Enzymatic Hydrolysis of Olive Husk

Enzymatic hydrolysis was performed following the method of Martín-Diana et al. [30 (link)], with some modifications. OH was resuspended in water (1:20 w/v). The solution was submitted at high hydrostatic pressure (HHP, 6 × 108 Pa, 5 min) using an HHP unit (Wave 6000/135, NC Hyperbaric, Burgos, Spain) with a vessel of 135 L and 200 mm diameter. After the batch was treated using a hydrothermal machine performed at 121 °C, 1.2 × 10⁵ Pa for 15 min using Ilpra Plus 100 autoclave equipment (Ilpra Systems, Barcelona, Spain). Subsequently, 1.5 M malic acid was used to adjust the pH to 5 prior to enzyme incorporation. Immediately thereafter, one part of batch was incubated with Novozymes food grade UltraFlo XL and in the other batch with Viscoferm, both at 1% (enzyme to OH dry weight ratio, w:w), and enzymatic hydrolysis was performed at 47 °C for 20 h using a temperature-controlled water bath with magnetic stirring at 1000 rpm (Unitronic Vaivén C, Selecta S. A., Spain), resulting in enzymatic hydrolysates EH1-OH and EH2-OH, with UltraFlo XL and Viscoferm, respectively. At the end of the incubation period, enzymes were inactivated in a water bath at 95 °C for 10 min. Insoluble residues were removed by filtration using a nylon filter (200 μm-mesh). Finally, OH soluble fractions were filtrated and stored at 4 °C and immediately analyzed.