RÚBIA CARVALHO GOMES CORRÊA

Título da Tese: Aplicações Biotecnológicas e Nutracêuticas de Pleurotus spp.

Orientadora: Profa. Dra. Rosane Marina Peralta

Data da Defesa: 18/03/2016

RESUMO GERAL

INTRODUCTION AND AIMS – The genus Pleurotus is a cosmopolitan group of mushrooms with high nutritional value and therapeutic properties, besides a wide array of biotechnological and environmental applications. The most important Pleurotus species cultivated in large scale are P. ostreatus and P. pulmonarius. However, the detection of novel bioactives in less explored Pleurotus species, together with the determination of their chemical structures and mechanisms of action, are demands that science is seeking to accomplish. P. ostreatoroseus is an example of a Brazilian edible mushroom whose chemical characterization and bioactivity remain underexplored. Besides, another current and important issue is the fact that mushroom processing industries generate an unused surplus of pretreated lignocellulosic fibers called spent mushroom substrate (SMS). This pretreated lignocellulosic biomass can be used to produce ethanol, a promising alternative energy source for the environmentally unfriendly crude oil. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Therefore, the principal aims of the three articles that comprise this thesis were: 1) To provide a critical review on aspects related to chemical compounds isolated from the genus Pleurotus with possible biotechnological, nutritional and therapeutic uses. In the review, only reports published after 2005 were considered. 2) To prepare bioactive formulations of the fruiting body and mycelium of P. ostreatusroseus (ethanolic extracts), to characterize them in terms of hydrophylic and lipophilic compounds, to evaluate their antioxidant, antiinflammatory and antimicrobial potential, and to confirm their non-toxicity in a primary cell culture of porcine liver cells. 3) To investigate the potential of SMS of P. pulmonarius in the generation of fermentable reducing sugars through saccharification of its polysaccharides by a mixture of commercial cellulase and β-glucosidase. METHODS –1) The bioactive formulations of P. ostreatoroseus were obtained by extracting its mycelium (produced by submerged culture) and basidioma with an ethanolic (70:30 in water) solution. The soluble materials were firstly concentrated with a rotary vacuum evaporator at 40 °C, and freeze-dried. Free sugars were determined by high performance liquid chromatography (HPLC) coupled to a refraction index detector (RI detector). Organic acids were determined by ultra-fast liquid chromatography (UFLC) coupled with a photodiode array detector (PDA). Phenolic acids determination was performed using UFLC. Double online detection was carried out in the DAD using 280 nm as the preferred wavelength and in a mass spectrometer (MS) connected to a HPLC system via the DAD cell outlet. Tocopherols were determined by HPLC coupled to a fluorescence detector. Regarding the antioxidant activity assays, the sample concentrations (mg·mL−1) providing 50% antioxidant activity or 0.5 absorbance (EC50) were calculated from the graphs of antioxidant activity percentages (DPPH, β-carotene/linoleate and TBARS assays) or absorbance at 690 nm (ferricyanide/Prussian blue assay) against the sample concentrations. Trolox was used as a positive control. For the anti-inflammatory activity assay, the bioactive formulations were subjected to further dilutions from 8 mg·mL−1 to 0.125 mg·mL−1 and the tests were performed in mouse macrophage-like cell line RAW264.7. The effect of the samples on the growth of porcine liver primary cells (PLP2), according to methodology established by the group, was evaluated by the sulforhodamine B (SRB) colorimetric assay. For the antibacterial activity assay, |Gram- 10 negative and Gram positive bacteria were used. The Gram-negative bacteria were: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Enterobacter cloacae; the Gram-positive were: Staphylococcus aureus, Bacillus cereus, Micrococcus flavus, and Listeria monocytogenes. The minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations were determined by the microdilution method. For the antifungal bioassays, the following microfungi were used: Aspergillus fumigatus, Aspergillus versicolor, Aspergillus ochraceus, Aspergillus niger, Trichoderma viride, Penicillium funiculosum, Penicillium ochrochloron, and Penicillium verrucosum var. cyclopium. The minimal inhibitory concentration (MIC) determination was performed by a serial dilution technique using 96-well microtitre plates. 2) P. pulmonarius was cultivated on corn cob solid state cultures. During 30 days of vegetative growth, at intervals of 5 days, samples of SMS were collected from the cultivation bags and analyzed in order to investigate the transformations that occurred until fruiting bodies formation. Evaluations of the mycelial growth and consumption of reducing sugars and determinations of enzyme activities (laccase, catalase, Mn peroxidase and superoxide dismutase, SOD) were done in the crude water extracts obtained from each sample. The hydrogen peroxide content was quantified via the 2′–7′-dichlorofluorescein-diacetate (DCFH-DA) assay. The corn cob fibers obtained before and after fungal cultivation were subjected to enzymatic hydrolysis using cellulase from Trichoderma reesei ATCC and βglucosidase from Aspergillus niger. One unit of enzyme activity was defined as the amount of enzyme capable of releasing 1 μmol of product per minute. Also, the characterization of corn cob before and after P. pulmonarius cultivation was performed by scanning electron microscopy and Fourier transform infrared spectroscopy. The influence of the growth of P. pulmonarius on the substrate was analyzed in terms of the percent diminutions in intensity of the lignin (1427 and 1515 cm-1 ) and carbohydrate peaks (1395, 1098 and 898 cm-1 ). MAIN RESULTS AND DISCUSSION – 1) The bioactive formulations contain at least five free sugars, four organic acids, four phenolic compounds and two tocopherols. The fruiting body-based formulation revealed higher reducing power, DPPH scavenging activity, β-carotene bleaching inhibition and lipid peroxidation inhibition in brain homogenates than the mycelium-based preparation, as well as higher anti-inflammatory and antimicrobial activities. The absence of hepatotoxicity was confirmed in porcine liver primary cells. These functional responses can be related to the levels of bioactive components including phenolic acids, organic acids and tocopherols. 2) After mushroom harvesting, the SMS was hydrolyzed with commercial cellulase and β-glucosidase resulting in 300 g reducing sugars/kg dry SMS, being more than 60% glucose. Our data suggest that the SMS obtained from P. pulmonarius cultured on corn cob is a promising pretreated lignocellulose fiber for the obtainment of cellulosic ethanol. CONCLUSIONS – 1) A series of compounds have already been precisely defined in Pleurotus spp., including polysaccharides, phenolics, terpenes and sterols. However, intensification of structure determination is highly desirable and demands considerable efforts. Further studies including clinical trials need to be carried out to ascertain the safety of these compounds as adequate alternatives to conventional drugs. Not less important is to extend the search for novel bioactives to less explored Pleurotus species. 2) Overall, and to the best of our knowledge, this was the first report of anti-inflammatory properties of P. ostreatoroseus fruiting body and mycelium extracts, and from the results obtained, a clear anti-inflammatory and antimicrobial potential of the tested samples can 11 be inferred. Therefore, these formulations can be used to prepare dietary supplements for nutraceutical purposes. 3) This study reveals the potential of the SMS from P. pulmonarius cultivation as a source of biologically pretreated lignocellulosic fibers, useful for the obtainment of cellulosic ethanol. Nowadays, the cost of production of cellulosic ethanol is relatively high. The use of SMS could be a strategy for improving the yield and reducing the costs of the process. KEY-WORS: Pleurotus ostreatoroseus, bioactive formulations, anti-inflammatory potential, chemical composition, biological pretreatment, enzymes, hydrogen peroxide, Pleurotus pulmonarius, saccharification, spent mushroom substrate.

Artigos Publicados Vinculados a Tese:

https://doi.org/10.1016/j.tifs.2016.01.012; https://doi.org/10.1039/C5FO00465A; https://doi.org/10.1007/s12223-016-0457-8