Shedding light on anti-inflammatory and antioxidant origin and function of biomolecules from therapeutic thermal muds of the Euganean district

The Euganean hot springs are home to one of Europe's most prestigious thermal centres. The Euganean Thermal District's spas offer a wide range of therapeutic treatments, including pelotherapy. The topical application of therapeutic muds, or peloids, is recognized by the Italian Health Care System as a valid therapy for various conditions, including arthroreumatic diseases by reducing perceived pain and inflammation. All these treatments are intrinsically linked to the region, as both the thermal water and the mud used for peloid preparation are sourced exclusively from the Euganean hydrogeological circuit and local lakes, respectively. To obtain therapeutic mud, a maturation process is required, and the modern procedure, codified by a protocol and followed by each spa in the District. Specifically, mud is collected from lakes in the Euganean Hills, partitioned among spas, and placed in ponds or tanks where it is covered by a layer of flowing thermal water at approximately 40 °C. Over a period of two months, a thick mat of microorganisms, embedded in their polysaccharide matrix, forms. This green microbial biofilm, with its coloration due to the presence of photosynthetic microorganisms like cyanobacteria, indicates proper mud maturation. The mud's effectiveness is primarily attributed to mechanical, thermal and chemical factors. The chemical effects involve not only inorganic soluble complexes and ions but also organic compounds that are either present in the raw mud or accumulated during maturation as a result of microorganism growth. Indeed, some studies, conducted by the Department of Biology in collaboration with the Pietro d’Abano Thermal Studies Center, investigated the biodiversity of the microbiota colonizing the mud surface during the maturation process, with a focus on the bioactive properties of various macromolecules. For example, the most abundant cyanobacterium growing at the codified maturation temperature of 40 °C, Phormidium sp. ETS-05 has been verified to play a role in therapeutic activity through the production of anti-inflammatory glycoglycerolipids, tested both in vivo and in vitro. This discovery led to a European patent in 2013 and established this species as the target organism in the maturation process, which should therefore be present to maximize the beneficial effects of pelotherapy. Nonetheless, subsequent studies highlighted variability in the maturation temperature, leading to different microbial communities where Phormidium ETS-05 is abundant in the range 37-47 °C and other species like Leptolyngbyaceae sp. ETS-13 and Thermospirulina andreolii ETS-09 become dominant at higher temperature. No studies have yet investigated the bioactive compounds produced by these cyanobacterial species. Additionally, recent studies have begun to consider the anti-inflammatory effects of other bioactive molecules, such as exopolysaccharides (EPSs). The polysaccharides of Phormidium sp. ETS-05 were the first to be evaluated for their chemical composition and bioactivity. The assessment of EPSs anti-inflammatory activity in the model organism zebrafish suggested that these compounds contribute to the beneficial properties of Euganean peloids. Subsequently, also the polysaccharides extracted from therapeutic mud matured at 40 °C, and colonized by the target species, were characterized and tested for their anti-inflammatory and antioxidant capacities. It is important to note that no studies have been conducted on the effects of temperature modification on the composition and properties of bioactive molecules.Based on this background, this thesis investigates the role of microbiota and the contribution of individual species to the properties of the final product, with a particular focus on the impact of temperature and the mechanisms of action of bioactive compounds. The goal is to better characterize this unique environment and unravel the origins of the therapeutic value of Euganean muds.