Optimization of Saponification Process for Quantification of Individual and Total Tocopherols and Phytosterols in Canola Oil Refining By-Products

Abstract

In this study, saponification variables were optimized using a face-centred central composite design-response surface methodology. The variables included reaction temperature (50°C–120°C), reaction time (30–180 min), KOH concentration (0.5–3 M) and pyrogallol concentration (0–1% w/v). Using this approach, individual and total tocopherols and phytosterols in canola oil refining by-products, including soapstock, oil from spent bleaching earth (SBE), and deodorizer distillate, were quantified, and effects of variables were evaluated. The obtained results suggest that the non-saponified extract in distillate presented a substantial level of tocopherols and phytosterols and their levels increased after saponification by ~60% and ~20%, respectively. Quadratic models were significant (model p < 0.001) for all responses with good fit (R ² ≥ 0.91) and predictability. Furthermore, the effects of variables on individual and total tocopherols and phytosterols were explained and potential mechanisms were described. For tocopherols, pyrogallol generally exerted a protective effect, whereas stronger alkali and prolonged reaction time tended to negatively affect tocopherols, with γ-tocopherol showing greater reduction compared to α-tocopherol in some matrices. For phytosterols, temperature and time generally increased phytosterols, while losses during hot alkaline conditions and liquid–liquid extraction could offset gains depending on the by-products. The optimized conditions were experimentally validated and the co-optimized condition (120°C, 180 min, 0.5 M KOH, 1% w/v pyrogallol) for distillate yielded ~12.7 g/kg total tocopherols and ~108.9 g/kg total phytosterols. These conditions provide a practical basis for matrix-specific saponification and reliable quantification of tocopherols and phytosterols in canola oil refining by-products.