Aqueous two-phase systems for the primary purification of olive pomace extracts obtained by autohydrolysis

Abstract

Olive pomace (OP), the main by-product of two-phase olive oil extraction, retains up to 99% of the fruit’s phenolic compounds, including hydroxytyrosol (HTY) and tirosol (TY), making it a promising source of natural antioxidants for food and pharmaceutical use. While autohydrolysis is an effective green method for extracting these compounds, it also co-extracts other substances like sugars, highlighting the need for selective purification. In this context, aqueous two-phase systems (ATPS), particularly those based on ethanol and salt, offer a sustainable, low-cost, and scalable alternative for this purpose. Although ATPS have been used with olive mill wastewater, their application to purify phenolic extracts from olive pomace remains unexplored. This study investigates ethanol/salt-based ATPS for the primary selective purification of phenolic compounds, particularly HTY and TY, from OP extracts obtained via autohydrolysis. OP consisting of a 50% mixture of Arbequina and Picual varieties, was obtained from a local olive oil mill using a two-phase extraction system. Autohydrolysis extraction was carried out under previously optimized conditions: 168 °C, 24 minutes, and a liquid-to-solid ratio of 20 mL per g of OP on a dry basis. Three different ethanol/salt-based ATPS were investigated: ethanol/sodium citrate (ATPS-1), ethanol/ammonium sulfate (ATPS-2), and ethanol/monobasic potassium phosphate/dibasic potassium phosphate (ATPS-3, molar ratio of dibasic to monobasic salt: 1.3). In all cases, the binodal curves were determined at 25 °C by the cloud point method and validated. The separation experiments were conducted at 25 °C, with a phase volume ratio of 1 between the top (ethanolic) and bottom (aqueous) phases, and the following tie-line lengths (TLL): 40% for ATPS-1, and 50% for the remaining systems (ATPS-2 and ATPS-3). In all cases, the same initial mass of OP extract was added. The experiments were carried out in triplicate. Total phenolic content (TPC), total soluble sugars (TS), HTY and TY content were determined. Figures 1 and 2 show the recovery results in the top phase, defined as the mass of the target compound (TPC, TS, HTY, or TY) relative to the initial mass of the corresponding compound in the extract, for the three tested ATPS. On the one hand, a differential separation between the compounds of interest (TPC and HTY) and TS is observed in all three ATPS, with a clear preference of the compounds of interest to migrate to the top (ethanolic) phase. In the case of TY, a differential separation with respect to TS was achieved only in the case of ATPS-2. On the other hand, ATPS-2 showed the highest recovery of TPC, HTY, and TY (approximately 90% in all three cases), with similar TS recoveries (approximately 50%) compared to the other two ATPS. The recovery of the compounds of interest in the top phase—which in the case of ATPS-2 contains around 27% (w/w) ethanol—could facilitate subsequent concentration and/or purification steps, given that ethanol is easier to remove than water (the sole solvent in the original extracts). In conclusion, the results demonstrate that all three ATPS evaluated are effective for achieving a primary separation between phenolic compounds and total sugars. Among them, ethanol/ammonium sulfate ATPS stands out due to its superior recovery of the target compounds (TPC, HTY, and TY), while maintaining comparable TS recoveries. Future work should be carried out to thoroughly evaluate different separation conditions using the ethanol/ammonium sulfate ATPS.