Ultrasound-Assisted Natural Deep Eutectic Solvent Extraction of Polysaccharides from the Filamentous Microalga Tribonema minus: Process Optimization, Mechanistic Insights, and Immunomodulatory Activity

Tribonema species are filamentous microalgae rich in bioactive polysaccharides; however, their dense “H”-shaped cell wall limits extraction efficiency. This study established an ultrasound-assisted natural deep eutectic solvent (UA-NADES) strategy for polysaccharide extraction from Tribonema minus, focusing on process optimization and mechanistic understanding. Among ten NADES formulations, the choline chloride–malonic acid system was selected as the optimal NADES. Under optimized ultrasonic conditions (450 W, 80 °C, 60 min), the polysaccharide yield was 2.98-fold that of hot-water (HW) extraction. This enhancement arose from the synergistic effects of ultrasound-induced cavitation and NADES-mediated hydrogen-bond interactions, which effectively promoted cell-wall disruption. Density functional theory (DFT) calculations suggested multiple hydrogen-bonding sites and strong electrostatic interactions between NADES components and glucose units, facilitating polysaccharide solubilization. Although UA-NADES-extracted polysaccharides (UA-NADES-TPs) and HW-extracted polysaccharides (HW-TPs) were both glucose-dominant glucans with β-glycosidic linkages, UA-NADES-TPs exhibited a higher total sugar content (65.98% vs. 53.12%) and a higher predominant molecular weight (5.54 vs. 3.95 kDa), indicating better preservation of the major fraction. UA-NADES-TPs also exhibited a more balanced immunomodulatory response in RAW264.7 macrophages, enhancing phagocytosis and TNF-α mRNA expression while markedly upregulating IL-10 transcription. Overall, UA-NADES extraction offers a viable route for obtaining structurally preserved bioactive polysaccharides from T. minus.