Structural and functional insights into ulvan from Ulva lactuca L.: multi-methodological characterization and in vitro fermentability

Ulva lactuca is a widespread macroalga that has attracted attention due to its role in green tides, phenomena linked to coastal eutrophication. Approved in the European Union as both food and food supplement, Ulva is rich in ulvan, a sulfated polysaccharide which represents a promising product for biomass valorization. This study investigates U. lactuca harvested in the Orbetello lagoon, where it grows naturally and requires costly disposal. The research aimed to provide insights for transforming this underexploited biomass into a source of bioactive compounds focusing on ulvan, by optimizing extraction, characterizing its chemical structure, and assessing its in vitro fermentability and prebiotic potential. Additionally, enzymatic hydrolysis was optimized to produce oligosaccharides suitable for biological applications. Extraction conditions were optimized using Response Surface Methodology (RSM). Oligosaccharides were produced by ulvan lyase PLSV_3875. Ulvan was characterized by 2D-NMR, HPAEC-PAD (monosaccharide composition), and GPC-RID (molecular weight). In vitro digestion (INFOGEST 2.0) and subsequent batch colonic fermentation were conducted, with samples collected over time for targeted and untargeted LC-MS/MS analyses. RSM optimal conditions (pH 2.0, 105 min, L/S ratio 60:1 mL/g) yielded 9.27% ulvan with 27.8% rhamnose, 20% sulfate, and average Mw of 88 kDa. Ulvan fermentation by gut microbiota resulted in SCFA production averaging 2.43 mg/L at 48 h) and modulation of tryptophan metabolism, generating healthpromoting catabolites such as indole 3-propionic acid and indole 3-acetic acid. RM-ASCA statistical analysis of untargeted data highlighted ulvan-specific features. Optimized enzymatic hydrolysis (25 mM KCl, 100 mM phosphate buffer, pH 7.5, 3 mg/mL ulvan) and kinetic characterization yielded oligosaccharides suitable for biological applications. The developed protocols for ulvan extraction and hydrolysis provide the basis for future research on Ulva biomass valorization. Ulvan shows promising fermentability and prebiotic potential, supporting its use as a dietary supplement or functional food ingredient.