This study proposes a novel and optimized protocol for polyphenol fortification in extra virgin olive oil (EVOO) and a rapid, non-destructive, quantitative method to detect exogenous polyphenols using near-infrared spectroscopy (NIR) combined with partial least squares discriminant analysis (PLS-DA). Hydroxytyrosol (HTyr)-based extracts of different origins were characterized by HPLC-DAD to select those most compatible with the native EVOO phenolic profile. Among the tested extracts, olive fruit–derived extracts exhibited the highest similarity to EVOO, providing total polyphenol contents up to 1530–1584 mg/kg, with hydroxytyrosol levels exceeding 900 mg/kg and tyrosol around 170 mg/kg after fortification. The dissolution and fortification process was optimized using experimental design, identifying the aqueous phase concentration and water/oil ratio as the only significant factors. Optimal conditions consisted of 0.2 g HTyr per 2 g water and a 2:5 ( w /w) aqueous-to-oil ratio, yielding reproducible and homogeneous fortification. A total of 399 NIR spectra were recorded from 133 samples corresponding to seven fortification levels (0–100%) across 19 mono-cultivar EVOOs, enabling qualitative discrimination of exogenous bio-phenols. Chemometric analysis demonstrated that, despite natural variability in polyphenol content among EVOOs—particularly at low fortification levels (0–25%)—the PLS-DA model achieved 100% classification accuracy, with strong predictive performance (R 2 = 0.97, RMSEC = 5.90, RMSECV = 5.29). The method successfully differentiated fortified EVOO even when total polyphenol concentrations overlapped with those of naturally high-phenolic oils. These results confirm that hydroxytyrosol in EVOO predominantly originates from natural varietal and processing factors rather than exogenous addition, while also demonstrating that NIR–PLS-DA can reliably detect fortification at very low levels. The proposed approach offers a robust, fast, and sustainable tool for EVOO quality control, authentication, and fraud prevention, supporting the development of HTyr-enriched EVOO with enhanced antioxidant capacity, improved oxidative stability, and extended shelf life for food industry applications. • An optimal process for exogenous polyphenol fortification of various HTyr extracts was developed. • The model suggest that HTyr is naturally derived from field applications and processing. • A rapid, reliable, and non-invasive method for phenolic detection in EVOO was developed. • NIR-based PLS-DA strategy achieved an R 2 of 0.97 and a 100% correct rate in classification. • The PLS-DA model effectively distinguished between natural and fortified EVOOs, even at low levels.
Mehany et al. (Wed,) studied this question.