Long-term cryopreservation of Lentinus crinitus strains by wheat grain technique.

Lentinus crinitus (Basidiomycota: Polyporales) is a saprophytic fungus with biotechnological importance described more than 20 years ago. However, there are few studies on the long-term preservation of this basidiomycete. Cryopreservation is a long-term storage technique that reduces the metabolic activity of microorganisms, but its success depends on the adjustment of the freezing process, the cryoprotectants, and the protective substrates for each species. This study aimed to assess the mycelial viability and genetic stability of L. crinitus strains cryopreserved at -86 °C for two years by the wheat grain technique using different cryoprotectants and freezing methods. Three strains of L. crinitus (U9-1, U13-5, and U15-12) were subjected to different concentrations and types of cryoprotectants (dimethyl sulfoxide, glycerol, glucose, and sucrose), freezing methods such as immediate freezing from 25 to -86 °C and progressing freezing from 25 to -86 °C in a freezing container with isopropyl alcohol to control the rate of cell freezing at -1 °C min-1, protective substrate (wheat grain and 2% malt extract agar), and cryopreservation period (1, 6, 12, and 24 months). After thawing, samples were evaluated for mycelial viability, time to mycelial recovery, mycelial stability, and genetic stability of the fungus. All techniques achieved effective cryopreservation at -86 °C, mainly with the wheat grain technique. All cryoprotectants (3.5% glycerol, 1.5% dimethyl sulfoxide, 25% sucrose, and 5% glucose), freezing methods (immediate and gradual), and protective substrate (wheat grain and malt extract agar) were effective for cryopreservation of the three L.crinitus strains in an ultra-low temperature freezer for two years. Mycelial viability, mycelial stability, and genetic stability of the fungus were not affected after two-year cryopreservation, evidencing the robustness of the long-term cryopreservation technique and the fungus.

Endophytic fungi of Brunfelsia uniflora: isolation, cryopreservation, and determination of enzymatic and antioxidant activity.

Brunfelsia uniflora (Pohl.) D. Don (Solanaceae), commonly known as manacá-de-cheiro, is widely distributed in Brazil and used by local indigenous peoples as an antirheumatic, antisyphilitic, depurative, emetic, vermifuge, and purgative agent. Several studies have examined the biological activities and phytochemical profile of Brunfelsia; however, few have focused on the diversity of endophytic microorganisms that colonize members of the genus. This study aimed to isolate and cryopreserve endophytic fungi from B. uniflora and determine their cellulase, laccase, and antioxidant activities. Endophytic fungi were isolated from B. uniflora stems, cultured on wheat grains, immersed in a 150 g L-1 aqueous sucrose solution, and cryopreserved at - 80 °C for 1 and 6 months. Cellulase activity was determined by a qualitative test using carboxymethylcellulose medium and laccase activity by a quantitative test based on the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Prior to antioxidant activity assays, fungi were grown in malt extract broth for production of mycelial biomass. A methanolic extract was prepared for evaluation of DPPH· scavenging activity, FRAP activity, and total phenolic content. A total of 46 endophytic fungal isolates were obtained from B. uniflora stems and classified into 24 groups according to morphological similarities. B. uniflora was shown to harbor different genera of ascomycete fungi as endophytic organisms. Mycelial viability was observed after 1 and 6 months of cryopreservation at - 80 °C. Fungi exhibited cellulase and laccase activities. Isolate CE23 had the highest laccase activity after 7 days of cultivation. Twelve isolates were found to have low total phenolic contents and DPPH· and FRAP activities.

Basidiocarp structures of Lentinus crinitus: an antimicrobial source against foodborne pathogens and food spoilage microorganisms.

Lentinus crinitus basidiocarps are an alternative to antimicrobials, but the stipe (24% basidiocarp) is discarded even with potential antimicrobial activity. This study evaluated the antimicrobial activity of L. crinitus basidiocarp pileus and stipe extracts against foodborne pathogens and food spoilage microorganisms. Basidiocarps of L. crinitus were grown in sugarcane bagasse and rice husks and the pileus and stipe methanolic extract was analyzed by broth microdilution method for antimicrobial activity against eight bacteria and eight fungi. The minimum bactericidal concentration values for pileus and stipe ranged from 0.40 to 0.50 mg mL- 1, for streptomycin from 0.10 to 0.50 mg mL- 1, and for ampicillin from 0.40 to 1.20 mg mL- 1. The minimum fungicidal concentration values for pileus and stipe ranged from 0.06 to 0.60 mg mL- 1, for bifonazole from 0.20 to 0.25 mg mL- 1, and for ketoconazole from 0.30 to 3.50 mg mL- 1. Extracts had bacteriostatic, bactericidal, fungistatic and fungicidal activity against all microorganisms, but with greater efficiency and specificity for some microorganisms. Both pileus and stipe are promising and sustainable alternatives for use in food, agricultural, and pharmaceutical industries.

Antimicrobial activity, chemical composition and cytotoxicity of Lentinus crinitus basidiocarp.

Lentinus crinitus (L.) Fr. (Basidiomycota: Polyporales) is a wild mushroom with several biotechnological applications; however, there are few studies on its chemical composition and antimicrobial activity. Therefore, this study aims to evaluate the chemical composition, cytotoxicity, and antimicrobial activity of L. crinitus basidiocarp. For that, its nutritional value (AOAC procedures) and its composition in some hydrophilic and lipophilic compounds (chromatographic techniques) were assessed. Moreover, the potential hepatotoxic effects were evaluated using a primary cell culture obtained from porcine liver, and its growth inhibitory capacity was also evaluated against four human tumour cell lines (spectrophotometric assays). The antimicrobial activity was evaluated by microdilution against eight bacteria and fungi. The basidiocarp has a high content of carbohydrates and, therefore, a relatively high energetic value. It is also rich in soluble sugars, ß-tocopherol, phenolic acids, mainly p-hydroxybenzoic acid, and organic acids, mainly malic acid. L. crinitus did not show cytotoxicity in non-tumour cells, but it did not inhibit the growth of human tumour cell lines either. The basidiocarp has a wide antimicrobial activity, inhibiting the growth of different species of bacteria and fungi. It showed minimum bactericidal and fungicidal concentration values similar to or lower than those verified by commercial antibiotics or food additives used as preservatives. The antimicrobial activity was more evident against Listeria monocytogenes, Salmonella enterica, and Penicillium ochrochloron, followed by Aspergillus ochraceus and Trichoderma viride, when compared to the controls. The results obtained in this study showed that L. crinitus basidiocarp has great potential to be used by the industry without toxicity risks.

Mycelial biomass cultivation of Lentinus crinitus / Crescimento micelial de Lentinus crinitus

Lentinus crinitus is a medicinal basidiomycete, little studied regarding the basic cultivation conditions, which is used in bioremediation and consumed by native Indians from the Brazilian Amazon. Also, it produces a fungal secondary metabolite panepoxydone that has been described as an essential regulator of the inflammatory and immune response. This study aimed to evaluate basic conditions of temperature, pH, and nitrogen concentration and source in the cultivation of L. crinitus mycelial biomass. In order to evaluate fungal growth temperature, 2% malt extract agar (MEA) medium, pH 5.5, was utilized from 19 to 40 °C. For pH, MEA had pH adjusted from 2 to 11 and cultivated at 28 °C. Urea or soybean meal was added to MEA to obtain final concentration from 0.5 and 16 g/L of nitrogen, pH of 5.5, cultivated at 28 °C. The best temperature growth varies from 31 to 34 ºC and the optimal one is 32.7º C, and the best pH ranges from 4.5 to 6.5 and the optimal one is 6.1. Protein or non-protein nitrogen concentration is inversely proportional to the mycelial biomass growth. Nitrogen concentrations of 2.0 g/L soybean meal and urea inhibit mycelial biomass growth in 11% and 12%, respectively, but high concentrations of 16.0 g/L nitrogen inhibit the growth in 46% and 95%, respectively. The fungus is robust and grows under extreme conditions of temperature and pH, but smaller adaptation with increasing nitrogen concentrations in the cultivation medium, mainly non-protein nitrogen.

Lentinus crinitus é um basidiomiceto medicinal consumido por índios nativos da Amazônia brasileira. Este fungo tem sido estudado quanto ao potencial de biorremediação de metais, mas ainda carece de estudos sobre às condições básicas de crescimento. L. crinitus produz panepoxidona - um metabólito secundário fúngico - descrito como regulador da resposta inflamatória e imune em células animais. Este trabalho teve como objetivo avaliar as condições básicas de temperatura, pH e concentração e fonte de nitrogênio para o crescimento micelial de L. crinitus. O fungo foi crescido em meio agar extrato de malte a 2% (MEA), pH 5,5 e mantido entre 19 e 40 °C. Para a avaliação de pH o MEA teve o pH ajustado de 2 a 11 e o crescimento foi realizado a 28 °C. As fontes de nitrogênio estudadas foram a uréia e o farelo de soja adicionado ao MEA para obter entre 0,5 a 16 g/L de nitrogênio, pH de 5,5, cultivado a 28 ° C. A melhor faixa temperatura para o crescimento micelial foi de 31 a 34 ºC com ótimo a 32,7 º C; a melhor faixa de pH de 4,5 a 6,5 e com ótimo de 6,1. A concentração de nitrogênio proteico ou não proteico é inversamente proporcional ao crescimento do fungo. Concentrações de nitrogênio de 2,0 g/L reduzem o crescimento da biomassa micelial em 11% e 12%, respectivamente e meios com nitrogênio de 16,0 g/L reduzem o crescimento em 46% e 95%, respectivamente. O fungo é robusto e cresce sob condições extremas de temperatura e pH, mas menor adaptação em meios com alta concentração de nitrogênio, principalmente não proteico.

Five-year cryopreservation at -80 °C of edible and medicinal basidiomycetes by wheat grain technique.

This research has focused on basidiomycete cryopreservation at -80 °C and developed a cryopreservation method based on the use of hard or medium-hard endosperm wheat grains as a mycelial carrier for cryopreservation. The aim of this study was to evaluate the mycelial viability of edible and medicinal basidiomycetes, using 13 strains of Agaricus spp. and eight strains of non-Agaricus spp., cryopreserved at -80 °C on hard endosperm wheat grain, with or without cryoprotectant agent (4% glucose), for two and five years. Two groups of basidiomycetes, Agaricus genus and other non-Agaricus genera, were cryopreserved at -80 °C by wheat grain technique for two and five years. The cryopreservation technique with hard endosperm wheat grain without cryoprotectant (preservation substrate), settled previously for A. subrufescens is efficient to cryopreserve other basidiomycetes such as Lentinus crinitus, Pleurotus ostreatus, Pleurotus eryngii, Schizophyllum commune, and Lentinula edodes, besides A. subrufescens strains.

Semisolid culture medium improves mycelial recovery of Agaricus subrufescens cryopreserved in cereal grains.

This study aimed to evaluate the effects of the solid and semisolid culture medium on the mycelial viability of A. subrufescens after 5-year cryopreservation at - 70 °C. Mycelia were grown in three types of whole or ground grains, with or without 5% glycerol addition in the substrate and/or in a cryotube. After 5 years of cryopreservation at - 70 °C, every treatment was thawed and recovered in malt extract culture medium with 15 (solid culture medium) or 5 g L-1 (semisolid culture medium) of agar. The semisolid recovery culture medium increased the mycelial viability recovery capacity of A. subrufescens cryopreserved for 5 years in grains with glycerol only in the cryotube, and specifically with medium-hard wheat grain without glycerol addition at all. Agar-based substrates such as malt extract agar, agar-ground grain, or the one with glycerol addition to the substrate were not effective to keep the mycelial viability, regardless of the recovery culture medium consistency. Hard and medium-hard endosperm wheat grains or hard endosperm rye grains with addition of glycerol as cryoprotectant only to the cryotube were effective to cryopreserve the fungus for 5 years without cryoprotectant addition in the substrate.

Cryopreservation at -75 °C of Agaricus subrufescens on wheat grains with sucrose

Abstract Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20 °C and at -75 °C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75 °C or at -20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75 °C. The fungus genome does not show alteration after two-year cryopreservation at -75 °C.

Cryopreservation at -75°C of Agaricus subrufescens on wheat grains with sucrose.

Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20°C and at -75°C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75°C or at -20°C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20°C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75°C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75°C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75°C. The fungus genome does not show alteration after two-year cryopreservation at -75°C.

Mycelial antineoplastic activity of Agaricus blazei.

Basidiocarp of Agaricus blazei (=Agaricus brasiliensis; =Agaricus subrufescens) is used as teas or capsules due to its antineoplastic effect but there are few reports of using mycelium for this purpose. The objective of this study was to evaluate the antineoplastic activity on sarcoma 180 cells implanted in mice of two forms of preparation of the mycelium from two A. blazei strains grown in culture medium with different concentrations of isolated soy protein. Mycelia were grown in Pontecorvo medium with different concentrations of isolated soybean protein (ISP). Mycelial hot water extract, moistened mycelial powder, hot water extract of green tea, Ifosfamida(®) (ifosfamide drug), and saline solution were administered daily by gavage in mice with sarcoma 180 cells to evaluate antineoplastic activity. It was concluded that antineoplastic activity was the same for both strains, except when used as moistened mycelial powder, which rules out the use of mycelial powder in capsules. Mycelial hot water extract had high antineoplastic activity with lower metabolic demand on the spleen and maintenance of normal blood parameters. Mycelial growth in different ISP concentrations had the same antineoplastic activity. Also the vegetative mycelium was as effective as the basidiocarp for sarcoma 180 tumor inhibition. Green tea was as effective as mycelial hot water extract.