Abstract

Microalgae are valuable sources of bioactive compounds with applications in health and nutrition. Tetradesmus obliquus is particularly promising due to its ability to produce lipids, carotenoids, and phytosterols, making it a candidate for integrated biorefinery approaches. Despite this potential, research on the extraction, characterization, and bioactivity of phytosterols from T. obliquus remains scarce. The present study optimized a sequential extraction to efficiently recover phytosterols and other high-value compounds. In the first step, supercritical CO2 (scCO?) at 306 bar and 40 °C extracted PUFAs reaching concentrations of 44.07 mg g?1 extract, and carotenoids, such as lutein and ?-carotene, reaching concentrations of 2.22 mg g?1 extract. The second step, using scCO? with 5% ethanol at 400 bar and 40 °C recovered phytosterols, with concentration of 1594.2 ± 0.002 µg g?1 extract for stigmasterol, 1072.7 ± 0.00 µg g?1 extract for campesterol and 597.5 ± 0.00 µg g?1 extract for ?-sitosterol. In terms of bioactivity, the carotenoid-rich extract demonstrated an antioxidant capacity of 0.191 ± 0.006 mmol Trolox eq g?1 extract by ABTS and 0.05196 ± 0.00165 mmol Trolox eq g?1 extract by ORAC. The bioactive properties of T. obliquus-derived phytosterols were evaluated, providing new insights into their potential applications. The phytosterol-rich extract exhibited significant ?-glucosidase inhibitory activity (IC50 = 111.1 µg mL?1), indicating potential for managing diabetes. To our knowledge, this is the first time that this bioactivity has been described for phytosterols obtained from T. obliquus. This study highlights the efficient and sustainable recovery of phytosterols from T. obliquus using an optimized biorefinery approach. © The Author(s), under exclusive licence to Springer Nature B.V. 2025.