Biodegradation of agave Comiteco bagasse by Pleurotus spp.: a source of cellulases useful in hydrolytic treatment to produce reducing sugars.

This study aimed to determine the production parameters of five strains of Pleurotus spp. during their cultivation on agave Comiteco bagasse, as well as the feasibility of using cellulolytic extracts to produce reducing sugars in the same bagasse. After cultivation, the basidiome production parameters varied between 41.2 and 65.7% (biological efficiency), 0.17 and 0.30 (yield), 0.60 and 0.90% (production rate), 16.4 and 41.1% (Bioconversion) and 9.4 and 21.3 g (mean mushroom weight). At day 15 of growth, P. djamor showed the highest ß-glucosidase activity (43.95 ± 4.5 IU/g); on day 33. The same strain had the highest endoglucanase activity (21.12 ± 0.5 IU/ml). Both extracts were partially purified, and the kinetic parameters Vmax and Km were estimated (20.83 µmole/ml sec and 232.01 µmole/ml for ß-glucosidase and 685.01 µmole/ml sec and 1,240.34 µmole/ml for endoglucanase). In the enzymatic hydrolysis assay, the highest concentration of reducing sugars (43.13 ± 1.09 g/L; 0.21 g/g bagasse) was obtained by a mixture of the two partially purified extracts acting synergistically after 48 h and with a pH adjustment. The results suggest that the use of agave Comiteco bagasse for cultivating edible mushrooms while obtaining cellulolytic extracts is an alternative treatment for waste reduction and valorization of agro-industrial by-products.

Nematocidal activity of hydroalcoholic extracts of spent substrate of Pleurotus djamor on L3 larvae of Haemonchus contortus.

The objective of this study was to evaluate and compare the in vitro lethal effect of the hydroalcoholic extract of the spent substrate of Pleurotus djamor ECS-123, obtained at 15 days of colonization (SPS) and at the first (SPS1) and second (SPS2) harvests, against infective larvae L3 of Haemonchus contortus. The in vitro lethal effect was evaluated by the L3 larval mortality test (LM) using six concentrations: 1.25, 2.5, 5, 10, 20, and 40 mg/mL, with ivermectin and thiabendazole (5 mg/mL) as controls. The first harvest extract (SPS1) of strain ECS-123 was subjected to liquid-liquid bipartition, which resulted in two fractions: aqueous (PdAcO) and ethyl acetate (PdAct). The chemical fractionation of PdAct with the highest mortality rate (80.11 %) was carried out with open-column chromatography, giving a total of 13 fractions, which were analyzed by thin-layer chromatography (TLC) and grouped into 5 mixtures (R1;1-3, R2;4-7, R3;8-9, R4;10-11 and R5;12-13). Subsequently, the mixtures were evaluated against H. contortus L3 larvae. Finally, the components of the mixtures with the highest nematocidal effects were evaluated by gas chromatography coupled to mass spectrometry (GC-MS). The data were analyzed with a completely randomized design through ANOVA using the generalized linear model (GLM) with the "R" program. The purification and characterization of R4 and R5 by GC-MS revealed the presence of the following compounds: veratryl alcohol, 4-hydroxy-3,5,5 trimethyl-4-[3-oxo-1-butenyl]-2- cyclohexen-1-one, caffeine and 5,6-dimethoxy-1(3 H) isobenzofuranone. This information allowed for the identification of nematocidal compounds in the degraded substrate of P. djamor, an activity that had not been reported previously.

Edible Mushroom Protein Content and Antioxidant Capacity Increase the Longevity of Anastrepha ludens Fruit Flies.

This study aimed to determine the effect of the protein and antioxidant contents of edible mushrooms on the longevity of the fruit fly (Anastrepha ludens). The contents of protein (Bradford assay), antioxidants (DPPH and ABTS assays), total phenols, and flavonoids in nine strains of different edible mushroom species were determined. Freeze-dried and finely ground complete mushroom fruiting bodies were used to feed the flies, with a concentration of 0.5% in the diet. Male and female fruit flies, both fertile and sterile, were used in this study. Two controls were used: the standard fly diet and a diet supplemented with cinnamon as a food rich in antioxidants. Differences in protein and antioxidant contents were found among the evaluated strains. Differences were also observed in the responses of female and male flies as well as between the responses of fertile and sterile flies. Overall, the sterile flies lived longer. The addition of mushrooms in the diet resulted in greater longevity than in the controls. The use of sterile flies allowed observation of the effect of proteins and antioxidants on reproduction and the subsequent effect of reproduction on longevity.

Nematicidal Effect of Shiitake (Lentinula edodes) Extracts Against Haemonchus contortus.

During the previous decades, the indiscriminate use of anthelmintics for the control of gastrointestinal nematodes has generated anthelmintic resistance in different parts of the world. It is necessary to search for new sustainable control alternatives, such as the use of extracts from plants and edible mushrooms. Therefore, the aim of the present study was to evaluate the nematicidal activity of extracts and fractions of the edible mushroom Lentinula edodes against Haemonchus contortus eggs and infective larvae. The basidiomata of L. edodes ECS-401 were provided by the Tropical Fungi Laboratory of El Colegio de la Frontera Sur and were extracted with ethyl acetate, methanol, and water. Fractionation of the most active extract was carried out by open column chromatography. The bioassays were performed in 96-well microtiter plates using 100 eggs/larvae, a final volume of 100 µL, and different concentrations of extracts/fractions (n = 4). Bioassay readings were taken at 48 h for egg hatching inhibition (EHI) and at 24, 48 and 72 h for larval mortality (LM). The ethyl acetate extract exhibited the highest percentage of EHI (100%). For the LM bioassay, the aqueous extract was the most active (69%), but its fractions did not show larvicidal activity. The chemical profile of the aqueous extract was analyzed by high-performance liquid chromatography, which showed the presence of phenols, flavonoids, and terpenes. L. edodes extracts showed ovicidal and/or larvicidal activity.

In Vitro and In Vivo Nematicide Effect of Extract Fractions of Pleurotus djamor Against Haemonchus contortus.

Sheep haemonchosis is a disease that causes anemia, diarrhea, weight loss, low production, and even death of young animals. This represents a negative economic impact on the livestock sector. For decades, chemicals have been used to control this parasitic disease; however, cases of anthelmintic resistance have increased around the world. For this reason, the search for control alternatives is necessary. Several studies have shown that edible mushrooms of Pleurotus genus have different medicinal properties, including nematicidal activity. In this study, the chromatographic fractionation of the hydroalcoholic extract of fruiting bodies Pleurotus djamor was carried out to identify and isolate the metabolites responsible for nematicidal activity. From the fractionation of the extract, the fraction PdR2 and the subfraction PdB were obtained, which were evaluated against Haemonchus contortus under in vitro conditions. The nematicidal effect of the fraction PdB in gerbils infected artificially with H. contortus was evaluated. Finally, the analysis of the PdB fraction by gas chromatography-mass spectrometry and nuclear magnetic resonance was carried out. The results showed that the PdB had 100% of egg hatching inhibition from 5 mg/mL. Regarding larvicidal activity, it presented >97.2% from 24 h to 20 mg/mL. The in vivo evaluation of the PdB fraction showed a reduction of H. contortus larvae of 92.56%. The compounds present in this fraction were the mixture of allitol and an unidentified terpene in a ratio of 9:1. The PdB fraction is a potential alternative for the control of H. contortus, where allitol and a terpene could be responsible for nematicidal activity.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues / Bacillus subtilis con actividades de endocelulasa y exocelulasa aislado en la fase termofílica del compostaje con residuos de café

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57 -#9702;C and pH 5.5 and the other, a temperature of 61 -#9702;C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24 h until 144 h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

El objetivo de este estudio fue aislar, seleccionary caracterizar bacterias con actividad celulolítica a partir de 2 diferentes pilas de compostaje de residuos de café, una con temperatura interna de 57°C y pH 5,5; la otra con temperatura interna de 61 °C y pH 9,3. Se utilizaron medios de cultivo con carboximetilcelulosa y celulosa cristalina como únicas fuentes de carbono. La actividad enzimàtica fue evaluada por formación de halos de hidrólisis, producción de azúcares reductores y zimogramas. De 20 cepas aisladas, 3 presentaron mayor actividad enzimàtica y fueron identificadas como Bacillus subtilis sobre la base de sus características morfológicas, fisiológicas y bioquímicas y del análisis de las secuencias de la región 16S del ADNr. Los extractos enzimáticos de las 3 cepas seleccionadas presentaron actividad de exocelulasa y de endocelulasa, con máximos de 0,254 y 0,519 U/ml, respectivamente; la actividad de estas enzimas se mantuvo incluso a pH ácido (4,8) o básico (9,3) y a temperaturas de hasta 60 °C. Las actividades enzimáticas halladas en este estudio se ubican dentro de las más altas reportadas para celulasas producidas por bacterias del género Bacillus. En los zimogramas se demostró actividad de endocelulasa desde las 24h hasta las 144h de incubación. Asimismo, se reporta por primera vez el efecto del pH sobre la actividad de endocelulasa observado por zimogramas. Los resultados de este estudio abren la posibilidad de hacer uso de estas enzimas en la obtención de sustratos fermentables para la producción de energía a partir de los residuos generados en grandes cantidades por la agroindustria del café.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues.

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57°C and pH 5.5 and the other, a temperature of 61°C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24h until 144h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

Self-Heating Pasteurization of Substrates for Culinary-Medicinal Mushrooms Cultivation in Mexico.

The aim of this study was to evaluate a self-heating pasteurization technique in preparing substrates for mushroom production. Seven species were used: Agrocybe aegerita, Auricularia fuscosuccinea, Pleurotus djamor, P. eryngii, P. ostreatus, Lentinula edodes, and Ganoderma lucidum. They were cultivated on grass, corncob, wood shavings, and a mixture thereof. The self-heating technique allowed for pasteurization of 3 of the substrates (grass, corncob, and the mixture). The preheating chamber comprised a drawer placed under the pasteurization crate. With this chamber, it was possible to increase inlet air temperatures by 4--5°C. The evaluated mushroom species responded in different ways to the pasteurization process. P. ostreatus (control) and P. djamor produced basidiomes when cultivated in all pasteurization substrates. A. aegerita and P. eryngii fruited only on corncob and the mixture, whereas A. fuscosuccinea fruited only on the pasteurized corncob. G. lucidum and L. edodes did not fructify on the pasteurized substrates.

Hongos comestibles y medicinales en Iberoamérica: investigación y desarrollo en un entorno multicultural / Edible and medicinal fungi in Ibero-America: research and development in a multicultural environment

Los macromicetos u hongos macroscópicos conforman un grupo especial del Reino de los Hongos que adquiere cada día mayor relevancia. En efecto, existe en el mundo un claro interés por el conocimiento y la domesticación de estos organismos, toda vez que se descubren cada día mayores posibilidades de beneficio para la humanidad a partir de ellos. Si de la totalidad de especies de hongos estimadas sobre la tierra, una cuarta parte pertenecen a este grupo (lo que hace una estimación de alrededor de 400 mil especies), es evidente que aún queda muchísimo por conocer. Los avances actuales de la humanidad han permitido cultivar sólo un centenar. Es claro entonces, que con 0.025% de estos organismos domesticados ­mas no todos ellos bien conocidos- la rama de la micotecnología dedicada al cultivo de los macromicetos aún no inicia su expansión, mas bien se encuentra en fase de latencia o incubación y se esperaría que abundantes descubrimientos y grandes desarrollos y beneficios se concretaran en un futuro no muy lejano, en base a investigación y se difundieran mediante capacitación, transferencia y apropiación del conocimiento. Los temas tratados durante el taller superaron el planteamiento inicial ya que, además de tratar estrictamente el tema de cultivo de hongos comestibles, se abordaron temas como la situación y el potencial de los hongos silvestres. También se trataron diferentes aspectos de varios hongos de interés medicinal y funcional, y aún temas sobre manejo postcosecha de los hongos, entre otros. Estos tópicos no estaban contemplados inicialmente, pero resultaron indispensables para conformar una mejor perspectiva sobre la situación que guarda el cultivo y el aprovechamiento de los macromicetos en Iberoamérica.

Use of spent substrate after Pleurotus pulmonarius cultivation for the treatment of chlorothalonil containing wastewater.

Lignocellulosic materials are used as substrate for the cultivation of the edible mushroom Pleurotus pulmonarius. After two or three flushes of mushrooms, the spent substrate is discarded although it still has an important enzymatic activity that can be used for several purposes. In this study, we sought to determine the technical feasibility of using spent substrate from P. pulmonarius to degrade chlorothalonil. Reaction mixture was prepared with 6 ml of pesticide aqueous solution (2 mg active ingredient/l) and 3 ml of enzymatic extract obtained from spent P. pulmonarius substrate. The enzymatic reaction (27 °C, pH 7.4) was conducted for 1 h with sampling at 15 min intervals. The effect of storage time and temperature (freezing or refrigerating) of spent substrate and enzymatic extract, respectively, on the activity over chlorothalonil was determined. Freshly obtained spent substrate extract was able to reduce 100% of the initial concentration of chlorothalonil (2 mg/l) after 45 min of reaction. Storage time had a negative effect on the stability of the enzymatic activity: with spent substrate stored for a week, chlorothalonil concentration was reduced in 49.5% after 1 h reaction and with substrate stored for two and three weeks, the degradation efficiency decreased to 9.15% and 0%, respectively. Cooling and freezing the spent substrate extract also had a negative effect on chlorothalonil degradation.