A Box-Behnken Design Study For Boosting A Hydroxytyrosol-Rich Phytocomplex From Fermented Olive Mill Wastewater

Hydroxytyrosol (HT), a strong antioxidant compound, naturally occurring in table olives and virgin olive oil (VOO), is gaining increasing attention in the nutraceutical sector for its numerous health benefits, including anti-inflammatory, neuroprotective, antiviral, and antibacterial effects [1]. However, HT is not easily produced in high amounts. Its chemical synthesis suffers from multiple limitations, as harsh reaction conditions, high energy consumption, and use of toxic organic solvents. Moreover, several laborious and time-consuming purification steps are required, notably reducing the final HT yield [1, 2]. Conversely, advanced biotechnological approaches, coupled with innovative extraction technologies, have emerged as highly eco-sustainable alternatives. Olive mill wastewater (OMW), the primary by-product of olive oil extraction, contains the majority of olive phenolic compounds and an HT content up to a hundred times higher compared to VOO itself [3, 4]. In this study, OMW was investigated as a valuable and economically appealing source of HT. Firstly, OMW was fermented with β-glucosidase-positive microbial strains, previously isolated from table olives [5] and belonging to the microbial culture collection of Di3A and ProBioEtna srl, a spin-off of University of Catania (Italy). Driven fermentation process was monitored at different timepoints by UHPLC/DAD chromatography, highlighting the dynamic changes occurring during OMW fermentation. Oligomeric olive polyphenols, including oleuropein (OLE) and HT glycosides, were detected only at the beginning of fermentation, while HT was the predominant phenolic constituent of fermented OMW, suggesting a hydrolytic bioconversion from OLE. Subsequently, a Box–Behnken experimental design (BBD) model was developed to determine the optimal ultrasound-assisted extraction (UAE) conditions yielding the extract with the highest HT content from the fermented OMW. Ethanol in water concentration (% v/v), ultrasonication time (s), and amplitude (%) were selected as independent variables. The resulting HT-enriched phytocomplex was qualitatively and quantitatively characterized by UPLC/DAD analysis. Results showed strong antioxidant activity (evaluated by FRAP, DPPH, and ABTS assays) and a good scavenging capacity against intracellular reactive oxygen species (ROS), namely superoxide (O2• ̶ ) and ROO•. In addition, the fermented OMW samples were tested for their antimicrobial activity against Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19114, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica Typhimurium ATCC 14026, and Candida albicans ATCC 10231. Overall, this preformulative study underscores the nutraceutical potential of a new HT-enriched phytocomplex from fermented OMW, promoting more sustainable nutraceutical production practices and setting the stage for following encapsulation studies and in vivo tests.