**5. Conclusions**

In the present study, the collagenous content of the demosponges *A. cannabina* and *S. carnosus* was exhaustively examined. The insoluble, intercellular, and spongin-like collagens were isolated from *A. cannabina* and *S. carnosus*, representing 12.6%, 3.0%, and 42.8% dry weight for the former and 5.0%, 1.9%, and 21.8% dry weight for the latter sponge. SEM and TEM observations confirmed the characteristic fibrous structures, while IR spectroscopic analysis verified the characteristic absorption bands for proteins of the collagen class. Moreover, the acid–base properties of the insoluble collagen were investigated by titration, placing the isoelectric point approximately at pH 7. Marine sponge collagen, as compared to that derived from terrestrial animals and other marine collagen sources, has been reported to differentiate in its characteristics, such as amino acid composition, which consecutively affects collagen's thermal behavior, isoelectric pH, solubility, and many other properties. In our case, the measured low imino acid content for the intercellular collagen, already reported being low in marine sources and even lower, specifically, in sponges, results in thermal stability comparable to that determined for collagen isolated from edible jellyfish and tropical fish. Indeed, the denaturation temperatures of the intercellular collagen isolated from *A. cannabina* and *S. carnosus* were determined by DSC studies at 25.4 ◦C and 32.9 ◦C, respectively, the first one being relatively lower than that reported for other marine organisms, while the second one being comparable to values observed for an array of collagens isolated from tropical fish. CD spectra indicated the existence of helical structures and the fact that the denaturation temperatures were dependent on the amount of imino acids. Marine collagen is considered as an equivalent, although safer, biomaterial than the one from terrestrial sources dominating the market nowadays. Our results suggest that the sponges *A. cannabina* and *S. carnosus* can be considered as an alternative source of collagen.

**Acknowledgments:** The authors wish to thank Aggeliki Panagiotopoulou for recording the CD spectra at the Circular Dichroism Laboratory at the Institute of Biosciences and Applications of NCSR "Demokritos". This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Author Contributions:** Vassilios Roussis, Efstathia Ioannou, Leto-Aikaterini Tziveleka, and Dimitris Tsiourvas conceived and designed the experiments; Leto-Aikaterini Tziveleka, Dimitris Tsiourvas, Panagiotis Berillis, and Evangelia Foufa performed the experiments; Leto-Aikaterini Tziveleka, Vassilios Roussis, Efstathia Ioannou, and Dimitris Tsiourvas analyzed the data; Vassilios Roussis and Efstathia Ioannou contributed reagents/materials/analysis tools; and Leto-Aikaterini Tziveleka, Efstathia Ioannou, and Vassilios Roussis wrote the paper.

**Conflicts of Interest:** The authors declare no conflict of interest.
