Tecnologia de cultivo para produção de proteases e prebióticos de cogumelo comestível da ordem Auriculariales

Abstract

Microorganisms represent an excellent source of proteases due to their great biochemical diversity. Among protease-producing fungi, few studies emphasize the relationship with the bioprocesses of mushrooms of the genus Auricularia. This research aimed to evaluate the qualitative activity of prebiotics and the production of extracellular proteases of Auricularia delicata DPUA1625 for the optimization of production and partial characterization of proteolytic enzymes under standardized conditions. The mushroom used in this study was provided by the DPUA Culture Collection and was subcultured on Potato Dextrose Agar (PDA) supplemented with 0.5% (w/v) yeast extract in Petri dishes. The cultures were maintained at 25ºC, in the absence of light, for 8 days. The mycelial growth of A. delicata was evaluated in seven culture media in order to select a medium for obtaining the inoculum culture and maintenance. After this procedure, protease production and mycelial growth were evaluated by submerged fermentation in cultures in GYP liquid medium (Glucose-Yeast Extract-Peptone, supplemented with 0.5% (w/v) gelatin); pH 6.4. Fermentation was carried out at 28 °C, at 150 rpm, for eight days. At the end of the bioprocess, the biomass was separated from the crude extract by vacuum filtration. The optimization of the culture conditions for protease production was performed by cultivation in two stages: initially, A. delicata was pre-cultivated in 50 mL of GYP for four days, at 28 °C, at 150 rpm, then the mycelial mass separated by vacuum filtration was transferred to the second stage culture and cultures were maintained under similar pre-cultivation conditions. Azocasein 1% (w/v) was used to determine the activity and partial characterization of the proteases. The qualitative activity The prebiotic activity was evaluated in liquid medium [MRS (Man Rugosa and Sharpe)], adding the crude extract (0.1%, 0.25% and 0.50%) recovered from the best fermentation process. Lactobacillus casei was used as standard. The cultures were maintained at 37 ºC. The partial result indicated that, among the media tested, PDA+YE agar was the medium in which A. delicata expressed the highest mycelial density. The optimum protease activity was observed at pH 6.0 and 60 ºC. These data indicate that A. delicata produces slightly acidic protease with importance for application in the food and beer industries.