4.7. Beta-Glucosidase Activity

Beta-glucosidase activity was measured determining the quantity of p-nitrophenol released from the p-nitrophenyl-beta-D-glucopyranoside (p-NPG) by the enzymatic activity of the tested strains. The activity was determined according to the method of Khoufi et al. [16], slightly modified. For the enzymatic assay, the incubation mixture contained 0.9 mL of 5 mM p-NPG (Sigma-Aldrich, Milan, Italy) in 50 mM citrate buffer (pH 4.8), and 0.1 mL of centrifuged broth from each overnight culture. The reaction was maintained at 50 °C for 2 h and stopped by the addition of 2 mL of Na₂CO₃ buffer (1.0 M). The amount of p-nitrophenol (p-NP) released was spectrophotometrically determined at 400 nm and quantified by using p-NP (Glentham, Life Science, UK) to obtain a standard curve. One unit (IU) of enzyme activity was defined as the amount of enzyme that produced 1 μmol of p-NP/min under the same conditions. The enzymatic results were expressed as IU/mL of sample by means of the following equation: IU/mL = Q/T × C/V, where Q is the quantity (µmole) of p-NP generated at the time T (min), V is the volume (mL) of the sample, and C is the total reaction volume (mL).