Green ultrasound-assisted extraction for efficient carotenoid profiling of calamansi (Citrofortunella microcarpa)

Calamansi (Citrofortunella microcarpa), a small citrus fruit valued for its unique flavor, is a rich source of bioactive carotenoids that have not yet been individually identified. An efficient carotenoid extraction method, together with knowledge of its carotenoid compositions, is essential to support broader utilization of this intriguing fruit. Herein, we present a sustainable and efficient carotenoid extraction method by integrating ultrasound-assisted extraction (UAE) with green solvent systems. Seven solvent systems were initially screened for their extraction efficiencies with and without UAE. Among them, a deep eutectic solvent, 1:1 menthol:camphor (Men:Cam), stood out as an effective solvent. Interestingly, UAE also dramatically improved the efficiency of edible oils, e.g., sunflower oil. Response surface methodology (RSM) was then employed to elucidate the correlations among parameters (solvent ratio, temperature, and time) that affect the extraction efficiency in selected systems. For example, the optimal condition for Men:Cam (1:2, 39 °C, 32 min) yielded predicted total carotenoid contents (1.87 ± 0.06 µg/mL) with only 10.4% deviation from experimental values, hence demonstrating the robustness of this approach. Importantly, liquid chromatography - mass spectrometry (LC-MS) revealed a unique carotenoid profile of calamansi juice, which consisted of lutein, β-cryptoxanthin, and their esterified derivatives (C10:0 - C18:0). Last but not least, greenness evaluation using the AGREE metric confirmed significant improvement of the optimized 1:1 Men:Cam system over the conventional extraction. Overall, this work demonstrates that UAE combined with green solvent systems is a practical tool for sustainable carotenoid extraction from valuable agricultural products.