Total fungal biomass was determined by measuring
the N-acetylglucosamine released by acid hydrolysis of the chitin present in
the fungal cell walls (10, 11). In brief, 0.5 g of dry fermented corn cob powder
was mixed with 1 mL of concentrated H2SO4. Acetyl acetone reagent (1 mL) was
added to the mixture, which was then placed in a boiling water bath for 20 min.
After cooling, 6 mL of ethanol was added, followed by 1 mL of Ehrlich reagent
(Sigma-Aldrich, Milwaukee, WI, USA) and incubated at 65°C for 10 min. After
cooling to room temperature, optical density (OD) was measured at 530 nm
against the reagent blank using N-acetylglucosamine (Sigma-Aldrich) as the external
standard.