*4.2. Sample Preparation*

*U. lactuca* was soaked in 80% alcohol for 18 h and heated at 70 ◦C for 4 h to remove pigment, protein, and some salt. The sample was centrifuged and dried. The polysaccharide was extracted by hot water extraction. The crude extract and water were dissolved in a ratio of 1:30 and reacted at 100 ◦C for 1 h. Ulvan crude extracts were filtered and centrifuged (4000 rpm, 10 min). The supernatants were collected and concentrated at reduced pressure. They were precipitated overnight at 4 ◦C with twice the volume of 95% ethanol. The polysaccharide was obtained by freeze-drying after collecting the precipitation and named UPE.

#### *4.3. Characterization of UPE*

The content of total sugar in UPE was determined by phenol–sulfuric acid method, and glucose was taken as the standard [38]. Protein content was determined by Kjeldahl method [39]. The uronic acid content in UPE was calculated by m-hydroxybiphenyl method [40]. The sulfate radical content was evaluated by barium sulfate turbidimetry [41].

The molecular weight of UPE was analyzed by gel permeation chromatography (GPC). Agilent 1260 HPLC was used and equipped with a PL aquagel-OH 60 column (300 mm × 7.5 mm; Tosoh, Shiba, Tokyo, Japan) and refractive index detectors. The dried polysaccharide samples were ground and mixed with potassium bromide to make the tablets and scanned by Nexus 70 infrared spectrometer. The monosaccharide components of UPE were determined by reversed-phase HPLC method. After acidolysis with trifluoroacetic acid, PMP was used for derivation. Monosaccharide analysis was performed on a ZORBAX Eclipse XDB-C18 separation column (4.6 mm × 250 mm) at 245 nm.
