Tecnologic Development on Pleurotus Cultivation: Specific Practices Used in Brazil

HIGHLIGHTS P. ostreatus and P. sapidus are the most productive species under the evaluated conditions. Different growing systems are suitable for the production of P. ostreatus var. Florida. Temperature control level affects differently the P. ostreatus var. Florida isolates. Environmental and strain factors affect yield and production parameters of P. ostreatus var. Florida.

Abstract In Brazil, Pleurotus is the most important mushroom produced especially P. ostreatus var. Florida. In this country as in many others, the great potential for mushroom cultivation remains unexplored. Therefore, it is very important to develop new studies that allow optimizing its production. The aims of the manuscript were: i) to evaluate the productivity of six different species of Pleurotus (P. citrinopileatus; P. djamor; P. ostreatus; P. ostreatus var. Florida; P. pulmonarius; P. sapidus); ii) to measure the effect of three different environmental conditions during cultivation of three isolates of P. ostreatus var. Florida. As results, P. ostreatus and P. sapidus were the most productive isolates under the evaluated conditions. Different environments produced variable effects according to the P. ostreatus var. Florida isolates, being possible to observe a highly plastic strain (POF 02/18), a highly sensitive strain (POF 03/18) and a strain with variable responses (POF 01/18).

Estudo do potencial anti-leishmania e anti-trypanosoma cruzi do ergosterol isolado do basidiomiceto pleurotus salmoneostramineus

Considerando a necessidade de novos tratamentos para doenças negligenciadas como a leishmaniose visceral e a doença de Chagas, o presente trabalho realizou o fracionamento do basidiomiceto comestível Pleurotus salmoneos tramineus na busca por substâncias potencialmente antiparasitárias. Dentre as frações ativas, foi isolado um composto denoninado ergosterol, o qual apresentou atividade anti-Leishmania (L.) infantum e anti-Trypanosoma cruzi. O ergosterol foi ativo contra amastigotas intracelulares de Leishmania (L.) infantum, com valor de Concentração Efetiva 50% (CE50) de 125 µM e de 129 µM contra formas tripomastigotas de Trypanosoma cruzi. O estudo da citotoxicidade em células de mamífero resultou em um valor de CE50 de 619 µM. Seu mecanismo de ação em tripomastigotas resultou uma rápida permeabilização da membrana plasmática, com a despolarização do potencial de membrana mitocondrial,levando o parasito à morte. Apesar disso, não se verificou aumento de espécies reativas de oxigênio no parasito, demonstrando que seu mecanismo de ação não envolve a indução de estresse oxidativo. A seleçãode metabólitos secundários antiparasitários presentes na natureza podefornecer futuros protótipos para o desenho de novos fármacos para doenças negligenciadas.

Considering the need for new treatments for neglected diseases as visceralleishmaniasis and Chagas disease, in this work we fractionated the ediblemushroom Pleurotus salmoneostramineus in the search for potentialantiparasitic compounds. Among the active fractions, it was isolated theergosterol, which showed anti-Leishmania (L.) infantum e anti-Trypanosomacruzi activities. The ergosterol was active against intracellular amastigotes ofLeishmania (L.) infantum and trypomastigotes of Trypanosoma cruzi, with50% Inhibitory Concentration (IC50) values of 125 µM and 129 µM,respectively. The cytotoxicity in mammalian cells resulted in an IC50 value of619 µM. Its mechanism of action in Trypanosoma cruzi trypomastigotesresulted in permeabilization of the plasma membrane, as well asdepolarization of mitochondrial membrane potential, leading to parasitedeath. Nevertheless, there was no increase in reactive oxygen species,demonstrating that its mechanism of action does not involve the induction ofoxidative stress in the parasite. The selection of antiparasitic secondarymetabolites present in nature can provide future prototypes for the design ofnew drugs for neglected diseases.

Morphology and mycelial growth rate of Pleurotus spp. strains from the Mexican mixtec region

Two native Pleurotus spp. strains (white LB-050 and pale pink LB-051) were isolated from rotten tree trunks of cazahuate (Ipomoea murucoides) from the Mexican Mixtec Region. Both strains were chemically dedikaryotized to obtain their symmetrical monokaryotic components (neohaplonts). This was achieved employing homogenization time periods from 60 to 65 s, and 3 day incubation at 28 °C in a peptone-glucose solution (PGS). Pairing of compatible neohaplonts resulted in 56 hybrid strains which were classified into the four following hybrid types: (R1-n xB1-n, R1-n xB2-1, R2-n xB1-n and R2-n xB2-1). The mycelial growth of Pleurotus spp. monokaryotic and dikaryotic strains showed differences in texture (cottony or floccose), growth (scarce, regular or abundant), density (high, regular or low), and pigmentation (off-white, white or pale pink). To determine the rate and the amount of mycelium growth in malt extract agar at 28 °C, the diameter of the colony was measured every 24 h until the Petri dish was completely colonized. A linear model had the best fit to the mycelial growth kinetics. A direct relationship between mycelial morphology and growth rate was observed. Cottony mycelium presented significantly higher growth rates (p < 0.01) in comparison with floccose mycelium. Thus, mycelial morphology can be used as criterion to select which pairs must be used for optimizing compatible-mating studies. Hybrids resulting from cottony neohaplonts maintained the characteristically high growth rates of their parental strains with the hybrid R1-n xB1-n being faster than the latter.

Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust

Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc) and manganese peroxidase (MnP). New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm-1 of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm-1, respectively). The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Criopreservação do gênero Pleurotus a -20 °c e a -70 °c / Criopreservación del género Pleurotus a -20 ºc y -70 ºc / Criopreservation of genus Pleurotus at -20 °c and -70 °c

O desenvolvimento de técnicas de criopreservação utilizando temperaturas comerciais (-20 °C) na conservação de Basidiomicetos é uma vantagem técnica e econômica para a produção de biomassa e/ou biocompostos, porém geralmente associada a problemas de danos celulares. O objetivo deste trabalho foi avaliar o uso de substratos para o crescimento do fungo associado a agentes crioprotetores na criopreservação a -20 ºC e a -70 ºC do gênero Pleurotus. O fungo, crescido em ágar-batata-dextrose ou em grãos de aveia, foi criopreservado a -20 °C e a -70 °C na presença dos crioprotetores glicerol, dimetilsulfóxido, glicose, sacarose, extrato de malte e polietilenoglicol. Foram discutidas as vantagens do substrato em associação com o crioprotetor, nas diversas temperaturas de crioproteção. Concluiu-se que o substrato utilizado para o crescimento do micélio influencia na viabilidade de recuperação do fungo criopreservado, em ambas as temperaturas, sendo que grãos de aveia promovem melhor recuperação do fungo e melhor vigor micelial. Os resultados sugerem uma simplificação técnica e redução de custos de manutenção de linhagens de Basidiomicetos.

The development of cryopreservation techniques using commercial temperatures (-20 °C) to preserve Basidiomycetes is an economical advantage for biomass and/or biocompound production; however, it is usually associated to cellular damages. The objective of this work was to evaluate the use of substrates for the fungus growth associated to cryoprotectants for the cryopreservation of Genus Pleurotus at -20ºC and -70ºC. The fungus, grown in either potato dextrose agar or oat grains, was cryopreserved at -20 °C or -70 °C with glycerol, dimethyl sulfoxide, glucose, sucrose, malt extract and polyethyleneglycol. The advantages of the substrate associated with the cryoprotectant at different cryopreservation temperatures were discussed. It was concluded that the substrate used for the mycelial growth positively influences the viability of the recovery of the cryopreserved fungus on both temperatures – oat grains promote better recovery and mycelial vigor. Those results suggest technical simplification and reduction of maintenance costs of culture collections of Basidiomycetes.

El desarrollo de técnicas de criopreservación utilizando temperaturas comerciales (-20ºC) en la conservación de Basidiomicetos es una ventaja técnica y económica para la producción de biomasa y/o biocompuestos, pero generalmente está asociada a problemas de daños celulares. El objetivo de esta investigación fue evaluar el uso de substratos para el crecimiento del hongo, asociado a agentes crioprotectores en la criopreservación a -20ºC y a -70ºC del género PleurotUs. Elhongo, crecido en agar-patata-dextrosa o en granos de avena, fue criopreservado a -20ºC y a -70ºC en la presencia de los crioprotectores glicerol, dimetilsulfóxido, glucosa, sacarosa, extracto de malte y polietilenoglicol. Fueron discutidas las ventajas del substrato en asociación con el crioprotector, en las diversas temperaturas de crioprotección. Se concluyó que el substrato utilizado para el crecimiento del micelio influencia en la viabilidad de recuperación del hongo criopreservado, en ambas las temperaturas, siendo que granos de avena promueven mejor recuperación del hongo y mejor vigor del micelio. Los resultados sugieren una simplificación técnica y reducción de costos de manutención de liñajes de Basidiomicetos.