniciado com os objetivos de avaliar a adaptação da stévia às condições ecológicas do interior norte de Portugal, a resposta da planta à aplicação de diferentes doses de azoto, fósforo e potássio, bem como dois regimes de corte: simples e duplo. Os ensaios de campo decorreram em Bragança (na Terra Fria Transmontana) e no vale da Vilariça (na Terra Quente Transmontana). Foi avaliada a sobrevivência das plantas ao inverno, a produção de matéria seca, a composição elementar dos tecidos e determinados diversos índices de vegetação. Os resultados revelaram que as plantas não sobreviveram ao inverno, apresentando mortalidade superior a 95%, significando que a stévia só poderá ser cultivada como se de uma cultura anual se tratasse, com instalação de novos campos todos os anos. A produção de matéria seca aumentou significativamente com a adubação azotada. Na modalidade mais produtiva (100kgN/ha) atingiram-se valores de matéria seca de folhas e total de 1560 e 4961kg/ha, respetivamente. A concentração de azoto nas folhas não variou significativamente com a dose de azoto aplicada, apesar dos aumentos registados na produção de matéria seca, o que deixa antever dificuldades na monitorização do estado nutricional azotado das plantas em campo e no estabelecimento de programas de fertilização para a cultura.

Garlic (Allium sativum L.) hás been used worldwide not only for its dietary interest, but also for medicinal purposes, in prophylaxis and treatment of diverse pathologies. Garlic presents an interesting nutritional profile as well as bioactive compounds, responsible for its biological properties, namely, antioxidant, antimicrobial, immunostimulatory, hypoglycemic, among others [1]. The present work aimed to study bulbils (cloves) of white garlic (WG) and black garlic (BG). Both white and black garlic samples were supplied by local producers from Trás-os-Montes region. Black garlic samples were obtained by applying a dehydratation procedure to /t. sativum bulbs with controlled humidity and temperature. Ali samples were studied regarding their chemical composition in organic acids, analysed by High Performance Liquid Chromatography (HPLC) coupled to a Diode Array Detector, and tocopherols determined by HPLC-fluorescence. Furthermore, their antioxidant activity was evaluated through DPPH (2, 2-diphenyl-l-picrylhydrazyl) radical scavenging activity, reducing power, p-carotene bleaching inhibition and TBARS (tiobarbituric acid reactive substances) formation inhibition assays. a-Tocopherol was the only vitamer identified in the studied samples, being quantified in BG samples at the highest concentration. These samples presented oxalic, malic and pyruvic acids, while WG samples showed also citric and fumaric acids, besides the ones previously mentioned. Malic and pyruvic acids were the main compounds present in BG and WG, respectively. BG samples presented higher antioxidant activity than WG samples, in ali the assayed methodologies. The knowledge on the chemical and bioactive composition of natural matrices is of great importance in the formulation of novel products. Therefore, the present study highlights that the processed garlic (BG), an innovative product, presented higher antioxidant properties than WG, and could be explored for bioactive purposes. The authors are graleful to lhe Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financiai support to CIMO (UID/AGR/00690/2013), A.F grant (SFRH/BPD/114753/2016) and L.B contract. N/A

The chestnut tree is practically the only cash crop grown in the mountain highlands of NE Portugal. Despite the many phytosanitary problems that this crop is currently facing, producers have been increasing the planted areas in response to favorable prices that the chestnut fruit has been reached in the market. The farmers have also been showing a new attitude regarding the cropping technique. In the past, the chestnut groves were a very marginal crop rarely fertilized. Nowadays, the producers seek to make rational fertilizations, hoping to balance the trees, allowing them to better cope with unfavorable biotic and abiotic stresses and stimulate productivity. However, studies on chestnut trees fertilization are rare. Little is known about the response of this species to liming or to fertilizers application. In this work, results are presented on the response of young chestnut plants to liming and nitrogen, phosphorus, potassium or boron application. Three years after planting, the results showed that the application of the nutrients N, P, K or B as fertilizer increased the concentration of each one of the respective nutrients in the leaves. However, the height of the plants was particularly reduced in the plots not fertilized with boron, followed by those not fertilized with potassium, in comparison to the plots receiving the respective nutrients. Boron and potassium appeared as the most limiting nutrients in this early phase of plant growth. info:eu-repo/semantics/publishedVersion

Tomato (Lycopersicon esculentum L.) is one of the most consumed vegetables in the world and probably the most preferred garden crop. It is a key component of the Mediterranean diet, commonly associated with a reduced risk of chronic degenerative diseases. Currently there are a large number of tomato cultivars with different morphological and sensorial characteristics and tomato-based products, being major sources of nourishment for the world’s population. Its consumption brings health benefits, linked with its high levels of bioactive ingredients. The main compounds are carotenoids such as β-carotene, a precursor of vitamin A, and mostly lycopene, which is responsible for the red colour, vitamins in particular ascorbic acid and tocopherols, phenolic compounds including hydroxycinnamic acid derivatives and flavonoids, and lectins. The content of these compounds is variety dependent. Besides, unlike unripe tomatoes, which contain a high content of tomatine (glycoalkaloid) but no lycopene, ripe red tomatoes contain high amounts of lycopene and a lower quantity of glycoalkaloids. Current studies demonstrate the several benefits of these bioactive compounds, either isolated or in combined extracts, namely anticarcinogenic, cardioprotective and hepatoprotective effects among other health benefits, mainly due to its antioxidant and anti-inflammatory properties. The chemistry, bioavailability and bioactivity of these bioactive compounds will be discussed, as well as the main mechanisms of action against cancer and other bioactivities including antioxidant, antiinflammatory, cardiovascular and hepatoprotective effects in humans. Possible applications of tomato bioactive compounds in the industry will also be proposed The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support to CIMO (PEst-OE/AGR/UI0690/2014) and REQUIMTE (PEst-C/EQB/LA0006/2014) and for the scholarship (SFRH/BD/92994/2013) granted to the first author. info:eu-repo/semantics/publishedVersion

The interest and demand for aromatic and medicinal plants have been growing due to their combined organoleptic and bioactive properties. However, in general these plants suffer natural contamination by fungi and associated toxins during growth as also in harvesting, storage and drying processes, which represents a threat to public health. The rigorous standards required by the industrial sector in terms of good quality of raw materials demand efficient decontamination procedures (1-3). Gamma radiation is assumed as an accredited methodology for the decontamination of medicinal and aromatic plants, with numerous advantages not only to the product itself but also to the consumer and the environment (4). In this study, efficient methods for detecting aflatoxins (AFB" AFB2, AFG1 and AFG2) and ocratoxin A (OTA), were optimized and validated, and afterwards, applied to spiked samples of Aloysia citrodora Pahiu submitted to gamma radiation treatment at different doses (I , 5 and I 0 kGy ), to evaluate the effectiveness of irradiation as a decontamination technique for dry plants. Mycotoxin levels were determined by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection, after immunoaffinity column (lAC) cleanup. All the applied gamma radiation doses conducted to a degradation of the studied mycotoxins. In relation to the control sample (0 kGy), the reduction rates in the irradiated samples ranged from 4.9 and 5.2% in OTA, 5.3 and 9.6% in AFBt. 12.3 and 13.5 in AFB2, 16.4 and 23.6 in AFG1 and, finally, 52.6 and 62.7% in AFG2. The gamma radiation dose of 5 kGy stood out as the best decontamination dose for AFB1 and AFG1, which are the most significant aflatoxins naturally found in food commodities. For OTA, AFG2 and AFB2 there was no significant difference in decontamination between doses. In conclusion, the extraction and analysis methods proved to be suitable for detection of aflatoxins and ocratoxin A in A. citrodora. Gamma radiation seems to be an effective technique for reducing aflatoxins G in A. citrodora, and eventually oth~r medicinal and aromatic plants. On the other hand, aflatoxins B and OTA are less affected by this treatment.

Cynara scolymus L. (artichoke) and Silybum marianum (L.) Gaertn (milk thistle), belonging to the Asteraceae family, are medicinal plants vo.ith well-reported antioxidant and hepatoprotective effects. Widely consumed as infusions, these plants can also be found in several formulations to allow an easier consumption. The bioactivity of infusions, pills, and syrups based on artichoke and milk thistle was previously reported by our research group [1 ,2] and among the various phytochemicals present in these dietary supplements, phenolic compounds are pointed out as the most responsible for their beneficial properties. With the aim of studying the antimicrobial activity and possible relation vo.ith the phenolic composition, two different formulations of each plant were assessed (pills and syrups). The phenolic profiles were obtained by HPLC-DAD-ESIIMS, and the antimicrobial activity was performed with clinical isolates from hospitalized patients, namely Escherichia coli, Escherichia coli spectrum extended producer of P-lactarnases (ESBL), Proteus mirabilis, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). Vanillic acid (5.58 J.tg/g) and luteolin-7-0-glucoside (2.2 J.tg/g) were the most abundant compounds in artichoke syrup, that did not reveal antimicrobial activity against the studied strains, which could be due to their low concentrations. On the other hand, artichoke pills presented a prevalence of 5-0-caffeoylquinic (28.2 J.tg/g), 1,3-dicaffeoylquinic (24 J.tg/g), and 4-0-Caffeoylquinic acids (13.3 J.tg/g); revealing the capacity to inhibit MRSA vo.ith a MIC value of 1.9 mg!g. Regarding milk thistle, isorhamnetin-0-deoxyhexoside-0-hexoside, isorhamnetin-3-0-rutinoside, and isorhamnetin-0-deoxyhexoside-0-dihexoside were the major compounds detected in the syrup, in concentrations of 7.26, 5. 75, and 3.64 J.tg/g, respectively. This formulation proved to be able to inhibit the growth of E. coli, ESBL, MRSA and P. aeruginosa, with MIC values ranging from 0.2 to 1.3 mg!mL. Hydroxylated silibinin (1.565 J.!g/g) was the major flavonoid found in the pills, that revealed antimicrobial activity against ESBL, with a MIC value of 15 mg!mL, but did not inhibit the growth of the remaining bacteria None of the studied samples was able to inhibit P. mirabilis at the studied concentrations (1000 and 26.4 mg!mL for the syrups of artichoke and milk thistle, respectively; 150 mg/mL for both pills). Overall, the studied syrups and pills of artichoke and milk thistle revealed to be a good source of phenolic compounds, with some of these formulations revealing antimicrobial activity.

Allium sativum L., vulgarmente designado de alho, é muito utilizado desde a antiguidade, quer para fins alimentares quer para fins medicinais. No presente trabalho, estudaram-se amostras de alho-negro, que corresponde a bolbilhos (dentes) de A. sativum sujeitos a um processo de fermentação em estufa com controlo de humidade e temperatura, o qual provoca alterações organoléticas. Determinou-se a atividade antibacteriana das amostras de alho em isolados clínicos de Enterococcus faecalis (isolado de urina); Listeria monocytogenes (isolada de líquido cefalorraquidiano); Staphylococcus aureus resistente à meticilina, MRSA (isolado de expectoração); Staphylococcus aureus sensível à meticilina, MSSA (isolados de exsudado de ferida); Acinetobacter baumannii e Pseudomonas aeruginosa (isolados de expectoração), Escherichia coli e Klebsiella pneumoniae (isoladas de urina) provenientes do Centro Hospitalar de Trás-Os-Montes e Alto Douro (unidade de Vila Real). Os ensaios foram feitos utilizando o método da microdiluição para cálculo da concentração mínima inibitória (CMI) e bactericida (CMB). Observou-se ação antibacteriana contra todos os isolados clínicos testados, dos quais destacamos os melhores resultados de CMI para MSSA, MRSA, E. faecalis e L. monocytogenes. Relativamente aos Gram negativo, E. coli apresentou o melhor resultado de CMI seguida de P. aeruginosa. A. baumannii e K. pneumoniae apresentaram valores de CMI elevados. Relativamente à ação bactericida, o melhor valor de CMB foi obtido para MRSA e E. coli, seguido de MSSA, P. aeruginosa e A. baumannii. Em suma, verifica-se que o alho-negro, resultado da fermentação de bolbilhos de A. sativum, apresenta potencial antibacteriano, com uma maior eficácia contra bactérias Gram positivo, particularmente contra MRSA. Allium sativum L., commonly known as garlic, is largely used since antiquity for dietary purposes but also for its medicinal interest. The present study studies samples of black garlic which refers to bulbils (cloves) of A. sativum, submitted to a controlled humidity and temperature fermentation process in incubator that produces organoleptic alterations. The antibacterial activity of the garlic samples was determined in clinical isolates of Enterococcus faecalis (isolated from urine); Listeria monocytogenes (isolated from cerebrospinal fluid); methicillin resistant Staphylococcus aureus, MRSA (isolated from sputum); methicillin susceptible Staphylococcus aureus, MSSA (isolated from wound exudate); Acinetobacter baumannii and Pseudomonas aeruginosa (isolated from sputum); Escherichia coli and Klebsiella pneumoniae (isolated from urine) from the Hospital Centre of Trás-Os-Montes and Alto Douro (unity of Vila Real). The tests were made using the microdilution method to calculate the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). It was observed antibacterial activity against all clinical isolates tested, of which we highlight the best MIC results against MSSA and MRSA followed by E. faecalis and L. monocytogenes. Regarding to Gram negative bacteria, E. coli presented the best MIC result followed by P. aeruginosa. A. baumannii and K. pneumoniae presented high MIC values. Regarding bactericidal action, the best value of MBC was obtained against MRSA and E. coli, followed by MSSA, P. aeruginosa and A. baumannii. Overall, it appears that the black garlic resulting from the fermentation of A. Sativum bulbils presents antibacterial potential, with better efficacy against Gram positive bacteria, particularly against MRSA. info:eu-repo/semantics/publishedVersion

Wild strawberry, Fragaria vesca L., belongs to Rosaceae family and is commonly found in roadsides and slopes [1]. The most consumed parts of this plant are its sweet small fruits, which constitute a source of vitamins and phenolic compounds, being also used in infusions due to their organoleptic properties and for the treatment of some intestinal disorders [2, 3]. In the present work, F. vesca fruits were evaluated for their nutritional value and further used in the preparation of infusions. The chemical composition of the fruits and corresponding infusions was determined in terms of soluble sugars, organic acids, tocopherols, folates (by HPLC coupled to different detectors), phenolic compounds (by HPLC-DAD/ESI-MS) and mineral elements (atomic absorption spectroscopy). Some of these bioactive compounds were correlated with antioxidant and antibacterial properties evaluated either in infusions as also in hydromethanolic extracts. Carbohydrates were the main macronutrients in the fruits, followed by fat and proteins. Regarding the fatty acids, polyunsaturated fatty acids showed higher prevalence, mainly due to the presence of D-linolenic (Cl8:3n3) and y-linolenic (Cl8:3n6) acids. Sucrose and citric acid were, respectively, the main sugar and organic acid found in the fruits and in its infusions. The microelement found in higher amounts in both samples was manganese, while potassium and calcium were the macroelements present in higher levels in the fruits and infusions, respectively. Both samples presented folates and tocopherols, being ytocopherol the main isoform detected in the fruits, while a-tocopherol was the only isoform quantified in the infusion. The hydromethanolic extract prepared from the fruits gave higher antioxidant and antibacterial activities, namely against Escherichia coli and Pseudomonas aeruginosa, than the infusion; it also showed capacity to inhibit the formation of bacterial biofilm. Both bioactivities are highly correlated with the presence of phenolic compounds, in which the major are ellagic acid derivatives (sanguiin hlO) followed by tlavan 3-ols ((+)catechin) and anthocyanin compounds (pelargonidin-3-glucoside). Although fruits of wild F. vesca are mainly consumed in fresh, this study also proves the potentiality of their infusions as a source of bioactive molecules and properties.

Tomato (Lycopersicon esculentum L.) is the second most important vegetable crop worldwide and a key component in the so-called “Mediterranean diet”. In the Northeastern region of Portugal, local populations still prefer to consume traditional tomato varieties which they find very tasty and healthy, as they are grown using extensive farming techniques. A previous study of our research team described the nutritional value of the round (batateiro), long (comprido), heart (coração) and yellow (amarelo) tomato varieties [1], but the phenolic profile was unknown until now. Thus, the objective of this study was to characterize the phenolic profiles of these four tomato farmers’ varieties by using HPLC-DAD-ESI/MS and evaluate its antioxidant capacity through four in vitro assays based on different reaction mechanisms. A cis p-coumaric acid derivative was the most abundant compound in yellow and round tomato varieties, while 4-O-caffeolyquinic acid was the most abundant in long and heart varieties. The most abundant flavonoid was quercetin pentosylrutinoside in the four tomato varieties. Yellow tomato presented the highest levels of phenolic compounds, including phenolic acids and flavonoids, but the lowest antioxidant activity. In turn, the round tomato gave the best results in all the antioxidant activity assays. This study demonstrated that these tomato farmers’ varieties are a source of phenolic compounds, mainly phenolic acid derivatives [2], and possess high antioxidant capacity [1]; being thus key elements in the diet to prevent chronic degenerative diseases associated to oxidative stress, such as cancer and coronary artery disease.

The demand for natural sweeteners has been gaining more and more importance due to the great controversy associated with the use of some synthetic sweeteners as cyclamates, aspartame and acesulfame-K. The steviol glycosides (E 960) are a group of natural sweeteners of generalized use; these compounds are obtained from Stevia rebaudiana Bertoni, a sweet plant native from South America (Carocho et al., 2015). However, Stevia rebaudiana Bertoni may have other uses to be exploited, in particular due to its antioxidant capacity. This plant is already produced in Portugal but it is important to evaluate if the plant chemical composition is maintained regardless of culture conditions. Therefore, in this study, stevia samples were cultivated in Braganca (northeastern of Portugal) in a field trial with defined culture conditions. After harvesting, the plants were submitted to two different treatments: kept fresh by freezing (-20°C) and oven-dried (30°C). The antioxidant profile of the samples was studied through evaluation of free radicals scavenging activity, reducing power, phenolic compounds (HPLC-DAD-ESI/MS), tocopherols (HPLC-fluorescence) and free sugars (HPLC-RI). Significant differences were observed: while oven-dried samples showed the highest antioxidant activity and phenolic compounds concentration (mainly 5-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid), the frozen fresh samples had the highest values of total tocopherols and total sugars. These results confirm that the plants grown in Bragança have excellent bioactive secondary metabolites responsible for the observed antioxidant capacity.

In the present study, the evaluation of chemical composition of various ecotypes of “stamnagathi” (Cichorium spinosum L.) was carried out. Seeds from selected ecotypes were collected in situ and sown in seed trays for plant propagation and consequent plant production. At the harvest stage, the dry matter, chlorophyll, sugars, fatty acids and organic acids content in leaves was measured. Regarding dry matter content, significant differences were observed among the ecotypes, with mean values ranging from 6.3 to 8.3%. Differences were also observed in chlorophyll a, b and total chlorophyll, with ecotypes B and D having the highest content comparing to ecotypes A and C. Sugars content differed among the ecotypes for all the detected sugars (glucose, fructose, sucrose) and the total sugars content. Ascorbic acid was detected only in the case of ecotypes B and C, whereas in the other two ecotypes only traces were detected. Finally, regarding tocopherols content, significant differences were observed for both α- tocopherol, with ecotype B having the highest content followed by ecotype C, and δ- tocopherol, where the highest content was observed for ecotype C. For total tocopherols, ecotype C had also the highest content comparing to the other ecotypes. Fatty acids content did not differ among the studied ecotypes, for both the content of the main fatty acids (α-linolenic, linoleic and palmitic acid) and the monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and total fatty acids (TFA) content. In conclusion, genotype has an important effect on nutritional and chemical composition of ‘stamnagathi’ ecotypes, and special interest rises for detecting and evaluating ecotypes with high quality profile for commercial cultivation. info:eu-repo/semantics/publishedVersion

After harvest, plants remain living organisms with the capacity to carry out metabolic processes. Thus, from the moment they are detached from the source of nutrients, they become entirely dependent on their own organic reserves [1]. Postharvest changes cannot be stopped, but they can be slowed within certain limits. Therefore, this study was conducted to evaluate the effects induced by storage in the profiles of sugars, organic acids and tocopherols of two leafy vegetables. Wild samples of watercress (Nasturtium officinale R. Br.) and buckler sorrel (Rumex induratus Boiss. & Reut.), from the Northeastern region of Portugal, were analyzed after harvest (control) and after storage in sterilized packages (using the passive modification mode) at 4ºC for 7 or 12 days, respectively. Analyses were performed by high-performance liquid chromatography (HPLC) using different detectors, i.e., a refraction index detector (RID) for free sugars, a photodiode array detector (PDA) for organic acids, and a fluorescence (FP) detector for tocopherols. The storage time decreased the levels of fructose, glucose and total sugars in both leafy vegetables and increased the total organic acids content. The decrease of these sugars can be related to its use by the plant to produce the required energy. Ascorbic acid was detected in buckler sorrel and decreased with storage; while the amount of malic acid increased in both species. Curiously, all the tocopherol isoforms increased in watercress, while buckler sorrel just present higher values of γ- and δ- tocopherols. In fact, the de novo synthesis of these bioactives compounds can be a plant strategy to fight against the reactive species that are produced during storage. The knowledge of the behavior of these compounds during storage that was achieved with this study [2] may contribute to the development of more effective preservation strategies for leafy vegetables.

Plants frequently suffer contaminations by toxigenic fungi, and their mycotoxins can be produced throughout growth, harvest, drying and storage periods. The objective of this work was to validate a method for detection of toxins in medicinal and aromatic plants, through a fast and highly sensitive method, optimizing the joint co-extraction of aflatoxins (AF: AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) by using Aloysia citrodora P. (lemon verbena) as a case study. For optimization purposes, samples were spiked (n=3) with standard solutions of a mix of the four AFs and OTA at 10 ng/g for AFB1, AFG1 and OTA, and at 6 ng/g of AFB2 and AFG2. Several extraction procedures were tested: i) ultrasound-assisted extraction in sodium chloride and methanol/water (80:20, v/v) [(OTA+AFs)1]; ii) maceration in methanol/1% NaHCO3 (70:30, v/v) [(OTA+AFs)2]; iii) maceration in methanol/1% NaHCO3 (70:30, v/v) (OTA1); and iv) maceration in sodium chloride and methanol/water (80:20, v/v) (AF1). AF and OTA were purified using the mycotoxin-specific immunoaffinity columns AflaTest WB and OchraTest WB (VICAM), respectively. Separation was performed with a Merck Chromolith Performance C18 column (100 x 4.6 mm) by reverse-phase HPLC coupled to a fluorescence detector (FLD) and a photochemical derivatization system (for AF). The recoveries obtained from the spiked samples showed that the single-extraction methods (OTA1 and AF1) performed better than co-extraction methods. For in-house validation of the selected methods OTA1 and AF1, recovery and precision were determined (n=6). The recovery of OTA for method OTA1 was 81%, and intermediate precision (RSDint) was 1.1%. The recoveries of AFB1, AFB2, AFG1 and AFG2 ranged from 64% to 110% for method AF1, with RSDint lower than 5%. Methods OTA1 and AF1 showed precision and recoveries within the legislated values and were found to be suitable for the extraction of OTA and AF for the matrix under study.

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Cichorium spinosum L. is a wild edible green, native to the Mediterranean basin, which usually grows in coastal areas and plateaus. In the present study, plant growth and mineral composition of C. spinosum was evaluated for two consecutive growing periods. Seeds of C. spinosum were sown in seed trays on 15th of October, 2013 and 17th of January, 2014 and young seedlings were transplanted on 12th of December, 2013 and 7th of March, 2014 respectively. Harvest took place at marketable size. Seed germination rate was lower than 50% at both sowing dates (46 and 38%, for 1st and 2nd sowing, respectively), whereas seedling emergence was complete after 11 and 18 days, for the 1st and 2nd sowing date, respectively. Leaf fresh and dry weight, and rosette diameter did not differ significantly, whereas number of leaves and total leaf area was higher in the 1st sowing. Regarding mineral composition, Ca, Mg and Mn content was higher in the 2nd sowing date, whereas K and Na in the 1st sowing date. Fe and Zn content was not affected by sowing date. Regarding nutritional value, ash content increased in the 2nd sowing date, whereas protein and carbohydrate content decreased. However, fructose and glucose and consequently total sugars content, and ascorbic acid content increased in the 2nd sowing, whereas α-tocopherol and total tocopherols, and chlorophyll b decreased. Fatty acids consisted mainly of linoleic, α-linolenic and palmitic acids, whereas no significant differences were observed between the two sowing dates. PUFA/SFA and n-6/n-3 ratio was higher than 0.45 and lower than 4.0, for both sowing dates. In conclusion, sowing date affected plant growth in terms of leaf number and leaf area, without affecting leaf fresh weight, whereas second sowing resulted in an increase in sucrose, glucose and total sugars content and ascorbic acid content that could improve the taste and nutritional value of the final product. info:eu-repo/semantics/publishedVersion

In the present study, the effect of ammonium fertilizer rate on plant growth and quality of Cichorium spinosum L. was examined. Five fertilizer treatments were applied regarding the ammonium nitrogen percentage of total nitrogen, namely (1) 14%, (2) 24%, (3) 34%, (4) 43%, and (5) 53% of total nitrogen applied in the form of ammonium nitrogen. All the treatments received the same total nitrogen rate. Seeds of C. spinosum were sown in seed trays containing peat, and young seedlings were transplanted in 2L pots containing peat and perlite in a ratio 1:1. Plants were harvested twice during growing period and when rosettes reached the marketable size. On the harvest days, plant features regarding plant development (the number of leaves, the fresh and dry weight of leaves, and rosette diameter), as well as quality mineral composition, SPAD index of leaves) were recorded. From the results it is suggested that fertilizer composition had a significant effect on plant growth and quality. In particular, fresh weight for both harvests, as well total fresh weight were higher in treatments 4 and 5, where 43 and 53% of the total nitrogen was applied as ammonium nitrogen. Dry weight did not differ significantly in the first harvest, while in the second harvest treatment 1 had the lowest dry weight. Diameter and number of leaves were the lowest in treatments 2 and 3, respectively, while significant differences were also observed between treatments regarding the quality features. info:eu-repo/semantics/publishedVersion

The biochemistry of cheese ripening involves mechanisms such as glycolysis, proteolysis and lipolysis. Fatty acids are released by the action of lipases from different sources, milk, rennet, bacteria, moulds included as secondary starters, and other exogenous lipases, during lipolysis [1]. The composition of the lipid fraction contributes positively to the flavour of cheese, for being precursors of more complex aroma compounds responsible for the characteristic “goaty flavour” of goat cheeses [2]. Goat milk is recognized by its easier digestibility, alkalinity, buffering capacity and certain therapeutic values in medicine and human nutrition [3]. A high total content of fatty acids is strongly linked to a rancid and tart off flavour in goat milk and may be considered undesirable in most cheese varieties [4]. In this sense, the purpose of the present study was to examine the composition and changes in fatty acids and saponification value of goat cheese during curing period (2, 7 and 12 months). Goat cheese was made in industrial unit of Cachão - Mirandela (Trás-os- Montes) with raw milk Serrana goats’ race, salt and rennet from animal origin. During the first two months, the samples were stored in a ripening chamber (9.5-11 °C and RH 75-85%). From the second month to one year, the samples were stored in a preservation chamber (10.5-12 °C and RH 75-85%). The fatty acids profile of the inner part of the cheese was analyzed by gas-chromatography coupled to flame ionization detection (GC-FID). The degree of saponification was determined both in the crust and inside the cheese by HCl titration of ethanol KOH solution of the samples. Twenty-six fatty acids (FA) were identified and quantified in the inner part of the cheese (total fat was 45-46 g/100 g during the curing period). Saturated fatty acids (SFA) did not change up to 7 months of curing, increasing only after 12 months, being palmitic (C16:0), stearic (C18:0), myristic (C14:0) and capric (C10:0) acids the most abundant FA in this class. Monounsaturated fatty acids (MUFA) decreased only after 12 months, and oleic acid (C18:1) was the predominant FA. In polyunsaturated fatty acids (PUFA) class, the most abundant were linoleic (C18:2) and linolenic (C18:3) acids, and followed the same tendency of MUFA. This is corroborated by an increase in the degree of saponification, either in the crust as in the inner part of the cheese, after 12 months of curing, probably related with the saturation of the fatty acids [3]. Extra-long curing can be done in cheeses produced with goat milk up to seven months of storage without changing the total fat and individual FA content.

Introduction Fragaria vesca L. (Rosaceae), wild strawberry, belongs to Rosaceae family and is commonly found in roadsides and slopes [1]. Its small sweet fruits can be consumed as sources of vitamin C, either in fresh or in infusions used for the treatment of intestinal disorders [2]. Objectives, Material and Methods Herein, the fruits of wild F. vesca were characterized in terms of nutritional value (carbohydrates, proteins, fat, ash, and energy value), dietary fiber content and fatty acids profile. Furthermore, both the fruits and corresponding infusions were characterized by their content in soluble sugars, organic acids, folates and tocopherols by HLPC coupled to RI, DAD and FL detectors, respectively, and also for their mineral elements composition, evaluated by EAA. Results Carbohydrates were the main macronutrient found in F. vesca fruits, followed by fat, ash and proteins. They also contain high levels of dietary fiber, mainly as soluble dietary fiber. Linolenic (C18:3n3) and γ-linolenic acids (C18:3n6) were the main fatty acids found in the fruits, highlighting a prevalence of polyunsaturated fatty acids (PUFA). The fruits and corresponding infusion showed sucrose, followed by fructose as main sugars. Citric acid was the most abundant organic acid in both samples; while oxalic and ascorbic acids were only detected in traces in the infusion. In terms of microelements, manganese was the most abundant in both forms. Potassium and calcium were the main macroelements found in the fruits and their infusion, respectively. In terms of vitamins, both samples presented folates (vitamin B9) and tocopherols (vitamin E), being γ-tocopherol the main isoform found, followed by α-tocopherol. By the way, in the infusion only α-tocopherol was detected. Conclusions Although fruits of wild F. vesca are mainly consumed in fresh, this study also proves the potentiality of their infusions as a source of bioactive molecules.

Human health, nutritional status and well being can be enhanced through consumption of foods containing specifically desired nutrients and bioactive agents [1]. Popularly known as St John’s Bread, Ceratonia siliqua L. (carob) has a long history of use in food (over 4000 years). It has a good nutritional value and its polyphenolic extract shows high antioxidant capacity and even higher antiradical activity than well-aged red wines. Its reducing power can also be four-fold higher than many well known potent antioxidant agents such as gallic acid, caffeic acid and catechin [2]. Nevertheless, preparing high quality nutritious food is critically dependent on availability of effective delivery systems. Such systems should preserve the specific nutritional, biological, chemical and functional properties of the sensitive constituent, and should effectively release the compounds, in a desired mode, after ingestion. Nowadays, the most promising technology that can allow overcoming the stated difficulties is microencapsulation [1]. In this context, a hydroethanolic (80:20, v/v) extract obtained from carob pulp by ultrasound extraction was microencapsulated for further use in the development of functional yogurts.

Under stress, human body produce more reactive oxygen and nitrogen species (ROS/RNS) than enzymatic antioxidants and non-enzymatic antioxidants (e.g., vitamin C, vitamin E, glutathione, carotenoids, and flavonoids), causing an imbalance that may lead to cell damage and health problems [1]. Antioxidants can block the complex networks of oxidative damage pathways at different levels, yielding an overall therapeutic effect. Consequently, antioxidants that are exogenously acquired from diet may have important functions in redox cell homeostasis and can be important for cellular function and disease prevention [2]. Antioxidant compounds might be isolated and characterized from different plant parts like roots, stem, bark, leaves, flowers, fruits and seeds [3]. Herein, different polar extracts (water, ethanol and water:ethanol 1:1) obtained from the green parts and three floral stages of Coleostephus myconis (Asteraceae) were evaluated for their antioxidant activity, using different in vitro chemical and biochemical assays. All the studied botanical parts showed high antioxidant potential, either considering their reducing power, radical scavenging activity and lipid peroxidation inhibition. Independently of the studied botanical part or antioxidant assay, the hydro-alcoholic extract gave the best results. Furthermore, significant differences were detected among the green parts and each of the flowering stages. In conclusion, the results highlighted the possibility of using C. myconis as a potential source of bioactive compounds with several possible application.

The interest of mushrooms hás been increasing over time due to their potential in prevention or treatment of different diseases. Mushrooms have been reported for their anticancer, antiinflammatory, and immunosuppressive and antimicrobial properties. In particular, several authors reported the antibacterial activity of extracts prepared from different mushroom species [1]. In spite of these undeniable qualities, mushrooms are one of the most perishable products and tend to loose quality immediately after harvest. Irradiation is recognized as a safe and effective presen/ation method, being used worldwide to increase the shelf life of foods (e. g, fruits and vegetables, spices, grains, meat or seafood, aromatic and medicinal plants and spices) [2, 3]. In the present study, the antibacterial properties of methanolic extracts prepared from Boletus edulis Buli. and Hydnum repandum L. Fr., previously submitted to gamma irradiation, were assessed against clinicai isolates with different resistance profiles (two Gram negative bacteria; Escherichia coli and Proteus mirabilis, isolated from urine, and two Gram positive bactéria: MSSA- methicillin-sensitive Staphylococcus aureus, isolated from wound exudate and MRSA- methidllin-resistant Staphylococcus aureus, isolated from expectoration) from hospitalized patients in Local Health Unit of Mirandela, Northeast of Portugal. The fruiting bodies were collected in Trás-os-Mõntes (Northeast of Portugal) in November 2012. Gamma irradiation was performed in experimental equipment with four Co-60 sources at 1 and 2 kGy doses. The 1 kGy dose does not seem to be the preferable choice to treat B. edufe and H. repandum(which have a better response to the 2 kGy dose). Among the tested extracts, those obtained from B. ecfufe presented the highest activity against ali the tested bacterial. The antimicrobial activity of the mushroom extracts did not suffer significant changes that might compromise applying irradiation as a possible mushroom conservation technology. As a general conclusion, the 2 kGy should be considered as the best choice in terms of antimicrobial activity maintenance. FCT (RECI/AAG-TEC/0400/2012).and COMPETE/QREN/EU - strategic projects PEst-OE/AGR/UI0690/2014 (CIMO) and PEst-C/EQB/LA0006/2014 (REQUIMTE); grants SFRH/BD/76019/2011 and SFRH/BPD/72802/2010 to A. Fernandes and J.C.M. Barreira, respectively.