*3.2. Jellyfish Samples*

*Rhizostoma pulmo*, Macrì 1778 [67] specimens were collected at Marina di Ginosa (Taranto, Italy) (Supplementary Materials, Video S1), in summers 2017–2018, by means of a nylon landing net with 3.5 cm mesh size, from an open type motorboat, and stored in refrigerated seawater in 100 L barrels for a maximum of 2 h. Specimens were adult both male and female jellyfish with a diameter ranging from 17 to 25 cm. Whole jellyfish were individually frozen in liquid nitrogen and stored at −80 ◦C. Frozen samples were then lyophilized in a freeze dryer (Freezone 4.5 L Dry System, Labconco Co. Thermo Scientific, Milan, Italy), at −55 ◦C for 4 days using a chamber pressure of 0.110 mbar and then stored at −20 ◦C until use. Each lyophilized jellyfish has been made homogeneous (oral arms and umbrella) by mixing its powder, and then the lyophilized powder from 5 individuals was pooled. Six different pools were considered as representative samples and used for independent experiments.

### *3.3. Protein Extraction and Sequential Hydrolysis*

Lyophilized tissues were ground into a fine powder with liquid nitrogen and 1 g was used as described below (Figure 9). Soluble proteins (SP) were extracted by insoluble material (IP) by gentle stirring of the sample with 16 volumes ( *w*/*v*) of PBS, (phosphate buffer saline) pH 7.4, at 4 ◦C for 2 h and then centrifuged at 9000× *g* for 30 min at 4 ◦C. Supernatant was separated from the insoluble material (IP) and subjected membrane fractionation as described below. Pellet was subjected to sequential enzymatic hydrolyses by pepsin (1 mg/mL) in 0.5 M acetic acid, using an enzyme/substrate ratio of 1:50 ( *w*/*w*) and stirred for 48 h at 4 ◦C. The digested sample was centrifuged at 9000× *g* for 30 min and the pepsin-hydrolyzed peptides (PHp) were stored for further separations. The pellet was washed two times with bi-distilled water, and subjected to a second digestion with collagenase (6 mg/mL in TES buffer 50 mM, pH 7.4 and 0.36 mM of CaCl2) using an enzyme/substrate ratio of 1:50 ( *w*/*w*), by stirring 5 h at 37 ◦C. Collagenase cuts the peptide sequences as –R-Pro-X-Gly-Pro-R where X is generally a neutral amino acid. After hydrolysis, the sample was centrifuged at 9000× *g* for 30 min, and the soluble collagenase-hydrolyzed peptides (HJCp) were stored for further treatments. The pellet of collagenase digestion was considered as not-hydrolysable material. Commercial calf skin collagen (Sigma) was used as control and subjected to the same sequential hydrolysis procedure.

**Figure 9.** Flow diagram showing the various steps of extraction, hydrolysis and fractionation of the proteins from *Rhizostoma pulmo*.

Soluble proteins derived from PBS extraction (SP), pepsin hydrolyzed peptides (PHp) and hydrolyzed collagen peptides (HJCp) were subjected to fractioning by membrane filtration.

### *3.4. Proteins Separation by Membrane Filtration*

All the obtained fractions (SP, PHp and HJCp) were separated by membrane filtration in fractions containing peptides with different molecular weight ranges. All the steps were performed at 4 ◦C. Each sample was filtered using Amicon® Ultra 30K device (Merck) by centrifugation at 4000× *g* to almost total filtration, the retentate contained compounds with MW higher than 30 kDa. The filtrates (containing compounds less than 30 kDa) were further fractionated using Amicon® Ultra 10K device (Merck) by centrifugation at 4000× *g* to obtain the 10–30 kDa fraction in the retentate. Finally, the filtrates containing compounds lower than 10 kDa were centrifuged using Amicon® Ultra 3K device (Merck) at 4000× *g* to obtain in the retentate fractions with 10 < MW < 3 kDa and MW < 3 kDa. Each sample was analyzed for protein content, antioxidant activity and cell culture test.
