Composti bioattivi e biodiversità dei cianobatteri dei fanghi termali euganei

Il Distretto Termale Euganeo, situato nel nord-est dell’Italia, rappresenta un importante centro per i trattamenti termali. La peloterapia Euganea è basata sull’uso del fango terapeutico ottenuto grazie al processo di maturazione. Il fango è estratto da laghi presenti nel territorio e viene di seguito distribuito alle spa. Qui viene posto all’interno di vasche e coperto da uno strato di acqua termale. Perché la maturazione avvenga con successo, la temperatura dell’acqua dovrebbe essere mantenuta attorno ai 40 °C. Nel corso di alcuni mesi un microbiota ricopre la superficie del fango, dandogli la colorazione verde dovuta alla presenza di cianobatteri. L’efficacia dei trattamenti è dovuta al calore del fango e dell’acqua, alla presenza di composti inorganici e organici, quest’ultimi prodotti dal microbiota durante la maturazione. Diversi studi hanno riguardato i microorganismi che crescono sui fanghi; ma mancano informazioni sulla biodiversità complessiva e solo alcune specie sono state isolate e caratterizzate. Per caratterizzare il Distretto Termale Euganeo sono stati considerati diversi aspetti: il microbiota e gli organismi che lo compongono, l’attività di composti estratti dalle singole specie isolate o prodotti dall’intero microbiota e la valutazione delle condizioni ottimali di crescita degli organismi per la produzione di tali molecole. Il microbiota del Distretto è stato analizzato: la biodiversità trovata è risultata alta, con la presenza di diverse specie da isolare e caratterizzare. Il risultato dello studio ha inoltre dimostrato che il microbiota che colonizza i fanghi è simile dai 37 ai 47 °C, con la maggior presenza della specie target Phormidium sp. ETS-05. Quando la maturazione è attuata a temperature più basse o più alte, si osserva un cambio nella composizione della popolazione, determinando la riduzione della biodiversità e dell’abbondanza di cianobatteri. È stato condotto l’isolamento di nuovi ceppi: sono stati ottenuti due cianobatteri filamentosi da diverse spa dell’area euganea, caratterizzati poi secondi i principi dell’approccio polifasico che si basa su informazioni morfologiche, biochimiche e molecolari per ottenere un corretto riconoscimento tassonomico. Inoltre è studiata una nuova specie del Distretto caratterizzata recentemente, Thermopirulina andreolii ETS-09, specie tipo del genere. Mancando test fisiologici, sono state valutate sia temperature che intensità luminose per stabilire quelle ottimali o limitanti. Le condizioni sono state correlate con la produzione di composti a valore aggiunto. Per l’identificazione di nuovi composti bioattivi sono state analizzate molecole di natura polisaccaridica. Phormidium sp. ETS-05, è stato studiato sia per la produzione di esopolisaccaridi (EPS) che per le loro proprietà. Gli EPS sono stati testati in vitro e in vivo per verificare l’assenza di tossicità, e in vivo per verificare l'azione antinfiammatoria usando l’organismo modello zebrafish (Danio rerio). Considerato l’interesse per questi polimeri, Phormidium sp. ETS-05 è stato coltivato variando le condizioni di crescita per implementare la produzione di EPS. Il ceppo è stato cresciuto utilizzando diversi spettri di luce, temperature e intensità luminose. La produzione di EPS è stata correlata a quella della biomassa. I polisaccaridi sono stati estratti anche dal fango terapeutico prodotto da una spa al termine del processo di maturazione. Per verificare la loro efficacia antiinfiammatoria e antiossidante, sono stati condotti diversi esperimenti in vivo usando larve di zebrafish. La riduzione dello stress infiammatorio è stata valutata grazie al recupero del corretto sviluppo larvale e misurando la diminuzione dell’attività di specifici marcatori dell’infiammazione in linee transgeniche.The Euganean Thermal District, located in North-East Italy, is an important center for thermal treatments. The Euganean pelotherapy relies on the use of a therapeutic mud or peloid obtained through a maturation process. Mud collected from local lakes is supplied to spas where is placed in tanks or ponds and covered by flowing thermal water that naturally gushes in the area. For the maturation to succeed, thermal water should be set at about 40 °C. During few months, a microbiota covers the surface of the mud giving it a peculiar green coloration due to the presence of cyanobacteria. The efficacy of the treatment is due to the heat of mud and water, the presence of inorganic components and organic compounds produced by the microbiota during the maturation process. Microorganisms growing on muds have been subject to a number of studies, however information on the total biodiversity is lacking and only few species have been isolated and characterized so far. Several aspects were considered to characterize the Euganean Thermal District: its microbiota and the organisms of which it is composed; bioactivity of compounds extracted either from single isolated species or produced by the whole microbiota; assessment of the optimal growth conditions to produce those molecules. The microbiota of the District have been surveyed: the biodiversity found was high with many species yet to be isolated and characterized. The result of the study demonstrated in addition that between 37 and 47 °C a similar microbiota colonizes the muds, with the dominant presence of target species Phormidium sp. ETS-05. Change in the population, with a reduction in cyanobacterial biodiversity and abundance, is instead observed when maturation in conducted at lower or higher temperatures. Considering the relevance of cyanobacteria in the maturation process and the importance of studying thermophiles from a biotechnological perspective, the isolation of new strains was pursued. Two filamentous cyanobacteria were obtained and resulted to be new species, named ETS-13 and ETS-31. Both were isolated from mature muds collected in different spas of the Euganean area and were characterized following the principles of the polyphasic approach including morphological, biochemical and molecular surveys, for a correct taxonomic recognition. In addition, a new species recently identified and characterized from the District, Thermopirulina andreolii ETS-09, was considered. Physiological studies were lacking, considering both temperatures and light intensities optimal or limiting for the species growth. All conditions were correlated with the production of value-added compounds. As regards the identification and characterization of new bioactive compounds extracted either from single isolated species or produced by the whole microbiota, molecules of polysaccharidic nature have been considered. Phormidium sp. ETS-05, the target species of mud maturation was studied for its exopolysaccharides (EPS) production and properties. EPS were tested in vitro and in vivo to verify the lack of toxicity and afterwards studied for the anti-inflammatory activity in vivo using zebrafish. Considering the interest in these polymers, Phormidium sp. ETS-05 was cultivated under different laboratory conditions with the purpose to boost EPS yield. The strain was grown using diverse light spectra, temperatures and light intensities, and EPS production was correlated with biomass production. Polysaccharides were extracted also from the therapeutic mud produced by a spa at the end of the maturation process. Efficacy tests were performed in vivo using zebrafish larvae to verify their anti-inflammatory and antioxidant activities. Reduction of the inflammatory stress was assessed both from the recovery of the correct larvae development and from the decrease of the activity of specific markers of inflammation using transgenic lines

Oral Palatability and Owners’ Perception of the Effect of Increasing Amounts of Spirulina (<i>Arthrospira platensis</i>) in the Diet of a Cohort of Healthy Dogs and Cats

The nutraceutical supplementation of Spirulina (Arthrospira platensis) in dogs and cats has not yet been investigated. The aim of this study was to evaluate if the dietary supplementation of increasing amounts of Spirulina for 6 weeks is palatable to pets and to assess the owner’s perception of such supplementation. The owners of the 60 dogs and 30 cats that participated in this study were instructed to daily provide Spirulina tablets starting with a daily amount of 0.4 g, 0.8 g, and 1.2 g for cats as well as small dogs, medium dogs, and large dogs, respectively, and allowing a dose escalation of 2× and 3× every 2 weeks. The daily amount (g/kg BW) of Spirulina ranged from 0.08 to 0.25 for cats, from 0.06 to 0.19 for small-sized dogs, from 0.05 to 0.15 for medium-sized dogs, and from 0.04 to 0.12 for large-sized dogs. Each owner completed a questionnaire at the time of recruitment and the end of each 2-week period. No significant effect on the fecal score, defecation frequency, vomiting, scratching, lacrimation, general health status, and behavioral attitudes was detected by the owners’ reported evaluations. Most animals accepted Spirulina tablets either administrated alone or mixed with food in the bowl. Daily supplementation of Spirulina for 6 weeks in the amounts provided in this study is therefore palatable and well tolerated by dogs and cats

In vivo anti-inflammatory and antioxidant effects of microbial polysaccharides extracted from Euganean therapeutic muds.

Therapeutic thermal mud produced by spas of the Euganean Thermal District (Italy) is used as a treatment for arthro-rheumatic diseases. Its production involves the growth of a specific microbiota embedded in a polysaccharidic matrix. Polysaccharides (Microbial-PolySaccharides, M-PS) released in the mud by the resident microorganisms were extracted and analyzed. The monosaccharidic composition analysis showed the presence of galacturonic acid, mannose, xylose, ribose and glucose and a high percentage of sulfated groups in the polymers. To assess their involvement in the therapeutic efficacy of the mud, the M-PS were tested using the model organism zebrafish (Danio rerio). The anti-inflammatory and antioxidant activities were evaluated after confirming the lack of toxic effects during development. Inflammatory state was induced chemically with copper sulfate, or through tail fin amputation procedure and UVB exposure. Recovery from inflammatory condition after exposure to M-PS was always observed with specific morphometric analyses, and further supported by qPCR. Genes linked with the inflammatory and oxidative stress response were investigated confirming the M-PS treatment's efficacy

Anti-Inflammatory Activity of Exopolysaccharides from Phormidium sp. ETS05, the Most Abundant Cyanobacterium of the Therapeutic Euganean Thermal Muds, Using the Zebrafish Model

The Euganean Thermal District (Italy) represents the oldest and largest thermal center in Europe, and its therapeutic mud is considered a unique product whose beneficial effects have been documented since Ancient Roman times. Mud properties depend on the heat and electrolytes of the thermal water, as well as on the bioactive molecules produced by its biotic component, mainly represented by cyanobacteria. The investigation of the healing effects of compounds produced by the Euganean cyanobacteria represents an important goal for scientific validation of Euganean mud therapies and for the discovering of new health beneficial biomolecules. In this work, we evaluated the therapeutic potential of exopolysaccharides (EPS) produced by Phormidium sp. ETS05, the most abundant cyanobacterium of the Euganean mud. Specifically, Phormidium EPS resulted in exerting anti-inflammatory and pro-resolution activities in chemical and injury-induced zebrafish inflammation models as demonstrated using specific transgenic zebrafish lines and morphometric and expression analyses. Moreover, in vivo and in vitro tests showed no toxicity at all for the EPS concentrations tested. The results suggest that these EPS, with their combined anti-inflammatory and pro-resolution activities, could be one of the most important therapeutic molecules present in the Euganean mud and confirm the potential of these treatments for chronic inflammatory disease recovery