A prospective study on bioactive properties of wild mushrooms mycelium grown in vitro under different conditions

Wild mushrooms have been extensively studied for their value as sources of high quality nutrients and of powerful physiologically bioactive compounds [1,2]. The present study was designed to evaluate the in vitro development of two wild edible mushroom species: Pleurotus eryngii (DC.) Quél. and Suillus belinii (Inzenga) Watling, by testing different solid (Potato Dextrose Agar medium –PDA and Melin-Norkans medium- MMN) and liquid culture media (Potato dextrose broth- PDB and Melin-Norkans medium- MMN). Each strain of mushroom produces a special type of mycelium and this range of characteristics varies in form, color and growth rate. S. bellinii presents a pigmented and rhizomorphic mycelia, whereas, P. eryngii has depigmented and cottony mycelia. The mycelium isolated and grown in PDA showed a faster radial growth compared to the mycelium isolated and grown in both solid and liquid incomplete MMN medium. P. eryngii exhibited a rapid growth and a higher mycelia biomass in both medium compared to S. belinii. Moreover, the obtained mycelia will be characterized in terms of well-recognized bioactive compounds namely, phenolic acids and mycosterols (mainly ergosterol), by using high performance liquid chromatography coupled to diode array and ultraviolet detectors, respectively. These compounds will be correlated to mycelia bioactivity: i) antioxidant activity, evaluated through free radicals scavenging activity, reducing power and lipid peroxidation inhibition in vitro assays; ii) anti-inflammatory activity, assessed through nitric oxide production inhibition in murine macrophages (RAW 264.7 cell line); iii) cytotoxic activity, evaluated either in human tumor cell lines (MCF-7- breast adenocarcinoma, NCIH460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma) as also in a non-tumor porcine primary liver cells culture established in-house (PLP2). Overall, our expectation is that the bioactive formulations obtained by in vitro culture can be applied as nutraceuticals or incorporated in functional foods.

The use of wild fruits of Fragaria vesca L. in preparations with bioactive properties: chemical characterization, antioxidant, antibacterial and antibiofilm activities

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.

Gamma radiation preserves chemical and bioactive properties of Boletus edulis wild mushrooms

Boletus edulis Bull: Fr. is an edible mushroom quite appreciated for its organoleptic and nutritional properties. However, the seasonality and perishability cause some difficulties in its distribution and marketing in fresh form; losses associated with this type of food during marketing can reach 40% [1]. Irradiation is recognized as a safe and effective method for food preservation, being used worldwide to increase shelf life of fresh and dehydrated products (e.g. fruits, vegetables and spices) [2]. In particular, gamma irradiation has already been applied to cultivated mushrooms (especially Agaricus, Lentinula and Pleurotus Genus) and proved to be an interesting conservation technology [3]. However, the studies with added-value wild species are scarce. In this work, the effects of gamma irradiation on chemical and antioxidant properties of wild B. edulis, were evaluated. Fruiting bodies were obtained in Trás-os-Montes, in the Northeast of Portugal, in November 2012. The irradiation was performed in experimental equipment with 60Co sources at 1 and 2 kGy. All the results were compared with nonirradiated samples (control). Macronutrients and energy value were determined following official procedures of food analysis; fatty acids were analyzed by gas-chromatography coupled to flame ionization detection (GC-FID), while sugars and tocopherols were determined by high performance liquid chromatography (HPLC) coupled to refraction index (RI) and fluorescence detectors, respectively. Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of β- carotene bleaching and inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. The nutritional profiles were not affected in high extension. Fatty acids and sugars were slightly affected, decreasing with the increasing doses. The performed assays for antioxidant activity, indicate that irradiated samples tended to have lower scavenging activity and reducing power, but higher lipid peroxidation inhibition. Despite the detected differences in individual compounds, the results of nutritional parameters, the most relevant in terms of mushroom acceptability by consumers, were less affected, indicating an interesting potential of gamma-irradiation to be used as an effective conservation technology for the studied mushrooms.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Postharvest changes in profiles of sugars, organic acids and tocopherols in leafy vegetables monitored by chromatographic techniques

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.

Numerical prediction of thermal and dynamic characteristics of a fireinduced ceiling-jet

The aim of this thesis is to test the ability of some correlative models such as Alpert correlations on 1972 and re-examined on 2011, the investigation of Heskestad and Delichatsios in 1978, the correlations produced by Cooper in 1982, to define both dynamic and thermal characteristics of a fire induced ceiling-jet flow. The flow occurs when the fire plume impinges the ceiling and develops in the radial direction of the fire axis. Both temperature and velocity predictions are decisive for sprinklers positioning, fire alarms positions, detectors (heat, smoke) positions and activation times and back-layering predictions. These correlative models will be compared with a 3D numerical simulation software CFAST. For the results comparison of temperature and velocity near the ceiling. These results are also compared with a Computational Fluid Dynamics (CFD) analysis, using ANSYS FLUENT.

Nutritional and biochemical profiling of Leucopaxillus candidus (Bres.) singer wild mushroom

The wild mushroom Leucopaxillus candidus (Bres.) Singer was studied for the first time to obtain information about its chemical composition, nutritional value and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. candidus methanolic extract was tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). L. candidus was shown to be an interesting species in terms of nutritional value, with high content in proteins and carbohydrates, but low fat levels, with the prevalence of polyunsaturated fatty acids. Mannitol was the most abundant free sugar and β-tocopherol was the main tocopherol isoform. Other compounds detected were oxalic and fumaric acids, p-hydroxybenzoic and cinnamic acids. The methanolic extract revealed antioxidant activity and did not show hepatoxicity in porcine liver primary cells. The present study provides new information about L. candidus.