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.