Mushrooms and Fungi and Their Biological Compounds with Antidiabetic Activity: A Review.

Mushrooms have been used by humans for centuries as food and medicine because they have been shown to affect certain diseases. Mushrooms for medicinal purposes have been consumed in the form of extracts and/or biomass of the mycelium or fruiting body. The beneficial health effects of mushrooms are due to their content of bioactive compounds (polysaccharides, proteins, ergosterol, lectins, etc.). On the other hand, diabetes is one of the metabolic diseases that affects the population worldwide, characterized by hyperglycemia that involves a defective metabolism of insulin, a hormone secreted by ß cells and that mainly stimulates glucose absorption by the cells. However, it also affects the metabolism of carbohydrates, fats and proteins; poor control of this disease leads to serious damage to eyesight, kidneys, bones, heart, skin, blood vessels, nerves, etc. It has been reported that the consumption of some mushrooms helps control and treat diabetes, since among other actions, they promote the secretion of insulin by the pancreas, help reduce blood glucose and have α-glucosidase inhibitory activity which improves glucose uptake by cells, which are effects that prescription medications have for patients with diabetes. In that sense, this manuscript shows a review of scientific studies that support the abilities of some mushrooms to be used in the control and/or treatment of diabetes.

Isolation of Fungi from a Textile Industry Effluent and the Screening of Their Potential to Degrade Industrial Dyes.

Six fungal strains were isolated from the textile industry effluent in which they naturally occur. Subsequently, the fungal strains were identified and characterized in order to establish their potential decolorizing effect on textile industry effluents. The strains of interest were selected based on their capacity to decolorize azo, indigo, and anthraquinone dyes. Three of the strains were identified as Emmia latemarginata (MAP03, MAP04, and MAP05) and the other three as Mucor circinelloides (MAP01, MAP02, and MAP06), while the efficiency of their decolorization of the dyes was determined on agar plate and in liquid fermentation. All the strains co-metabolized the dyes of interest, generating different levels of dye decolorization. Plate screening for lignin-degrading enzymes showed that the MAP03, MAP04, and MAP05 strains were positive for laccase and the MAP01, MAP02, and MAP06 strains for tyrosinase, while all strains were positive for peroxidase. Based on its decolorization capacity, the Emmia latemarginata (MAP03) strain was selected for the further characterization of its growth kinetics and ligninolytic enzyme production in submerged fermentation under both enzyme induction conditions, involving the addition of Acetyl yellow G (AYG) dye or wheat straw extract, and no-induction condition. The induction conditions promoted a clear inductive effect in all of the ligninolytic enzymes analyzed. The highest level of induced enzyme production was observed with the AYG dye fermentation, corresponding to versatile peroxidase (VP), manganese peroxidase (MnP), and lignin peroxidase (LiP). The present study can be considered the first analysis of the ligninolytic enzyme system of Emmia latemarginata in submerged fermentation under different conditions. Depending on the results of further research, the fungal strains analyzed in the present research may be candidates for further biotechnological research on the decontamination of industrial effluents.

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.

In silico Design of Laccase Thermostable Mutants From Lacc 6 of Pleurotus Ostreatus.

Fungal laccase enzymes have a great biotechnological potential for bioremediation processes due to their ability to degrade compounds such as ρ-diphenol, aminophenols, polyphenols, polyamines, and aryldiamines. These enzymes have activity at different pH and temperature values, however, high temperatures can cause partial or total loss of enzymatic activity, so it is appropriate to do research to modify their secondary and/or tertiary structure to make them more resistant to extreme temperature conditions. In silico, a structure of the Lacc 6 enzyme of Pleurotus ostreatus was constructed using a laccase of Trametes versicolor as a template. From this structure, 16 mutants with possible resistance at high temperature due to ionic interactions, salt bridges and disulfide bonds were also obtained in silico. It was determined that 12 mutants called 4-DB, 3-DB, D233C-T310C, F468P, 3-SB, L132T, N79D, N372D, P203C, P203V, T147E, and W85F, presented the lowest thermodynamic energy. Based on the previous criterion and determining the least flexibility in the protein structures, three mutants (4-DB, 3-DB, and P203C) were selected, which may present high stability at high temperatures without affecting their active site. The obtained results allow the understanding of the molecular base that increase the structural stability of the enzyme Lacc 6 of Pleurotus ostreatus, achieving the in silico generation of mutants, which could have activity at high temperatures.

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.

Integral Use of Amaranth Starch to Obtain Cyclodextrin Glycosyltransferase, by Bacillus megaterium, to Produce ß-Cyclodextrin.

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins (CDs) from starch and related carbohydrates, producing a mixture of α-, ß-, and γ-CDs in different amounts. CGTase production, mainly by Bacillus sp., depends on fermentation conditions such as pH, temperature, concentration of nutrients, carbon and nitrogen sources, among others. Bacillus megaterium CGTase produces those three types of CDs, however, ß-CD should prevail. Although, waxy corn starch (CS) is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch (AS) could be used to accomplish those purposes. AS has high susceptibility to the amylolytic activity of CGTase because of its 80% amylopectin content. Therefore, the aim of this work was evaluate the AS as carbon source for CGTase production by B. megaterium in a submerged fermentation. Afterwards, the CGTase was purified partially and its activity to synthesize α-, ß-, and γ-CDs was evaluated using 1% AS as substrate. B. megaterium produced a 66 kDa CGTase (Topt = 50°C; pHopt = 8.0), from the early exponential growth phase which lasted 36 h. The maximum CGTase specific activity (106.62 ± 8.33 U/mg protein) was obtained after 36 h of culture. CGTase obtained with a Km = 0.152 mM and a Vmax = 13.4 µM/min yielded 40.47% total CDs using AS which was roughly twice as much as that of corn starch (CS; 24.48%). High costs to produce CDs in the pharmaceutical and food industries might be reduced by using AS because of its higher α-, ß- and γ-CDs production (12.81, 17.94, and 9.92%, respectively) in a shorter time than that needed for CS.

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.

Phylogenetic analysis of ß-xylanase SRXL1 of Sporisorium reilianum and its relationship with families (GH10 and GH11) of Ascomycetes and Basidiomycetes.

In this paper, the amino acid sequence of the ß-xylanase SRXL1 of Sporisorium reilianum, which is a pathogenic fungus of maize was used as a model protein to find its phylogenetic relationship with other xylanases of Ascomycetes and Basidiomycetes and the information obtained allowed to establish a hypothesis of monophyly and of biological role. 84 amino acid sequences of ß-xylanase obtained from the GenBank database was used. Groupings analysis of higher-level in the Pfam database allowed to determine that the proteins under study were classified into the GH10 and GH11 families, based on the regions of highly conserved amino acids, 233-318 and 180-193 respectively, where glutamate residues are responsible for the catalysis.

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.

Medium selection and effect of higher oxygen concentration pulses on Metarhizium anisopliae var. lepidiotum conidial production and quality.

Rice and oat flours were analyzed as media for the production of conidia by M. anisopliae var. lepidiotum. The presence of peptone increased conidia yield regardless of the substrate used; however, the highest yield was achieved on oat flour media. The effect of oxygen on conidia production using oat-peptone medium was also studied at two levels: Normal atmosphere (21% O(2)) and Oxygen-rich pulses (26% O(2)). Maximum conidia production (4.25 x 10(7) conidia cm(-2)) was achieved using 26% O(2) pulses after 156 h of culture, which was higher than 100% relative to conidial levels under normal atmosphere. Conidia yield per gram of biomass was 2.6 times higher with 26% O(2) (1.12 x 10(7) conidia mg(-1)). Conidia quality parameters, such as germination and hydrophobicity, did not show significant differences (P < 0.05) between those treatments. Bioassays parameters, using Tenebrio molitor adults, were analyzed for conidia obtained in both atmospheres and data were fitted to an exponential model. The specific mortality rates were 2.22 and 1.26 days(-1), whereas lethal times for 50% mortality were 3.90 and 4.31 days, for 26% O(2) pulses and 21% O(2) atmosphere, respectively. These results are relevant for production processes since an oxygen increase allowed superior levels of conidia by M. anisopliae without altering quality parameters and virulence toward Tenebrio molitor adults.

Laccases of Pleurotus ostreatus observed at different phases of its growth in submerged fermentation: production of a novel laccase isoform.

The production of laccases during the lag, exponential and stationary phases of growth of Pleurotus ostreatus in submerged fermentation was evaluated. Laccase activity was positively correlated to the growth of the fungus. The specific growth rate was 0.02 h(-1) and the highest amount of dry biomass (7.8 gl(-1)) was obtained after 480 h of growth. Four laccase isoforms were secreted by the fungus, and tentatively named L(I)1, L(I)2, L(I)3 and L(I)4. L(I)2, L(I)3 and L(I)4 were produced during the stationary phase (between 408 and 456 h approximately) while L(I)1 was produced during the lag, exponential and stationary phases of growth. Maximal laccase activity (12 200 Ul(-1)) was observed at the beginning of the stationary phase (at 432 h of growth). L(I)1 was purified by preparative isoelectric focusing and partially characterized. L(I)1 had a molecular mass of 43.7 kDa as determined by SDS PAGE, a Km and Vmax of 90 microM and 1.18 DeltaAbs min(-1) respectively and an isoelectric point of 2.3. L(I)1 showed activity over a broad range of pH and temperature, which may make it useful in the biodegradation of phenolic compounds present in wastewater from several industrial processes.

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies.

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.