4.2.1. Fucoidan Origin

Dried flakes of the brown algae SL were obtained from Icelandic Blue Mussel & Seaweed (Bláskel, Iceland) in June 2017. LD (June 2017) and FE (March 2016) were collected by Coastal Research & Management (Kiel, Germany) in the Kiel Canal. LD and FE seaweeds were washed in demineralized water, lyophilized and grround to a size of approximately 0.25–3 mm.

## 4.2.2. Alginate Lyase Expression and Purification

The alginate lyase SigmALy was purchased from Sigma-Aldrich (Steinheim, Germany). The alginate lyase SALy A1-II' from *Sphingomonas* sp. was expressed and purified in *Escherichia coli*, as previously described in Manns et al. [24] with modifications described in Nguyen et al. [15].

#### 4.2.3. Enzyme Assisted Extraction of Brown Seaweed Polysaccharides

The enzymatic extraction of fucoidan was performed in 55 mM phosphate, 15 mM citrate bu ffer pH 6 with 5% substrate concentration. The enzymes were added with ratio 5% (v/w) for Cellic ®CTec2/3 (Novozymes A/S, Bagsværd, Denmark), 0.35% (w/w) for alginate lyases. The treatment was performed at 40 ◦C at 100 rpm. The reaction was stopped by boiling at 90 ◦C for 10 min and cooling on ice. The supernatant was collected by centrifugation 10 min at 19000 rpm. The fucoidans were precipitated and isolated by addition of 96% EtOH to a final concentration of 72% (except for the SL\_F1-F3, where this step was performed after the alginate precipitation). Residual high molecular weight alginates were precipitated either by a two-step acid precipitation (pH 4 followed by pH 2 adjustment with HCl and neutralization with NaOH to pH 7 followed by dialysis in demineralized water using a 3 kDa membrane) or by precipitation with 2% CaCl2 (except the LD\_SiAT2, FE\_SiAT2 and SL\_SiAT2 crude extract alginate was not further precipitated). The fucoidans were isolated by centrifugation and lyophilized.

4.2.4. SL Fucoidan Fractionation by Anion-Exchange Chromatography

The SL fucoidans were fractionated as previously described in Nguyen et al. [15]. The fucoidan solution (5 g in 100 mL) was applied to a DEAE-Macroprep column (2.6 cm × 40 cm) in Cl- form. The unbound materials were washed from the column with NaCl 0.1 M and the fucoidans were eluted in concentration gradient of NaCl from 0.1–2 M. The eluates were combined into fractions based on the results of total carbohydrate analysis by phenol-sulfuric acid method [38]. The fractions were passed through a 10 kDa membrane to up-concentrate and remove salt, followed by lyophilization.

Fucoidans were solved in Ampuwa bidest (Fresenius, Schweinfurt, Germany) at concentrations of 1 mg/mL with exception of the three SL fraction SL\_F1, SL\_F2 and SL\_F3 which were soluble at 5 mg/mL. Right before stimulation, used extracts were filtered with 0.2 μm Sarstedt filter (Nümbrecht, Germany) and a dilution series with appropriate medium was performed to ge<sup>t</sup> the final concentrations of 1, 10, 50 and 100 μg/mL.

#### *4.3. Chemical Composition and Size Distribution Analysis*

The monosaccharide composition of the fucoidans was determined as previously described [22]. The size distribution of the fucoidans was determined by HP-SEC as previously described in Nguyen et al. [15] and the pullulan standard was fitted with a Weibull decay model by the following equation: y = a \* (1 − EXP(−(x/b) ˆ (c))). The sulfate content of the crude extracts were determined as previously described [15] and sulfate content of the SL\_F1, F2 and F3 extracts was performed as previously described [15].
