**2. Results**

## *2.1. Chemical Characterization of the Fucoidans*

All tested fucoidans and the used extraction and purification methods were designated according to a code as seen in Table 1 In brief, the dried seaweed material was enzymatically extracted with commercial cellulase preparations Cellic®CTec2 or Cellic®CTec3 mixes ("2" or "3" in extraction code) and additional alginate lyase from Sigma-Aldrich SigmALy (SiAT) or alginate lyase SALy expressed from *Sphingomonas* sp. (SAT) were added (making the extracts SiAT2, SiAT3 as well as SAT2 and SAT3). The extracts with an additional "ad" in the code were precipitated with acid (HCl). Other extracts were precipitated with CaCl2. F1–F3 stands for an additional three-step fractionation process with chromatographic isolation (IEX).


**Table 1.** Overview of the used fucoidans, including algae species, extraction method and code used in this manuscript. For the explanation of the extraction methods refer to Section 2.1.

> \* Nguyen et al., 2020 [15].

Three crude extracts were made with Cellic®CTec2 and SigmALy from the three different algae (LD\_SiAT2, SL\_SiAT2 and FE\_SiAT2). The residual alginate was not precipitated in these extracts and the alginate content was high (mannuronic acid plus guluronic acid) 87.2%, 80.3% and 67.5% and the fucose content was low 3.9, 12.3 and 15.5% respectively, as determined by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) (Table 2; [22]) (chemical composition with standard deviations in Table A1).


**Table 2.** Overview of monosaccharide and uronic acid composition in mol%. (Fuc-Fucose, Ara/Rham-Arabinose/Rhamnose, Gal-Galactose, Glc-Glucose, Xyl-Xylose, Man-Mannose, GuluA-guluronic acid, GluA-glucoronic acid, ManA-mannonic acid); GuluA + ManA was calculated together and equals mol% alginates; the highest values are marked in bold.

\* Nguyen et al., [15]. To extract fucoidans in a more gentle way in order to retain the molecule as intact as possible, enzymes were employed in the extraction procedure. Crude fucoidan extracts were prepared using different enzyme cocktails, with either the cell wall degrading enzyme mix Cellic®CTec2 or 3 from Novozymes A/S, enzyme cocktails developed to degrade polysaccharides from terrestrial plant cell walls. In addition, alginate lyases were also added, since alginate is a brown seaweed specific polysaccharide that is not degraded by the Cellic®CTec2 or 3 [23]. Two different alginate lyases were used (refer Section 4.2.2), which have different specificities [24]. Fucoidans have previously been purified using different enzymes, including the cellulase enzyme Celluclast [25] The Cellic®CTec2 used here contains extra β-glucosidases and lytic cellulose monooxygenases (1.14.99.54, 1.14.99.56, AA9) as well as other proprietary proteins compared to Celluclast. In addition, it has specifically been shown that Cellic®CTec2 can degrade laminarin [23]. Furthermore, this new method includes the novel use of two different alginate lyases for purification of fucoidans.

In order to see the highest values of each substance instantly, we marked these values in bold. Not all alginate was degraded by the alginate lyase SigmALy, therefore the following extracts were prepared with different enzyme mixes and with further alginate precipitation with acid followed by neutralization of pH and dialysis (FE\_SiAT2ad, FE\_SiAT3ad, FE\_SAT2ad, FE\_SAT3ad). Since it contains the highest amount of fucoidan FE was used for these extractions. The resulting extracts had a comparable lower content of alginates 37.1, 50.4, 21.3 and 28.0% and higher content of fucose 36.1, 35.9, 52.2, 48.3%, respectively, compared to the previous extract FE\_SiAT2. Furthermore, it was evident that the use of the alginate lyase SALy was more efficient in degrading the alginate than SigmALy, in particular the mannuronic acids (Table 2). These optimizations of using SALy and alginate precipitation was used to prepare highly pure and fractionated fucoidans from *S. latissima* by ion-exchange chromatography (IEX), the purification method was described previously [15], while the bioactivity is tested here. SL was chosen, since previous work has shown that SL fucoidan has biological effects [19]. A further optimization was used, were the alginate was precipitated by CaCl2 instead of acid, this method is believed to be gentler and is therefore likely to preserve the fucoidan structure better than the use of acid. Crude extracts from SL and FE using this method contained comparable total fucose yields and sulfate content, compared to a mild chemical extraction using acid [15]. In comparison to the LD extract three fractions of fucoidans were obtained by IEX, SL\_F1, SL\_F2 and SL\_F3. The first elution SL\_F1 contained almost exclusively alginates (90.9%) of lower molecular weight (4 kDa; [15]) while the fucose content was 5%. The SL\_F2 and SL\_F3 extracts contained very low amounts of alginates (6.9% and 0.8%) and high fucose content (64.7 and 63.3%), respectively. Furthermore, the SL\_F2 and SL\_F3 extracts contained a considerable amount of galactose, which is a likely constituent of SL fucoidan (12.2 and 26.9%) [15].

The sulfate content was previously determined for the SL fractions and corresponds well to the high amount of fucoidan present in the F2 and F3 fractions. The sulfate content for SL\_F1, F2 and F3 was 6.6 ± 3.6, 35.6 ± 2.5 and 46.4 ± 3.5% respectively [15].

The size of the fucoidans was determined using high performance size-exclusion chromatography (HP-SEC). The calculated size of the fucoidans was between ~250 to over 800 kDa, with a general broad estimated size distribution (Table 3). Fucoidans from FE were generally around 350 kDa with an estimated distribution from 200–500 kDa, while the crude extracts of SL and LD were smaller, with a size around 250 and 320 kDa, respectively. The SL\_F1 fraction contained mostly low molecular weight alginates with a size around 10 kDa, a peak which was also present in all crude extracts. The comparably low amount of fucoidans made it hard to determine the size in the SL\_F1 extract. The size distribution was comparable and very large for SL\_F2 and F3, and ranged from 100–1000 kDa, with a calculated size of over 800 kDa.


**Table 3.** Size and size-distribution of fucoidans determined by HP-SEC.

\* Nguyen et al., 2020 [15].
