Exploring the potential of ulvan from Ulva lactuca L.: extraction optimization by design of experiment, chemical characterization, and evaluation of in vitro prebiotic activity

Ulva sp. is a green algae associated with the so-called “green tides”, caused by water eutrophication. The main biomolecule of interest this green algae is the sulfated polysaccharide ulvan. It has gelling properties and has shown anticoagulant, anti-hyperlipidemic, and immunomodulatory activities in in vitro and in vivo models .Its biological and chemo-physical properties are directly related to its chemical structure, which strongly depends on the extraction conditions . Particularly, Ulva lactuca L. has been found to grow spontaneously in aquaculture tanks in Orbetello lagoon (OL), Tuscany, Italy, and can be considered a by-product of fish farming. Thus, this study firstly focused on the optimization of ulvan extraction from U. lactuca L. collected in OL and its chemical characterization, which is crucial for understanding the biological activities of the ulvans and to define their future possible uses. To do so, a Response Surface Methodology was applied to investigate the effect of pH, extractant/sample ratio and time of extraction on yield, purity and sulfation degree of ulvan. Purity was evaluated by quantitative 1H-NMR as rhamnose percentage on dialyzed polysaccharides. Sulfate and neutral/acidic sugars were quantified by turbidimetric assay and HPAEC-PAD respectively. Then, the optimized extraction conditions based on the maximization of yield and purity were chosen and applied to produce ulvan for in in vitro test of prebiotic activity. To evaluate it, the isolated polysaccharide was subjected to an in vitro enzymatic digestion following the INFOGEST 2.0 protocol. The undigested ulvan underwent colonic microbial fermentation using an in vitro static fermentation system . Slurries were collected at different time points (0, 12, 24, 48h). pH, short chain fatty acids, and tryptophan-related metabolites were quantified and untargeted metabolomics was performed. Therefore, this comprehensive approach, integrating extraction optimization, chemical characterization and colonic microbial fermentation will provide insights on ulvan’s impact on the function of microbial communities and its potential future applicability for upcycling Ulva biomass towards the development of functional food with prebiotic effects.