Influence of Direct Saponification Conditions on Sterol Yield and Composition of the Unsaponifiable Fraction of the Microalga Isochrysis galbana and the Seaweed Ascophyllum nodosum

ABSTRACT Saponification is a key step for recovering the unsaponifiable fraction (USF) of lipids, which contains bioactive sterols, tocopherols, and carotenoids. This study optimized direct saponification conditions (50–300 g L −1 KOH; 60°C–100°C) to recover sterol‐rich USFs from the microalga Isochrysis galbana and the brown seaweed Ascophyllum nodosum , and compared this one‐step approach with conventional lipid extraction followed by saponification. Changes in carotenoid profile and in vitro antioxidant activity were secondary outcomes. Sterols and carotenoids were characterized by GC–MS and LC–MS/MS, respectively, and antioxidant capacity by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) assays. Direct saponification gave a USF profile broadly comparable to the two‐step method. In I. galbana , brassicasterol was the dominant sterol (up to 3 mg g −1 dry weight, DW), whereas fucosterol was dominant in A. nodosum (up to 1.7 mg g −1 DW). Measured sterol content increased with KOH concentration up to 200 g L −1 but declined at higher alkalinity or elevated temperatures, indicating a trade‐off between hydrolysis and degradation. Under hot alkaline conditions, intact fucoxanthin was not detected in the USFs, while β‐carotene and putative oxygenated (hydroxy−/keto‐) carotenoids became more prominent among the detected species, indicating pigment transformation. The USFs exhibited measurable antioxidant activity, particularly in the ABTS assay (IC 50 0.13 and 0.36 mg mL −1 for A. nodosum and I. galbana , respectively); these activities likely co‐varied with sterol and carotenoid contents, reflecting overall fraction composition rather than a single dominant contributor. Overall, ≈200 g L −1 KOH at 80°C–100°C produced the highest sterol yields while transforming thermolabile carotenoids.