Unraveling the prebiotic effect of Ulva lactuca L. by in vitro colonic fermentation and metabolome investigation

Introduction Ulva lactuca L., also known as sea lettuce, is a green marine seaweed distributed worldwide. It is involved in environmental concerning processes known as “green tides”; a phenomenon exacerbated by anthropogenic activities leading to water eutrophication [1]. Ulva is approved in Italy for the production of food supplements. Ulva contains unique polysaccharides called ulvan, which consist of disaccharides repeating units of rhamnose-3-sulfate (Rha3S) linked to D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-xylose (Xyl). Ulvan has shown antihyperglycemic effect in aged diabetic mice [2] and anti-inflammatory and immunoregulatory activities in a murine model of DNBS-induced colitis [3]. However, the biological mechanisms involved in these activities and the influence on the human gut metabolome remain unexplored. Thus, the aim of this study was to evaluate the prebiotic effects of purified ulvan through in-vitro colon fermentation and analysis of changes in gut microbial metabolome. Experimental Ulva lactuca was collected from an aquaculture tank belonging to Pescatori Orbetello cooperative, in the Orbetello lagoon, Tuscany, Italy. Ulvan extraction was conducted under acidic conditions for 105 minutes, followed by EtOH precipitation and freeze-drying. The dried crude extract was resuspended in water, dialyzed and freeze-dried again to obtain purified ulvan. The polysaccharide underwent in vitro enzymatic digestion following the INFOGEST 2.0 protocol. The undigested ulvan was then subjected to colonic microbial fermentation using an in-vitro static fermentation system [4]. The soluble fibre inulin and a growth control (no polysaccharide added) were included as positive and negative controls, respectively. Samples were collected at 0, 12, 24, 48 hours of fermentation. Short chain fatty acids (SCFA) and tryptophan-related metabolites were quantified using GC-FID and UHPLC-QQQ, respectively. Untargeted metabolomics was performed on methanolic extracts of fermented samples in both positive and negative ionization modes using an UHPLCQTOF instrument. Results Overall, the production of SCFA, acetic, propionic and butyric acids, consistently increased over time (Figure 1a), with ulvan showing higher levels than the growth control and similar levels to inulin, except at the end of the fermentation (48h). The production of branched-chain fatty acids (BCFA), iso-butyric, valeric, iso-valeric acids, was comparable between ulvan and the growth control, but slightly lower for inulin (data not shown). SCFA are primary end-products of carbohydrate fermentation, suggesting that ulvan is metabolized by the colonic microbiota. Among the 12 quantified tryptophan-related metabolites, a significant higher production of kynurenine was observed for ulvan over time (Figure 1b). Kynurenine pathway dysregulation is involved in immunoregulatory disfunction [5]. Interestingly, ulvan produced higher levels of indole-3-propionic acid (IPA) at 48h (mean values growth control/inulin/ulvan 0.07/0.03/0.12 μmol/L). IPA acts as an agonist on pregnane X receptor, enhancing gut epithelial barrier functions [6]. Untargeted metabolomics analysis showed a distinct metabolite profile for ulvan samples, both in positive and negative ionization modes (Figure 2). Current on-going studies are investigating these metabolites differences and their potential roles to unravel the gut microbiota functions. Conclusions This work represents the first comprehensive investigation of ulvan’s impact on human gut metabolome. It provides insights into ulvan’s potential applicability for upcycling Ulva biomass towards the development of functional food with prebiotic effects. Primary findings demonstrate ulvan’s ability to modulate the gut microbiota, producing beneficial metabolites such as SCFAs and IPA. Further studies are necessary to explore the specific roles of these metabolites in the gut and their general implications for human health.