Extraction of fucoidan from F. vesiculosus

F. vesiculosus was chosen for extraction as this species was the source of the commercial fucoidan (Merck, Denmark). Fresh F. vesiculosus was collected in September 2021 at the Aarhus Bay coast in the proximity of Aarhus (Denmark). The seaweed was washed with distilled water to remove sand and impurities and lyophilized (Christ, Gamma, 1–16, LSC). The dried seaweed was ground into powder (0.5 mm mesh, Retsch, ZM200) and was vacuum packed and stored at −20 °C until further processing. Seaweed polysaccharides were extracted as specified by Ptak et al. (2019) with modifications (Suppl. Fig. S1). Briefly, seaweed powder (60 g) was resuspended in 600 ml EtOH (95% v/v) and was stirred for 4 h at room temperature. The suspension was centrifuged, 4600 r/m, 30 min, 20°C (Heraeus, MULTIFUGE 3 S-R). The pellet was washed with 100 ml acetone 99.5 (v/v%), centrifuged and dried in an oven (Memmert, UF55) at 35°C overnight. The dried material was vacuum packed and stored at −20°C before extraction. Before extraction, dried pellets (1.5 g) were mixed with 45 ml 0.1 M HCl, and microwave extracted (Anton Paar Multiwave 3000) with a power-gradient: ramp from 0 to 300 W for 5 min, hold at 300 W for 15 min (fan 1), followed by 0 W for 5 min (fan 3). The maximum temperature was set to 90°C, if 90°C was reached, power turned off automatically. After microwave assisted extraction, all suspensions were mixed and centrifuged at 4600 r/m and 20°C for 30 min. The pellet was discarded and the supernatant was kept at 4°C until purification. To precipitate alginate, CaCl₂ was added to a final concentration of 2 (% w/v) and the mixture was stored at 4 °C overnight. The mixture was centrifuged at 4600 r/m 20°C for 30 min and the pellet was discarded. The suspension was dialyzed in dialysis bags (Spectra/Por 3, MWCO: 3500 Da) for 48 h in Mili Q water with water changes every 12 h. To precipitate polymers, EtOH was added to a final concentration of (72% v/v) and precipitation occurred overnight at 4°C. Fucoidan extract was harvested by centrifugation at 4600 r/m and 20°C for 30 min. The pellet was freeze-dried (Christ, Gamma, 1–16, LSC) and the Fucus extract (EXT) was stored at −20°C.