Production of hydroxytyrosol rich extract from Olea europaea leaf with enhanced biological activity using immobilized enzyme reactors

“As olive leaves constitute the main by-product of the olive oil industry with important environmental and economic impact, there is an increasing demand for its valorization. In the present work, we report the development and application of immobilized enzyme batch bioreactors for the chemo-enzymatic treatment of an aqueous Olea europaea leaf extract rich in oleuropein to produce an extract enriched in hydroxytyrosol and other oleuropein hydrolysis products. To this end, a robust biocatalyst was developed through the immobilization of β-glucosidase on chitosan-coated magnetic beads which exhibited high hydrolytic stability after 240 h of incubation at 37 °C. The biocatalyst was successfully used in both a rotating bed-reactor and a stir-tank reactor for the modification of the olive leaf extract leading to high conversion yields of oleuropein (exceeding 90%), while an up to 2.5 times enrichment in hydroxytyrosol was achieved. Over 20 phenolic compounds (from different classes of phytochemicals such as flavonoids, secoiridoids, and their derivatives) were identified, in the extract before and after its modification through various chromatographic and spectroscopic techniques. Finally, the biological activity of both extracts was evaluated. Compared to the non-modified extract, the modified one demonstrated 20% higher antioxidant activity, seven-fold higher antibacterial activity, and enhanced cytotoxicity against leiomyosarcoma cells.”

Highlights:

• “The chemo-enzymatic modification of OLEx catalyzed by the immobilized bgl [β-Glucosidase from almonds] on chitosan magnetic beads (CMB) was performed in two different reactor types. Α rotating-bed-reactor (RBR) Spinchem RBR equipped with a V2 vessel and an S2 rotating bed purchased from SpinChem (Umeå, Sweden) was linked with a circulating water bath (Fig. 2a). A stir-tank bioreactor (STR) was obtained from Bioengineering consisting of a 2.5-L storage tank with a constant-temperature water circulating system (custom made) (Fig. 2b).”
• “For this purpose, a stirred tank and a rotating bed bioreactor were applied. Results regarding the reaction rate, the turnover number (TON), and the total turnover number (TTN) of both reactors revealed that rotating bed reactors are more efficient for bioprocesses with biocatalysts based on supports with mechanical fragility.”
• “To the best of our knowledge, this is the first report on the application of immobilized enzyme rotating-bed and stir-tank bioreactors for the preparation of a ΗΤ-enriched aqueous olive leaf extract with enhanced biological activities.”