*2.4. Analytical Methods*

Sugar concentration during CW hydrolysis and fermentation process were quantified by high performance liquid chromatography (HPLC) analysis (1200 series Agilent, Santa Clara, CA, USA) equipped with a differential refraction detector and an Aminex HPX-87H column (300 mm length × 7.8 mm internal diameter). The mobile phase was 10 mM H2SO4. The analysis was performed under isocratic conditions at a flow rate of 0.6 mL/min and 65 ◦C column temperature [33]. Injection volume was 10 µL and run time for samples was 25 min. Before injection, samples were diluted to appropriate concentration and filtered through a 0.22 µm Whatman**®** (Maidstone, UK) membrane filter.

Protease activity was evaluated by the production of free amino nitrogen (FAN) after hydrolysis of 7.5 g L−<sup>1</sup> of casein in 0.2 M phosphate buffer (pH 6.0) at 55 ◦C for 30 min. One unit (U) of proteolytic activity was defined as the amount of enzyme required for the release of 1 µg FAN in one minute under the above conditions [34]. FAN concentration was determined in both hydrolysis and fermentation using the ninhydrin colorimetric method [35].

Production of β-galactosidase was measured by the o-nitrophenol-β-d-galactopyranoside (ONPG) assay according to Raol et al. [29] with slight modifications. Briefly, 0.1 mL of crude extract was added to 0.4 mL of ONPG (3.0 mM) dissolved in sodium citrate buffer (50 mM, pH 5.0) and incubated at 50 ◦C for 10 min. The reaction was terminated by the addition Na2CO<sup>3</sup> (0.1 M) and the release of o-nitrophenol was estimated spectrophotometrically at 420 nm at a final volume of 3.0 mL. A calibration curve was prepared with o-nitrophenol under the same conditions. One unit (U) of β-galactosidase was defined as the amount of enzyme catalyzing the release of 1 µmol of o-nitrophenol per min according to the absorbance measurement.
