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- Conservation of wild mushrooms through electron beam irradiationPublication . Fernandes, Ângela; Antonio, Amilcar L.; Rafalski, Andrzej; Barreira, João C.M.; Oliveira, Beatriz; Martins, Anabela; Ferreira, Isabel C.F.R.The high perishability is a characteristic of the mushrooms consumed in fresh. Therefore, it is mandatory the application of effective conservation technologies to preserve and protect their chemical composition and nutritional value. Drying processes are widely used, but do not avoid the development of bacteria and fungi which have the ability to survive for long periods of time in dry foods, causing the loss of some nutrients and leading to food browning and oxidation of lipids and vitamins [1]. Irradiation appears as an alternative to food preservation assuring and maintaining its quality [2]. In this work, the effects of electron beam irradiation and storage time on nutritional and chemical parameters of wild samples of Macrolepiota procera (Scop.) Singer, previously submitted to a drying process (oven at 30 ºC), were assessed. The wild mushroom samples were collected in Trás-os-Montes; electron beam irradiation (doses 0.5, 1 and 6 kGy) was carried out in the INCT- Institute of Nuclear Chemistry and Technology in Warsaw, Poland and the analyses were performed over the storage period (0, 6 and 12 months). The results were compared with a control (non-irradiated samples). The nutritional value was determined according to the official procedures of food analysis, while the profiles of fatty acids, tocopherols, mono and oligosaccharides were obtained by chromatographic techniques [1]. The irradiation showed a better capacity to maintain the nutritional and chemical profile, in comparison with the storage time. Effectively, the storage time had a significant effect in all parameters, but fatty acids undergone significant changes both with irradiation doses and storage time. Electron beam irradiation can be considered a suitable technique for conservation of mushrooms for long periods of time, attenuating the changes caused by the drying treatment.
- Mycorrhization and micropropation of chestnut (Castanea sativa Mill.) seedlings as tools to obtain high added-value phenolic compoundsPublication . Dias, Maria Inês; Pinela, José; Pereira, Carla; Ferreira, Patrícia; Oliveira, Maria de Fátima; Martins, Anabela; Afonso, Andreia; Barros, LillianMiChestnut3 is a project of the company DEIFIL whose main objective is to produce more resistant and productive hybrid chestnut (Castanea sativa Mill.) seedlings through micropropagation and mycorrhization techniques. In this work, in addition to the agronomic traits of the micropropagated mycorrhizal chestnut seedlings, it was also important to evaluate the changes induced by mycorrhization in the phenolic profile of these plants. Phenolic compounds are plant secondary metabolites involved in plant-microbe interactions/symbiosis and act as signaling molecules in the establishment of arbuscular mycorrhizal symbioses, as well as in plant defense mechanisms [1]. According to the literature, considerable increases in phenolic compounds in host plants as a result of arbuscular mycorrhizal fungus inoculation have been reported during the progression of the infection [1]. Therefore, this work was carried out to study the impact of the type of fungal inoculum and the period of mycorrhization (before or after potting) on the qualitative and quantitative profile of phenolic compounds of the roots and leaves of the chestnut seedlings produced by DEIFIL. After collection and lyophilization of the plant material, hydroethanolic extracts were prepared and the phenolic compounds were characterized by HPLC-DAD-ESI/MS [2]. Ellagic acid derivatives and Oglycosylated flavonoids were the major phenolic compounds in both plant roots and leaves, which agreed with previous reports [2,3]. A statistical analysis showed that the type of inoculum and period of mycorrhization significantly (p<0.05) affected the phenolic profile of the chestnut hybrids. In general, the mycorrhizal seedlings with the fungi Amanita caesarea and Boletus edulis were those that presented the highest levels of phenolic compounds. Relationships between the levels of these signaling compounds and the agronomic performance of chestnut seedlings were also found. The results bring new perspectives into the future production of a hybrid chestnut tree resistant to ink disease in the main traditional Portuguese varieties
- Bioactive compounds in Gyromitra esculenta and Helvella lacunosa wild mushrooms from Northeast of PortugalPublication . Leal, Ana Raquel; Barros, Lillian; Martins, Anabela; Ferreira, Isabel C.F.R.The search for foods that can improve health or reduce the risk of disease, has been steadily gaining interest. Mushrooms could be examples of these foods because they are appreciated worldwide for their nutritional properties [1] and bioactive compounds [2]. The chemical characterization of wild species is very important, in order to promote their consumption and conserve their habitats. This feature might place mushrooms in the pharma-nutrition interface. The present study describes the bioactive compounds in two wild mushrooms ( Gyromitra esculenta and Helvelfa lacunosa edible after proper pre-boiling and cooking) collected in Bragança (Northeast Portugal). The individual profiles of organic acids and phenolic compounds were obtained by high performance liquid chromatography coupled to photodiode array detection (HPLC-PDA); tocopherols and free sugars were characterized by HPLC-fluorescence and HPLCRI (refraction index), respectively, and the fatty acids profile was obtained by gas chromatography coupled to flame ionization detection (GC-FID). Gyromitra esculenta was the species with the highest levels of free sugars (6.13 g/1 OOg dw), tocopherols ( 112.83 IJg/1 OOg dw) and phenolic compounds (3.74 mg/1 OOg dw). The major fatty acid found in this specie was linoleic acid (prevalence of PUFA), while Helvella lacunosa presented the highest level of MUFA (prevalence of oleic acid). The Helvella lacunosa sample revealed the highest levels of total organic acids (6.93g/1 00 g dw). Overall, wild mushrooms can be sources of bioactive compounds to be included in nutritional balanced diets.
- Expanding current knowledge on chemical composition and antioxidant activity of the Genus LactariusPublication . Vieira, Vanessa; Barros, Lillian; Martins, Anabela; Ferreira, Isabel C.F.R.Despite the presence of toxic compounds in inedible mushrooms, the question whether the chemical nutrients and non-nutrients compositions in edible and inedible Lactarius species are similar remains unanswered. To answer this question, Lactarius citriolens Pouzar and Lactarius turpis (Weinm.) Fr., two inedible species, were studied in order to obtain information about their chemical composition and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. citriolens and L. turpis methanolic extracts were tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). The composition of macronutrients varied among the two species, but the profiles were similar between them and among other Lactarius species; L. citriolens gave the highest energy contribution, saturated fatty acids and organic acids, while the L. turpis sample was richer in free sugars, mono- and polyunsaturated fatty acids, tocopherols and phenolic compounds. L. turpis methanolic extract showed the highest antioxidant activity. The absence of hepatoxicity of the methanolic extracts was confirmed in porcine liver primary cells (in vitro conditions). The present study provided new information about wild L. citriolens and L. turpis, comparing their chemical composition and antioxidant properties with other Lactarius species, and expanding the knowledge about this genus.
- Evaluation of the chemical interactions in co-culture elements of Castanea sativa Miller mycorrhizationPublication . Carvalho, Filipa Arminda; Ferreira, Isabel C.F.R.; Barreira, João C.M.; Barros, Lillian; Martins, AnabelaIn the early steps of mycorrhizal associations an oxidative burst might occur through the rapid production of high amounts of reactive oxygen species in response to external stimuli, increasing the production of antioxidants in plant and/or mycelia. Herein, the effect of mycorrhizal association among Pisolithus arhizus or Paxillus involutus and Castanea sativa Miller (stems and roots) was studied for different co-culture periods, defined for a better comprehension of the chemical interactions in the early stages of mycorrhization (6, 24 and 72 h). Knowledge on the specificities of plant-host interaction provided information about the most suitable fungus to be included in the development of new management strategies, and the fungal species that induced the strongest response with increasing antioxidant activity (free radical scavenging activity, reducing power and lipid peroxidation inhibition) and production of antioxidant compounds (phenolics and tocopherols) and sugars. P. involutus seemed to be the most adequate fungus to mycorrhize with C. sativa. Considering bioactive compounds production, P. arhizus was more efficient since allowed an increase in the contents of sugars and tocopherols in all co-culture elements.
- Anti-inflammatory potential of mushroom extracts and isolated metabolitesPublication . Taofiq, Oludemi; Martins, Anabela; Barreiro, M.F.; Ferreira, Isabel C.F.R.Background: In the recent years natural resources are being in focus due to their great potential to be exploited in the discovery/development of novel bioactive compounds and, among them, mushrooms can be highlighted as alternative sources of anti-inflammatory agents. Scope and approach: The present review reports the anti-inflammatory activity of mushroom extracts and of their bioactive metabolites involved in this bioactive action. Additionally the most common assays used to evaluate mushrooms anti-inflammatory activity were also reviewed, including in vitro studies in cell lines, as well as in animal models in vivo. Key findings and conclusions: The anti-inflammatory compounds identified in mushrooms include polysaccharides, terpenes, phenolic acids, steroids, fatty acids and other metabolites. Among them, polysaccharides, terpenoids and phenolic compounds seem to be the most important contributors to the anti-inflammatory activity of mushrooms as demonstrated by numerous studies. However, clinical trials need to be conducted in order to confirm the effectiveness of some of these mushroom compounds namely, inhibitors of NF-κB pathway and of cyclooxygenase related with the expression of many inflammatory mediators.
- Chemical composition of Boletus pinophilus and Clitocybe subconnexa: preservation with gamma irradiationPublication . Fernandes, Ângela; Antonio, Amilcar L.; Barreira, João C.M.; Martins, Anabela; Oliveira, Beatriz; Ferreira, Isabel C.F.R.The short shelf life of mushrooms is a barrier for their distribution and, therefore, there has been extensive research to find technologies that ensure the preservation of mushrooms, maintaining their organoleptic and nutritional properties (1]. Irradiation has proved its technological feasibility to be safely used in the reduction of food losses, being recognized by international organizations as a valid conservation alternative in extending shelflife of many foods. The aim of the present work was to validate the use of 2 kGy dose of gamma radiation to maintain chemical composition of wild mushrooms. Boletus pinophilus Pihit & Dermek and Clitocybe subconnexa Murrill wild samples were obtained in Tnis-os-Montes; subsequently, the samples were divided in two groups: control (non-irradiated, 0 kGy) and irradiated (2 kGy). The irradiation of the samples was performed in a 6°Co experimental chamber. Moisture, protein, fat, carbohydrates and ash were determined following the standard procedures [2]. Free sugars and tocopherols were determined by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI) and a fluorescence detector, respectively; fatty acids were determined by gas-liquid chromatography with flame ionization detection (GC-FID) [3]. The protein and ash content was preserved in both groups, although the sugars and tocopherols decreased in the irradiated samples. Sugars and fatty acids showed significant changes after irradiation treatment, particularly in B. pinophillus, nevertheless, the magnitude of the obtained differences did not seem to be sufficient to affect the chemical profiles of the assayed mushrooms. Overall, the detected chemical changes might be considered as allowable, in view of the high advantages offered by gamma irradiation at decontamination and/or disinfestation level.
- Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: identification of phenolic acids with cytotoxic potentialPublication . Vaz, Josiana A.; Almeida, Gabriela M.; Ferreira, Isabel C.F.R.; Martins, Anabela; Vasconcelos, M. HelenaMushrooms are a possible rich source of biologically active compounds with potential for drug discovery. The aim of this work was to gain further insight into the citotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterized regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the Clitocybe alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI50 concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 µg/ml) for 48h caused an increase in the levels of wt p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, we found evidence for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.
- In vitro culture of Coriandrum sativumPublication . Dias, Maria Inês; Cardoso, Susana M.; Martins, Anabela; Sousa, Maria JoãoCoriander (coriandrum sativumL.) is a plant from the Umbellifera family. In Portugal, the use of coriander plants in gastronomy is very common, like in all the countries in the Mediterranean area. This plant species has also several other applications than as an aromatic plant, such as medicinal, being recommended for dyspeptic complaints, loss of appetite, convulsion, insomnia and anxiety. Moreover, the essential oils and various extracts from coriander have been shown to possess antibacterial, antioxidant, antidiabetic, anticancerous and antimutagenic activities among others, it has also been used as a flavoring agent in food products, perfumes and cosmetics. To study the potential use of this plant all over the year it is Necessary to establish an in vitro system production and to evaluate the better conditions for its growth. In vitro coriander cultures were started from seeds of Coriandrum sativum from a commercial origin. Seeds were inoculated in MS medium containing different concentration Of IBA and BAP. After 6 months of in vitro culture, the plants were separated in two lots named Lot A and B differentiated by their pigmentation (clones with differentiation in flavonoids accumulation), being lot B the less pigmented and lot A the one who presented an higher purple coloration, under the same in vitro growth conditions(nutrition, temperature and light). The growth rates of both lots were determined through fresh and dried weights and evaluating how pigmentation affects these parameters. The mdedium with better growth rates was MS with 0,1mg/L BA and 0,1mg/L BAP; The B lot grew better but have the stationary phase after 3 weeks while the A lot was still growing after 4 weeks but grew slower comparing wit lot B. These results will be used in further studies concerning the essential oils production in each lot.
- In vitro interaction of Amanita muscaria and Phytophthora cinnamomi: possible biocontrol effectPublication . Rodrigues, Paula; Martins, AnabelaInk disease, caused by the Oomycetes Phytophthora cinnamomi Rands. and Phytophthora cambivora (Petri) Buism., is a major soilborne problem in European chestnut (Castanea sativa) stands all over South Europe. Chemical treatments are not effective and comprise serious environmental and economic costs, so biological control is under study as a possible solution for this problem. Ectomycorrhizal (ECM) fungi are generally considered as potential biocontrol agents, for several of them have shown a positive effect on growth and survival of infected plants. The mechanisms underlying these effects are, however, still unknown. It is possible that they result from a chemical antagonism or a physical barrier of the ECM fungus over the pathogen, but other hypotheses involve active responses by the plant. Amanita muscaria is an important ECM fungus of C. sativa in the Northeast of Portugal. The present study intended to investigate a possible antibiosis effect of A. muscaria (Am) over P. cinnamomi (Pc). For this purpose, we used isolates of Am and Pc collected from a local chestnut orchard. The interaction between the organisms was tested by the dual culture technique on Petri dish, using mycelial inoculum, in two different culture media: MMN and PDA. The following dual cultures were tested: Am + Am, Pc + Pc, Am + Pc (inoculated simultaneously) and Am7→Pc (Pc inoculated 7 days after the inoculation of Am). Interaction was analysed daily for a period of seven days (given the rapid growth of Pc), and was based on radial growth and morphological features of both organisms. The growth of P. cinnamomi was heavily constrained by the presence of A. muscaria (in both Am + Pc and Am7→Pc dual cultures), and more spores were formed, when compared with Pc + Pc cultures. The results suggest a heavy antagonistic effect of the ECM fungus over the pathogen. Other ECM fungi are currently under study for the analysis and comparison of possible different effects over P. cinnamomi.