Ulva lactuca L. of Orbetello lagoon: multivariate optimization of the extraction of ulvan polysaccharides

Ulvan is a sulfated polysaccharide extracted from green algae of genus Ulva (family Ulvaceae) that has gelling properties and has shown anticoagulant, antiviral, anti-hyperlipidemic, immunomodulatory and antioxidant activities, offering great potential for the development of functional food. Ulvan is mainly built on disaccharides repeating sequences composed of sulfated rhamnose and glucuronic acid, iduronic acid or xylose. The biological and chemo-physical properties of ulvan are directly related to its chemical structure; thus, it is crucial for its application to understand how the chemical composition of the extract is influenced by the extraction conditions. This work involved Ulva lactuca L. samples collected in August 2022 in an aquaculture tank of Orbetello lagoon (GR, Italy), where the algae grow spontaneously and can be considered a by-product of fish farming. The aim of the study was the optimization of the extraction conditions for ulvan in order to maximize yields and purity of the polysaccharides extracted. A Free Wilson model was used to determine the effects of the pH, extractant/sample ratio and time of extraction on yields, purity and sulfation degree. The coefficients of the model were estimated with a symmetrical screening matrix (pH: 2–3.5–5; extraction time: 30–105 – 180 min; extractant/sample ratio: 20–50–80 mL/g). Purity was evaluated by quantitative 1H-NMR as rhamnose percentage on dialyzed polysaccharides and turbidimetric assay was applied for the quantification of sulfate; uronic acids and neutral sugars were determined by HPAEC-PAD. Light Scattering techniques were applied to evaluate the hydrodynamic diameter, correlated to the size distribution of polysaccharides in water, and the zeta-potential, that provided an indirect measure of the surface charge. Graphic analysis of effects showed that yields pre- and post-dialysis were not significantly affected by changing the pH. They were mainly influenced by the extractant/sample ratio, with the use of higher volumes of extractant leading to higher yields (up to 14.88%). The rhamnose content was dependent on the extraction time and on the extraction pH, with a higher degree of purity obtained at pH 2, confirming the higher selectivity for ulvan extraction at low pH values. The sulfate percentage was in the range of 11-20% and was strongly influenced by the extraction time; an increase of the time corresponded to a decrease of the response. Considering the results of the screening phase, a Response Surface Methodology (RSM) study was carried out for obtaining a map of the predicted responses in the experimental domain under study. RSM has never been applied before for this species and enabled to identify the multivariate zone where high quality ulvan with high yields could be obtained. Further studies should be done to investigate how pH can modulate ulvan structure.