After this, the enzyme was added. The enzymatic hydrolysis was performed under optimal conditions of the particular enzyme at 50 °C for 24 h in an orbital mixer incubator. The enzyme was inactivated by boiling the sample at 100 °C for 10 min and cooling immediately in an ice bath. The extract was centrifuged at 8000 g for 20 min at 4 °C. The supernatant was then adjusted to pH 7.0, freeze-dried by a freeze-dryer (Virtis, Stone Ridge, NY, USA), and stored at −20 °C until ready for fermentation