Recovery of Proteases and Protease Inhibitors from Ganoderma spp. Cultivated in Amazonian Lignocellulose Wastes.

BACKGROUND: Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain (G. lingzhi) and an Amazonian isolate (Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study. METHODS: Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). RESULTS: Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDS-- PAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor. CONCLUSION: Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities.

Aqueous Extracts of Fermented Macrofungi Cultivated in Oilseed Cakes as a Carbon Source for Probiotic Bacteria and Potential Antibacterial Activity.

Plant biomass colonized by macrofungi can contain molecules with bioactive properties with applications to human/animal health. This work aimed to verify antibacterial activities from aqueous extracts from oil seed cakes of Jatropha curcas (JSC) and cottonseed (CSC), fermented by macrofungi for probiotic bacteria cultivation. Coriolopsis sp., Tyromyces sp., Panus lecomtei, and Pleurotus pulmonarius were cultivated in solid and submerged media. The aqueous extract of unfermented JSC was more efficient than glucose for the growth of all probiotic bacteria. Extracts from four macrofungi fermented in CSC favored Lactobacillus acidophilus growth. In solid fermentation, macrofungi extracts cultivated in JSC favored Bifidobacterium lactis growth. All fungi extracts showed more significant growth than carbohydrates among the four probiotic bacteria evaluated. Regarding antimicrobial activities, no fungal extract or bacterial supernatant showed a more significant inhibition halo for enteropathogenic bacteria than ampicillin (control). Extracts from P. lecomtei and Coriolopsis sp. in CSC showed inhibition halos for Salmonella enterica. Supernatants from L. acidophilus, B. lactis, and Lactobacillus rhamnosus resulted in more significant inhibition of Staphylococcus aureus than the control, which indicates possible antimicrobial activity. Unfermented JSC supernatant showed better results for bacterial growth, while supernatants and aqueous extracts from CSC fermentation can be used for probiotic bacteria culture.

Bioactives and Extracellular Enzymes Obtained from Fermented Macrofungi Cultivated in Cotton and Jatropha Seed Cakes.

This work focused on obtaining fermented oil cake (cotton or Jatropha) via macrofungi growth with potential characteristics for animal feed formulations, such as the presence of extracellular enzymes, bioactive (ergosterol and antioxidants), and detoxification of antinutritional compounds. The concentration of phorbol esters was reduced by four macrofungi in Jatropha seed cake (JSC) to non-toxic levels. At least two macrofungi efficiently degraded free gossypol in cottonseed cake (CSC). Fermentation with Coriolopsis sp. INPA1646 and Tyromyces sp. INPA1696 resulted in increased ergosterol concentrations, antioxidant activity reduction, and high activity of laccases and proteases. Bromatological analysis indicated high crude protein concentrations, with partial solubilization by fungal proteases. Fermented products from Coriolopsis sp. and Tyromyces sp. in JSC or CSC can be considered important biological inputs for monogastric and polygastric animal feed.

Production of Lignocellulolytic Enzymes and Phenolic Compounds by Lentinus strigosus from the Amazon Using Solid-State Fermentation (SSF) of Guarana (Paullinia cupana) Residue.

The Amazon rainforest has a rich biodiversity, and studies of Basidiomycete fungi that have biomolecules of biotechnological interest are relevant. The use of lignocellulosic biomass in biotechnological processes proposes an alternative use, and also adds value to the material when employed in the bioconversion of agro-industrial waste. In this context, this study evaluate the production of lignocellulolytic enzymes (carboxymethylcellulases (CMCase), xylanase, pectinase, laccase) as well as phenolic compounds and proteases by solid-state fermentation (SSF) using the fungus Lentinus strigosus isolated from Amazon. The guarana (Paullinia cupana) residue was characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). SSF was carried out with 60% humidification of the residue, at 30 °C, for 10 days. The lignocellulosic biomass presented fragmented structures with irregular shapes and porosities, and was mainly constituted by cellulose (19.16%), hemicellulose (32.83%), and lignin (6.06%). During the SSF, significant values of CMCase (0.84 U/g) on the 8th day, xylanase (1.00 U/g) on the 7th day, pectinase (2.19 U/g) on the 6th day, laccase (176.23 U/mL) on the 5th day, phenolic compounds (10.27 µg/mL) on the 1st day, soluble proteins (0.08 mg/mL) on the 5th day, and protease (8.30 U/mL) on the 6th day were observed. In general, the agro-industrial residue used provided promising results as a viable alternative for use as a substrate in biotechnological processes.

Nutritional and Bioactive Properties of an Amazon Wild Oyster Culinary-Medicinal Mushroom, Pleurotus ostreatus (Agaricomycetes): Contributions to Functional Food and Human Health.

A wild Amazonian strain of Pleurotus ostreatus (Jacq.: Fr.) P. Kumm. was cultivated using local agroindustrial wastes-açai seeds (AS) and elephant grass straw (EGS)-as substrates and evaluated for its nutritional composition and bioactivities. Basidiomata presented higher contents of protein (27.19%) and dietary fiber (18.57%) when grown on AS, while lipids (2.26%), nonfiber carbohydrates (53.21%), and metabolizable energy (304.02 kcal/100 g) were higher on EGS substrate. Methanolic extracts of P. ostreatus grown on AS also provided a higher phenolic content (31.24 mg gallic acid equivalents/g extract) and greater antioxidant activity, scavenging 82.60% and 91.13% of DPPH· and ABTS·+ radicals, respectively, while chelating ability of Fe2+ was higher on EGS mushroom extracts (74.34%). Hemagglutinating activity of 1,997 HA U/mg protein was observed solely in the aqueous extracts of AS-grown mushrooms. Higher proteolytic activity was observed in aqueous extracts from mushrooms grown on EGS (219.10 U/mg protein), and their saline extract was the sole one with fibrinolytic activity (3.14 mm2). Both substrates and extractions yielded similar activity of protease inhibitors, with higher inhibition of serine than cysteine proteases. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis profiling showed protein bands related to lectins, proteases, fibrinolytic enzymes, and protease inhibitors. Thus, this wild Amazonian strain has great nutritional potential and produces biomolecules that can contribute to important applications in food, health, and industry.

Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum in submerged culture processes.

The antimicrobial activity of the metabolites produced by Fusarium oxysporum PR-33 in submerged culture was evaluated against Gram-positive and Gram-negative bacteria and yeasts. Metabolites were determined by HPLC-DAD-MS/MS. An extract was obtained following the removal of mycelium by centrifugation and lyophilisation of the supernatant. The compounds in this extract demonstrated broad-spectrum antimicrobial action, with rates of inhibition between 60 and 80%, depending on the species and extract tested. The major compounds of the extracts were identified as fusarinolic acid and its isomer [56.9% flask extract (FE)] and 59.2% bioreactor extract (BE), dehydrofusaric acid (35.7% FE and 31.6% BE), and fusaric acid (6.5% FE and 1.1% BE). Fusaric acid has been shown to be responsible for antimicrobial activity. The cytotoxicity of the extracts was evaluated in culture of HEK-293 and SH-SY5Y animal cells and toxicity of these extracts was verified even in the lowest tested concentrations. Therefore, our results indicate that the compounds identified exhibit potential as antimicrobial agents.

Composição mineral de substratos à base de resíduos de bananeira durante o cultivo de Pleurotus ostreatus / Mineral composition of substrates based on banana waste during the Pleurotus ostreatus cultivation

O objetivo deste trabalho foi avaliar a composição mineral (macro e micronutrientes) dos substratos [(inicial e residual (pós-colheita)] à base de diferentes combinações de resíduos (folha, pseudocaule e pseudocaule + folha) e cultivares de bananeira - Musa spp. (Thap Maeo, Prata Anã, Pelipita e Caipira), durante 49 dias de cultivo da linhagem POS 09/100 de Pleurotus ostreatus. Verificaram-se que todos os substratos à base de resíduos de diferentes cultivares de bananeira apresentaram quantidades satisfatórias de nutrientes para o cultivo de P. ostreatus, tanto na fase inicial de cultivo como na final.(AU)

The objective of this study was to evaluate the mineral composition (macro e micronutrients) of the substrates [initial and residual (postharvest)] based on different combinations of waste (leaf, pseudo-stem and pseudo-stem + leaf) and banana cultivars - Musa spp. (Thap Maeo, Prata Anã, Pelipita and Caipira) during 49 days for the cultivation of POS 09/100 strain of P. ostreatus. It was verified that all of the substrates based on different combinations of waste and banana cultivars presented satisfactory amounts of nutrients for the cultivation of P. ostreatus, both in the initial phase of cultivation and in the end.(AU)

Application of Chinese Jun-Cao technique for the production of Brazilian Ganoderma lucidum strains

Ganoderma lucidum is a medicinal mushroom traditionally used in China against a wide range of diseases such as cancer and also for its prevention. In this work, commercial Chinese strains G. lucidum were compared to wild Brazilian strains aiming to determine the cultivation potential through the use of Jun-Cao. Six formulations were tested and the strains presented good response to the applied method. In general, the mixture between the grass and wood was well suited for the basidiomycetes, contributing to the preparation of substrates that generated better results in Jun Cao.

Centesimal composition and physical-chemistry analysis of the edible mushroom Lentinus strigosus occurring in the Brazilian Amazon.

The centesimal composition and the physical and chemical analyses of Lentinus strigosus, an edible mushroom occurring in the Brazilian Amazon and produced in alternative substrates based on wood and agroindustrial residues, were evaluated. For this purpose, the C, N, pH, soluble solids, water activity, protein, lipids, total fiber, ash, carbohydrate, and energy levels were determined. The substrates were formulated from Simarouba amara Aubl. ("marupá"), Ochroma piramidale Cav. Ex. Lam. ("pau-de-balsa") and Anacardium giganteum ("cajuí") sawdust and Bactris gasipaes Kunth ("pupunheira") stipe and Saccharum officinarum (sugar cane bagasse). The results indicated that the nutritional composition of L. strigosus varied with the substrate of cultivation; the protein levels found in mushrooms grown in the different substrates (18-21.5%) varied with the substrate and was considered high; the soluble solids present in the mushrooms could have a relation with complex B hydrosoluble vitamins. L. strigosus could be considered as important food owing to its nutritional characteristics such as high protein content, metabolizable carbohydrates and fibers, and low lipids and calories content.

Applicability of the use of waste from different banana cultivars for the cultivation of the oyster mushroom

The objective of this research was to evaluate the oyster mushroom Pleurotus ostreatus- (Jacq.: Fr.) Kumm. cultivation in substrates based on different combinations of wastes (leaf, pseudo-stem and pseudo-stem + leaf) and banana cultivars - Musa spp. (Thap Maeo, Prata Anã, Pelipita and Caipira) during 49 days. Organic matter loss in the substrate by action of the fungus was also evaluated during that period. It was verified that the pseudo-stem waste provided the best averages of biological efficiency among all cultivars tested and best rates were obtained by Thap Maeo (61.5%). The highest organic matter loss (OML) was obtained from pseudo-stem + leaf wastes (Prata Anã - 78.6%; Thap Maeo - 67.6%; Pelipita - 64.8%; Caipira - 60.6%). Therefore, the use of those wastes showed itself viable for P. ostreatus cultivation due to its availability and low cost, besides decreasing discards to environment.

Applicability of the use of waste from different banana cultivars for the cultivation of the oyster mushroom.

The objective of this research was to evaluate the oyster mushroom Pleurotus ostreatus- (Jacq.: Fr.) Kumm. cultivation in substrates based on different combinations of wastes (leaf, pseudo-stem and pseudo-stem + leaf) and banana cultivars - Musa spp. (Thap Maeo, Prata AnãPelipita and Caipira) during 49 days. Organic matter loss in the substrate by action of the fungus was also evaluated during that period. It was verified that the pseudo-stem waste provided the best averages of biological efficiency among all cultivars tested and best rates were obtained by Thap Maeo (61.5%). The highest organic matter loss (OML) was obtained from pseudo-stem + leaf wastes (Prata Anã 78.6%; Thap Maeo - 67.6%; Pelipita - 64.8%; Caipira - 60.6%). Therefore, the use of those wastes showed itself viable for P. ostreatus cultivation due to its availability and low cost, besides decreasing discards to environment.

Aproveitamento de resíduos madeireiros para o cultivo do cogumelo comestível Lentinus strigosus de ocorrência na Amazônia / Use of wood residues for the cultivation of edible mushroom Lentinus strigosus in the Amazon

Este trabalho teve como objetivo a utilização de resíduos madeireiros do estado do Amazonas para o cultivo de Lentinus strigosus. de ocorrência na região. A linhagem foi procedente da coleção do Instituto Nacional de Pesquisas da Amazônia - INPA. Utilizou-se separadamente serragens de Simarouba amara (marupá), Ochroma piramidale (pau de balsa) e Anacardium giganteum (cajuí) suplementadas com farelo de arroz e de trigo e CaCO3 (80:10:8:2), respectivamente, ajustando-se a umidade em torno de 75 por cento. Os substratos (500g) foram acondicionados em sacos de polipropileno, esterilizados a 121 ºC , durante 30 minutos, inoculados e incubados em câmara climatizada a 25 ± 3 ºC e UR de 85 por cento, até emissão dos primórdios, com redução de temperatura de 25 para 23 ± 1 ºC e aumento de UR para 85-90 por cento, no período de "frutificação". O crescimento micelial ocorreu de 12 a 20 dias, com surgimento de primórdios com cerca de 15 a 25 dias após a inoculação. A produção de basidiocarpos ocorreu três a cinco dias após a emissão dos primórdios. Foram avaliados: eficiência biológica (EB, por cento), rendimento (g kg-1) e perda da matéria orgânica (PMO, por cento). As serragens suplementadas foram eficientes no cultivo de L. strigosus, apresentando EB de 38, 48 e 59 por cento; rendimento de 98, 119 e 177 g kg-1; e PMO de 42, 59 e 48 por cento, para marupá, pau de balsa e cajuí, respectivamente. Assim, há um potencial de aproveitamento desses resíduos na Amazônia, bem como uma provável utilização da linhagem selvagem, podendo contribuir para melhoria das condições sócio-econômicas da população regional e sustentabilidade dos recursos da biodiversidade.

The objective of this work was to use wood residues from the state of Amazonas for the cultivation of Lentinus strigosus. found in that region. The strain came from the collection of the National Institute of Amazonian Research. Sawdust species Simarouba amara, Ochroma piramidale and Anacardium giganteum were separately used in the preparation of the substrates, supplemented with rice and wheat bran and CaCO3 (80:10:8:2), respectively, with humidity adjusted to approximately 75 percent. Substrates (500 g) were packed using bags polypropylene, sterilized at 121 ºC for 30 minutes, inoculated and incubated in an acclimatized chamber at 25 ± 3 ºC and relative air humidity of 85 percent until primordia emission, with temperature reduced from 25 to 23 ± 1 ºC and air relative humidity increased to 85-90 percent in the fruiting period. Micelial growth occurred within 12 to 20 days and primordia appeared within 15 to 25 days after the inoculation. Harvest occurred three to five days after primordia emission. Biological efficiency (BE, percent), yield (g kg-1) and loss of organic matter (PMO, percent) were evaluated. Supplemented sawdust were efficient in the cultivation of L. strigosus, presenting BE of 38, 48 and 59 percent, yield of 98, 119 and 177 g kg-1 and PMO of 42, 59 and 48 percent for Simarouba amara, Ochroma piramidale and Anacardium giganteum, respectively. The results show the potential use of those residues in the Amazon, as well as the possibility of using the wild strain, to contribute to the improvement of the social and economical conditions of the regional population and sustainability of the biodiversity resources.

Mineral composition of raw material, substrate and fruiting bodies of pleurotus ostreatus in culture / Composición mineral de la matéria prima, el substrato y los cuerpos de fructificación de Pleurotus ostreatus / Composição mineral da matéria prima, do substrato e dos corpos de frutificação de Pleurotus ostreatus

In a culture of a Pleurotus ostreatus (oyster mushroom) strain, macro and micronutrients of the raw material and the initial and spent substrates were evaluated. Substrates were formulated with sawdust from Simarouba amara Aubl. and Ochroma piramidale Cav. ex. Lam., crushed Bactris gasipaes Kunth and crushed Saccharum officinarum (sugar cane). Samples were solubilized by means of acid digestion (nitric-peridrol). Ca, Mg, Fe, Cu, Zn and Mn were determined by atomic absorption spectrophotometry, Na and K by atomic emission, and P by colorimetry. The mineral composition of the fruiting body varied with the substrates, which made possible the production of a fruiting body rich in K, P, Mg and Fe. Potassium was the mineral with the highest content in the fruiting body in all substrates tested (36.83-42.18g·kg-1). There was an increase of protein and mineral content in the spent substrate in relation to the initial one.

En un cultivo del hongo Pleurotus ostreatus fueron analizados los macro y micronutrientes de la materia prima y de los sustratos inicial y residual (post-cosecha). Los sustratos analizados fueron formulados a partir de aserrín de de Simarouba amara Aubl. y de Ochroma piramidale Cav. ex. Lam., y de bagazos de Bactris gasipaes Kunth y de Saccharum officinarum (caña de azúcar). Las muestras fueron solubilizadas por digestón ácida (nítrico-peridol). Los elementos Ca, Mg, Fe, Cu, Zn y Mn fueron determinados por espectrofotometría de absorción atómica, Na e K por emissión atómica, y el P por colorimetría. La composición mineral del hongo varió con el sustrato de cultivo y los sustratos posibilitaron la producción de un hongo rico en K, P, Mg e Fe. El potasio fue el mineral de mayor contenido en el hongo en todos los sustratos ensayados (36,83-42,18g·kg-1). Hubo un aumento del contenido proteico y de minerales en el sustrato residual en relación al inicial.

Foram analisados macro e micronutrientes da matéria-prima, do substrato inicial, residual (pós-colheita) e do cogumelo no cultivo do Pleurotus ostreatus. Os substratos analisados foram formulados a partir de serragem de Simarouba amara Aubl. (marupá) e de Ochroma piramidale Cav. ex. Lam. (pau de balsa) e dos bagaços de Bactris gasipaes Kunth (pupunheira) e de Saccharum officinarum (cana-de-açúcar). As amostras foram solubilizadas mediante digestão ácida (nitrico-peridrol). Os elementos Ca, Mg, Fe, Cu, Zn e Mn foram determinados por espectrofotometria de absorção atômica, Na e K por emissão atômica, e o P por colorimetria. A composição mineral do cogumelo variou com o substrato de cultivo e os substratos possibilitaram a produção de um cogumelo rico em K, P, Mg e Fe. O potássio foi o mineral de maior conteúdo no cogumelo em todos os substratos testados (36,83-42,18g·kg-1). Houve um aumento do conteúdo protéico e de minerais no substrato residual em relação ao inicial.

Fungos emboloradores e manchadores de madeira em toras estocadas em indústrias madeireiras no município de Manaus, Amazonas, Brasil / Mould and stain fungi in logs stored in wood industries of Manaus, Amazonas, Brazil

Visando conhecer a diversidade e a incidência de fungos emboloradores e manchadores de madeira da região Amazônica, foi realizado um levantamento em 12 espécies florestais, estocadas em quatro indústrias madeireiras de Manaus. De cada espécie florestal selecionaram-se cinco toras, das quais retiraram-se amostras de onde procedeu-se isolamento dos fungos. Foram isolados 106 fungos associados às essências florestais, representados por nove gêneros e por dezesseis espécies. Paecilomyces variotii e Lasiodiplodia theobromae foram as espécies fúngicas mais freqüentemente associadas às espécies florestais. Ceiba pentandra e Hura crepitans foram as essências florestais que apresentaram maior diversidade de fungos, com sete espécies cada uma. Hymeneae courbaril foi a que apresentou menor diversidade de fungo, com apenas uma espécie.

In order to know a diversity and incidence of wood mould and wood stain fungi from Amazonian wood, the survey and identification of these kinds of fungi were carried out in twelve wood species from four wood industries of Manaus, Amazonas. Five logs were randomly selected. Wood samples were prepared and transported to the Wood Pathology Laboratory at the Forest Product Research Center of the National Institute for Amazonian Research, where the isolation and identification of the fungi were conducted. One hundred and six fungi associated to the wood species were isolated, represented by nine genera and sixteen fungi species. Paecilomyces variotti and Lasiodiplodia theobromae were the more representative fungi, associated with ten and seven wood species respectively. Ceiba pentandra e Hura crepitans were the wood species with highest diversity of fungi, with seven species each. Hymenae courbaril was the wood that presented the lowest diversity of fungi, with only one fungus.

Influência da estrutura anatômica de Brosimum rubescens Taub. Moraceae (Pau-rainha) no processo de microdistribuição do CCA -A

In the present study, the wood treatability of the Amazon species Brosimum rubescens Taub., Moraceae (pau-rainha) was evaluated. The tests were carried out on three different positions of the tree stem (lower end, middle and upper end) with CCA type A at 2% concentration, using the Full-Cell process, also known as "Bethell" process. The data showed that the sapwood is moderately difficult to preserve, whereas the heartwood is refractory. The results of absorption at different positions of the tree stem did not present significant differences. There was a significant difference between sapwood and heart-wood, with the retention ranging from 7,95 to 8,84 kg/m3 for sapwood and from 0,13 to 0,27 kg/m3 for heartwood. Although the vessels are considered structures of major importance related to conduction, the fibres played a relevant role in the process of preservative distribution. The rays and the axial parenchyma, even in the sapwood, were not efficient due to extractive deposits present in these elements. These results suggest that the refractory property of the heartwood, as well as the limited permeability of the sapwood of this specie is related to the extractive contents present in the wood, and especially due to the presence of tyloses inside the vessels.

No presente estudo foi avaliada a tratabilidade da espécie madeireira Brosimum rubescens Taub., Moraceae (pau-rainha), da região amazônica. Os testes foram feitos em três diferentes alturas (base, meio e ápice do fuste), com preservante CCA, tipo A, a 2% de concentração, utilizando-se o processo de impregnação sob pressão, através do método de célula cheia "Bethell". Os dados mostraram que o grau de tratabilidade do alburno é "moderadamente difícil" enquanto que o cerne é "refratário". Os resultados de absorção das toras estudadas nas diferentes alturas da árvore não apresentaram diferença significativa. Houve diferença significativa entre cerne e alburno, com intervalos de retenção de 7,95 a 8,84 kg/m3 para o alburno e de 0,13 a 0,27 kg/m3 para o cerne. Embora os vasos sejam considerados estruturas de maior importância em relação a condução, as fibras obtiveram papel relevante no processo de distribuição do preservante. O raio e o parênquima axial, mesmo no alburno, apresentaram-se ineficientes quanto a condução, devido a deposições de extrativos presentes nesses elementos. Os resultados sugerem que a refratabilidade do cerne, bem como a permeabilidade limitada do alburno desta espécie está relacionada com o conteúdo de extrativos presentes na madeira e, em especial, devido à presença de tiloses nos vasos.