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.

Micoencapsulation of bioactive plant extracts: a tool to functionalize cottage cheeses

Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (chamomile) are two examples of plants with reported antioxidant and antimicrobial properties, which can be related with their composition in phenolic compounds [1,2]. Furthermore, according to previous results of our research group, the direct incorporation of the aqueous extracts showed capacity to maintain the nutritional properties of the cottage cheeses, up to 7 days of storage, while improving the antioxidant potential. However, after 14 days, a decrease in the antioxidant properties was observed [1,2], which can be related with factors such as light, moisture, temperature and pH, that can cause bioactive compounds degradation. Therefore, the aim of the present study was to prepare microcapsules with the aqueous extracts of fennel and chamomile for incorporation in cottage cheese samples, in order to protect the bioactive molecules present in the extracts, such as phenolic compounds, and prevent the decrease of the antioxidant activity observed after the 14 days period. The microspheres were prepared using an atomization/coagulation technique. Sodium alginate was used as the matrix material to produce the microspheres that were characterized through optical microscopy (OM), during and after atomization, for inspecting morphology. The encapsulation efficiency (EE) was determined by HPLC-DAD by an indirect method by analysing the coagulation solution. FTIR was also used to attest the presence of the extract inside of the alginate matrix. These microencapsulated extracts were incorporated in cottage cheese samples that were further characterized in terms of nutritional properties and antioxidant potential right after incorporation, and after 7 and 14 days of storage at 4•c. The EE was estimated as -100% and the FTIR analysis confirmed the presence of the extracts inside the microspheres. The results showed that the incorporation of the microencapsulated extracts did not cause changes in the nutritional value of cottage cheeses (through a comparison with control samples without extracts). The predominant fatty acids were palmitic (C16:0) and oleic (CI8:0) acids. The order of abundance of fatty acids was as follows: saturated fatty acids (SF A)> monounsaturatcd fatty acids (MUF A)> polyunsaturated fatty acids (PUF A). Regarding free sugars, lactose was the only sugar identified and quantified in all samples. Regarding the antioxidant activity, the samples functionalized with the microencapsulated extracts showed a higher preservation of this property even after the 7th day of storage. Overall, the incorporation of the protected plant extracts in dairy foods can be a strategy to provide health benefits to consumers.

Comparative study of bioactive potential of Fueniculum vulgare Mill. and Matricaria recutita L. phenolic rich extracts to functionalize cottage chesse

Synthetic additives used in a wide variety of food products have been associated to some toxic effects. This conducted to an increasing interest of consumers for natural additives, including food preservers [1]. Many aromatic herbs have been used to prepare bioactive extracts with benefits to the consumer's health. Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (chamomile) are examples of popular herbs rich in phenolic compounds with documented antioxidant and antimicrobial properties [2,3]. The present work confirms the antioxidant (DPPH scavenging activity, reducing power and lipid peroxidation inhibition) and antimicrobial (against bacteria such as Bacillus cereus and Salmonella Typhimurium and fungi such as Aspergillus niger, A. versicolor and PenicilliumfimicuJosum) activities of fennel and chamomile extracts, obtained by decoction. The chemical characterization of the extracts, performed by HPLC-DAD-ESIIMS, revealed the presence of five flavonoids (mainly qercetin-3-0- glucoside) and twelve phenolic acids (mainly 5-0-caffeolyquinic acid) for fennel extract and the presence of nine flavonoids (mainly luteolin-0-glucuronide) and ten phenolic acids (mainly di-caffeoyl-2,7- anhydro-3-deoxy-2-octulopyranosonic acid) for chamomile extract. Due to their high antioxidant and antimicrobial activities, both extracts were then incorporated (at DPPH scavenging activity EC25 value: 0.35 mg/mL and 0.165 mg/mL for fennel and chamomile, respectively) in cottage cheeses (prepared by Queijos Casa Matias Lda) as natural additives with two objectives: to increase the shelf-life of the cottage cheeses and to provide bioactive properties to the final products. The results showed that the use of these natural extracts did not alter significantly the nutritional characteristics of the cottage cheese in comparison with control samples (cottage cheese without extracts), but improved its antioxidant potential (more evident in the samples with chamomile extract). After 14 days of storage, only the control samples showed signs of degradation. Overall, the present study highlights the preservation potential of fennel and chamomile extracts in cottage cheeses, improving also their bioactivity.

Contribution of plant phytochemicals to organic homeostasis: focus on the aging process and opportunistic infections

Aging process is conceived as a normal stage during human life cycle, but it is also considered a hot topic among scientists and medical community. Alarming rates of premature aging and oxidative stress-related diseases have increasingly affect human individuals. Stress, pollution and exposition to chemical substances are considered the main triggering factors for those conditions; in addition, they also suppress the immune system and, therefore, improve organic vulnerability and occurrence of opportunistic infections [I]. Apart from the associated morbidity and mortality, the increasing rates of antimicrobial resistance improve the severity of the clinical conditions [2]. Botanical preparations possess a multitude of bioactive properties, namely acting as antimicrobials, antioxidants, and homeostasis modulators. Thus, upcoming alternatives, mainly based in plant phytochemicals, are necessary to improve the wellbeing as also life expectancy of individuals. The present study aims to evaluate and to compare both antioxidant and antimicrobial properties of plant extracts rich in phenolic compounds. Among the tested plants, Glycyrrhiza glabra L. (licorice) evidenced the most pronounced free radicals scavenging and antimicrobial effects, followed by Salvia officina/is L. (sage), Thymus vulgaris L. (thyme) and Origanum vulgare L. (oregano). Eucalyptus globulus Labill. (blue gum) and Juglans regia L. (walnut) also showed a high effect, while Pterospartum tridentatum (L.) Willk. (carqueja) and Rubus ulmifolius Schott (elm leaf blackberry) displayed moderate effects, and lastly, Tabebuia impetigirwsa (Mart. ex DC) Standley (pau d'arco), Foeniculum vulgare Miller (fennel), Rosa canina L. (rose hips) and Matricaria recutita L. (chamomile) gave only slight effects. In general, the most pronounced bioactivities were observed in the plant preparations (infusion>decoction>hydromethanolic extract) with higher levels of phenolic compounds (both flavonoids and phenolic acids). The observed synergisms between the phenolic compounds present in the extracts highlight the use of phytochemicals as future health promoters. However, further studies are necessary to understand the effective mode of action of individual phenolic constituents as also the existence of polyvalence relationships between them.

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.

Nitrogen vs argon: what is the most effective atmosphere for shelf-life extension of buckler sorrel leaves?

Buckler sorrel (Rumex induratus Boiss. & Reut.) is an underutilized leafy vegetable with peculiar sensory properties and potential as a gourmet food. In the food industry, different packaging methods have been used for shelf-life extension, but it is important to know how the quality of minimally processed vegetable is affected by these treatments. Recently, nitrogen and argon have been used for food packaging. Nitrogen is low soluble in water and other food constituents and does not support the growth of aerobic microbes. In turn, argon is biochemically active and appears to interfere with enzymatic oxygen receptor sites. In this study, modified atmospheres enriched with nitrogen and argon were evaluated for shelf-life extension of buckler sorrel leaves. Wild samples were gathered in Bragança, Portugal, considering local consumers’ sites and criteria. Healthy and undamaged leaves were selected, rinsed in tap water, and a portion was immediately analyzed (non-stored control). The remaining fresh material was packaged in polyethylene bags under nitrogen- and argon-enriched atmospheres and a conventional control atmosphere (air). All packaged samples were stored at 4 ºC for 12 days and then analyzed. The headspace gas composition was monitored during storage. Different quality attributes were evaluated, including visual (colour), nutritional (macronutrients, individual sugars and fatty acids) and bioactive (hydrophilic and lipophilic molecules and antioxidant properties) parameters. Different statistical tools were used; the one-way analysis of variance (ANO VA) was applied for analyse the differences among treatments and a linear discriminant analysis (LDA ) was used to evaluate the effects on the overall postharvest quality. The argon-enriched atmosphere better prevent the samples yellowing. The proximate composition did not change significantly during storage. Samples in control atmosphere revealed higher protein and ash contents and lower levels of lipids. The non-stored control samples presented the higher amounts of fructose, glucose and trehalose. The storage time increased the palmitic acid levels and decreased the content in α-linolenic and linoleic acids. The γ- e δ-tocopherols were higher after the 12 days of cold storage. Probably, the synthesis of these lipophilic compounds was a plant strategy to fight against the abiotic stress induced by storage. Higher levels of total phenolics and flavonoids and increased reducing power and β-carotene bleaching inhibition capacity were also found in the stored control samples. Once again, this result may be attributed to the intrinsic plant-protection mechanisms. Overall, the argon atmosphere was more suitable for quality preservation and shelf-life extension of buckler sorrel.

Wild Morchella conica Pers. from different origins: a comparative study of nutritional and bioactive properties

Morchella conica Pers. is a species of fungus that belongs to the Morchellaceae family and was studied in order to obtain more information about this species, by comparing Portuguese and Serbian wild samples. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques. M. conica methanolic extracts were tested regarding antioxidant and antimicrobial properties. The absence of hepatotoxicity was confirmed in porcine liver primary cells.

Improving bioactive compounds extractability of Amorphophallus paeoniifolius (Dennst.) Nicolson

Elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson) is an underground, unbranched deciduous plant that produces a large tubercle (rhizome) with recognized health effects. In this study, the influence of solvent nature (water, water/etanol (1:1) and absolute ethanol) and processing type (fresh, lyophilized and boiled) on the antioxidant activity and bioactive compounds extractability of elephant foot yam was evaluated. Extracts were compared for their contents in total phenolics, flavonoids and tannins. Moreover, their antioxidant capacity was assessed by the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH▪) scavenging capacity assays. Phenolics (154 mg GAE/L) and tannins (109 mg GAE/L) were maximized in lyophilized samples extracted with the hydroalcoholic solvent, which attained also the highest FRAP value (711 mg FSE/L). In turn, flavonoids reached the highest yields in lyophilized samples (95 mg ECE/L) extracted with pure ethanol, as well as the highest DPPH▪ scavenging activity. These findings might have practical applications to define the best processing methodology regarding the enhancement of elephant foot yam, either for prompt consumption, as well as to develop food supplements or pharmaceutical related products.

Polyporus squamosus (Huds.) Fr from different origins: chemical characterization, screening of the bioactive properties and specific antimicrobial effects against Pseudomonas aeruginosa

Wild mushrooms are mainly collected during the rainy season and valued as a nutritious food and sources of natural medicines and nutraceuticals. The aim of this study was to determine the chemical composition and bioactive properties (antioxidant, antimicrobial and cytotoxicity) of Polyporus squamosus from two different origins, Portugal and Serbia. The sample from Portugal showed higher contents of as protein (17.14 g/100 g), fat (2.69 g/100 g), ash (3.15 g/100 g) and carbohydrates (77.02 g/100 g); the same sample gave the highest antioxidant activity: highest reducing power, DPPH radical scavenging activity, and lipid peroxidation inhibition in both β-carotene/linoleate and TBARS assay. These results could be related to its higher content in total tocopherols (1968.65 μg/100 g) and phenolic compounds (1.29 mg/100 g). Both extracts exhibited antibacterial activity against all the tested organisms. The samples from Serbia gave higher overall antibacterial activity and showed excellent antibiofilm activity (88.30 %). Overall, P. squamosus methanolic extracts possessed antioxidant, antimicrobial, antibiofilm and anti-quorum sensing activity, and without toxicity for liver cells. This investigation highlights alternatives to be explored for the treatment of bacterial infections, in particular against Pseudomonas aeruginosa. This study provides important results for the chemical and bioactive properties, especially antimicrobial activity of the mushroom P. squamosus. Moreover, to the authors’ knowledge this is the first report on sugars, organic acids, and individual phenolic compounds in P. squamosus.

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values ≤ 125.0 μg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 μg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 μg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Enhancing bioactive fatty acids of the meat from lambs reared in intensive systems through nutritional modulation

Tese de Doutoramento em Ciências Veterinárias, especialidade de Produção Animal

Bioactive compounds of citrus as health promoters

Citrus are a group of fruit species, quite heterogeneous in many aspects, including chemical composition of the fruit. Since ancient times, some citrus fruits were used to prevent and cure human diseases. In the recent decades, it has been demonstrated that fruits can actually help prevent and cure some diseases and above all, they are essential in a balanced diet. Citrus fruits, as one of the groups of fruit species, with greater importance in the world, have been studied for their effects on human health. Some species of citrus were referred as potential antioxidant based therapy for heart disease, cancer and inflammation. Fruit peels and seeds have also high antioxidant activity. The health benefits of citrus fruit have mainly been attributed to the high level of bioactive compounds, such as phenols (e.g., flavanone glycosides, hydroxycinnamic acids), carotenoids and vitamin C. These compounds are present in the fruit pulp and hence in the juice. But some bioactive compounds can be found in parts of the fruit which usually are not used for human food. The content of bioactive compounds depends on the species and cultivar, but also depends on the production system followed in the orchard. Citrus fruits, their derivatives and their by-products (peel, pulp and oil) are reach in different bioactive compounds and its maturity, postharvest and agroindustry processes influence their composition and concentration. The aim of this chapter was to review the main bioactive compounds of the different components of citrus and their relationship to health.