Bio-directed Chemical Study of Pleurotus ostreatus Spent Substrate and Its Nematicidal Activity.

PURPOSE: In the present study, the nematicidal activity of the chemical fractionation of the spent substrate of the edible mushroom Pleurotus ostreatus against eggs and L3 larvae of Haemonchus contortus was evaluated. METHODS: The hydroalcoholic extract of the spent substrate was subjected to a bipartition with ethyl acetate giving two fractions: one aqueous (F. Ac) and one organic (F. AcOET). Both fractions were evaluated against eggs and L3 larvae at different concentrations (5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and 2% methanol, PBS and thiabendazole (5 mg/mL) as controls. Chemical fractionation of F. AcOET was performed in open column chromatography where 76 fractions were obtained and when analyzed by thin layer chromatography (TLC) were grouped into 11 mixtures (R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11). These mixtures were evaluated at 10 mg/mL against eggs and L3 larvae. The data obtained were analyzed by ANOVA and a Tukey test in the SAS V9 program. RESULTS: The results showed that, in the evaluation of F. AcOET and F. Ac, for the inhibition of egg hatching, the highest percentages were 78.80 and 76.89% at 5 mg/mL, respectively. As for the percentage of larval mortality, F. AcOET obtained 60.91 and F. Ac 29.77% at 5 mg/mL. The results of the evaluations of the mixtures showed that mixtures R4, R5 obtained 100 and 95.41% larval mortality and mixtures R6 and R7 presented 100% inhibitory activity of egg hatching, so these were analyzed by gas chromatography mass spectrometry finding compounds such as vanillin, ß-sitosterol, ρ-methyl ρ-hydroxycinnamate and ρ-hydroxybenzaldehyde. CONCLUSION: The results of the present study demonstrate that the spent substrate of P. ostreatus has potential anthelmintic activity against H. contortus. Moreover, by reusing and taking advantage of this substrate, its environmental pollution effects can be reduced.

Nematicidal activity of a hydroalcoholic extract of the edible mushroom Neolentinus ponderosus on L3 larvae of Haemonchus contortus.

OBJECTIVE: In the present study, the in vitro and in vivo effect of the hydroalcoholic extract of Neolentinus ponderosus (EHNP) on L3 larvae of Haemonchus contortus was evaluated. MATERIAL AND METHODS: The N. ponderosus fungus was cultivated in potato dextrose liquid medium for 7 days at 120 rpm and 25 °C. Subsequently, the EHNP was obtained; in vitro bioassays were performed in 96-well plates. Furthermore, an in vitro confrontation with different concentrations of EHNP was carried out at 72 h against L3 larvae of H. contortus. The controls used were distilled water and ivermectin at 5 mg/mL. Subsequently, the in vivo activity of EHNP was evaluated using the gerbil against H. contortus L3 larvae as an experimental model. The experimental design consisted of four groups with: (1) distilled water, (2) fenbendazole at 20 mg/kg of body weight, (3) EHNP at a dose of 81 µg/mL, and (4) EHNP at a dose of 40 µg/mL. RESULTS: In vitro study showed 97% mortality of the parasite H. contortus at a concentration of 3.4 mg/mL and a lethal concentration (LC90) of 2 mg/mL EHNP. In the in vivo assessment the highest mortality was (49%) at 72 h at a concentration of 81 µg/mL bw. CONCLUSION: The result of the present study shows that EHNP has nematicidal activity in vitro and in vivo tests (close to 97% and 50%, respectively), the fungus N. ponderosus should be considered in future tests to elucidate the secondary metabolites through spectroscopic studies.

Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation.

This research was conducted to extend the knowledge on the differential regulation of laccase genes in response to dyes. In order to accomplish this, we analyzed both, the expression of five laccase genes by real time RT-qPCR, and also the laccase activity and isoforms patterns during the time-course of a Pleurotus ostreatus submerged fermentation supplemented with either acetyl yellow G (AYG) or remazol brilliant blue R (RBBR) dyes. For the purpose of obtaining a stable reference gene for optimal normalization of RT-quantitative PCR gene expression assays, we tested four candidate reference genes. As a result of this analysis, gpd was selected as reference index for data normalization. The addition of dyes had an induction effect on the enzymatic activity and also modified the zymogram profile. Fermentation with RBBR showed the highest laccase activity and number of isoforms along the course of the fermentation. Laccase gene expression profiles displayed up/down regulation along the fermentation time in four laccase genes (pox4, pox3, poxa1b and pox2), while pox1 was not expressed in either of the fermentation conditions. AYG addition caused the highest induction and repression levels for genes pox3 and poxa1b respectively. The expression level for all genes in the presence of RBBR were lower than in AYG, being in both conditions this response growth time dependent. These results show the influence of the nature of dyes on the induction level of laccase activity and on the differential regulation of the laccase genes expression in P. ostreatus.

Evaluation of the Antioxidant Activity of Aqueous and Methanol Extracts of Pleurotus ostreatus in Different Growth Stages.

Total polyphenols and flavonoids contents, as well as ferric reducing antioxidant power (FRAP), metal ions chelating activity, reducing power assay and scavenging activity of DPPH and ABTS radicals in aqueous and methanolic extracts obtained from mycelium, primordium, and fruiting body of Pleurotus ostreatus in both fresh as dry, were evaluated. The total polyphenol content of dried samples was higher in aqueous extracts obtained both in room temperature and boiling. The total polyphenol content of the fresh samples obtained at room temperature and boiling was higher in aqueous extract of mycelium and in the methanolic extract of the fruiting body. In general, flavonoids represented a very small percentage of the total polyphenol content. The antioxidant activity measured by the FRAP method of extracts from fresh samples were higher with respect to the dried samples. The results of the metal ion chelating activity indicate that all extracts tested had acted. The reducing power of all samples was concentration dependent. In general, the extracts of dried samples showed higher reducing power than the extracts of fresh samples and tend to show greater reducing power by aqueous than methanolic extracts. It was observed that the DPPH and ABTS radical scavenging activities were positively correlated to the concentration of the extract. The results suggested that antioxidant activity could be due to polyphenols, but mainly by different molecules or substances present in the extracts. Overall, the fruiting body of P. ostreatus showed the best results and the possibility of continuing to investigate its functional properties of this fungus is opened. This is the first report where the antioxidant activity of P. ostreatus in different growth stage was reported.

Influence of initial pH of the growing medium on the activity, production and genes expression profiles of laccase of Pleurotus ostreatus in submerged fermentation

Background: Enzymatic activity and laccase isoenzymes number of Pleurotus ostreatus grown in different pH values of the growing medium in submerged fermentation and incubated in buffer solutions of different initial pH values were determined. The expression profiles of five laccase genes (Lacc1, Lacc4, Lacc6, Lacc9 and Lacc10) in these cultures were also studied. Results: The highest laccases activity was obtained in cultures grown at initial pH of 4.5 and the lowest in cultures grown at initial pH of 8.5. Isoenzyme profiles were different in all the cases. Lacc1, Lacc4, Lacc6 and Lacc10 were expressed in all the cultures. Conclusions: The initial pH of the growing medium is an important factor for regulating the expression of laccase genes, having an effect on the activity and on the laccase isoenzymes number produced by P. ostreatus in SmF. This is the first report on the influence of different initial pH values of the growing medium on the laccases activity, laccase isoenzymes number and laccases expression profiles of P. ostreatus grown in submerged fermentation.

Ligninolytic activity patterns of Pleurotus ostreatus obtained by submerged fermentation in presence of 2,6-dimethoxyphenol and remazol brilliant blue R dye.

The degradation of 2,6-dimethoxyphenol (DMP) and decolorization of Remazol brilliant blue R dye (RBB), added to culture media of Pleurotus ostreatus developed in submerged fermentation, and the laccase, manganese peroxidase and veratryl alcohol oxidase activities produced in these systems were evaluated. Both compounds were removed from the culture medium mainly by enzymatic action. These compounds decreased the specific growth rate and the effect on the maximal biomass values was not important. The enzymatic activities were increased by DMP and/or RBB; however, the DMP showed a higher inducer effect on all enzymes than RBB. On the other hand, the RBB showed a larger inducer effect on manganese peroxidase activity than on the laccases and veratryl alcohol oxidase activities. These results show that DMP was a better inducer of ligninolytic enzymes than dye, and the process of dye decolorization and degradation of DMP requires the action of all enzymes of the ligninolytic complex.