*4.4. Enzymatic Digestions of Collagen and GAGs Extraction*

In total 200 μL of extracts (in dH2O) obtained either from Ch or Ec were mixed with 200 μL of pepsin (porcine pepsin, Sigma) solution (2.1 mg/mL in 6 mM HCl). The digestion was performed for 72 h at 37 ◦C. After the digestion, the samples were observed and photographed capsize-down to check the effect of the digestion. Samples were then centrifuged for 15 min at 17,000 *g* to better separate the collagenous part from the above supernatant. Both supernatants and the pellets were freeze dried and stored at −80 ◦C for further experiments. Control samples were obtained in parallel in the absence of pepsin in order to exclude possible changes induced by the temperature, acidity and dilution.

*GAG extraction*: 3 mL of the extracted collagen was mixed with 5 mL of digestion solution (5 mg/mL of papain in 1 M NaBH4, 0.05 M NaOH) and placed at 45 ◦C overnight. Materials were completely dissolved after 2 h and GAG precipitation was performed slowly adding trichloroacetic acid (strong reaction) to reach a 50% vol/vol concentration according to the procedure reported by Nandini et al. [63]. The powders obtained were stored at RT for further characterization steps.

*GAGs digestion*: 10 mg of precipitated GAG were resuspended in 300 μL of PBS; 30 μL of 5 U/mL chondroitinase ABC (Sigma) were then added and enzymatic digestion was performed at 37 ◦C during 24 or 72 h. At each endpoint GAG were separated from the enzyme as described by Silva et al. [64]; briefly: 150 μL of the GAGs/enzyme mix were heated for 25 min at 70 ◦C and then centrifuged at 12,000 *g* for 25 min. The pellet was discarded while supernatant containing digested GAGs was recovered, freeze-dried and kept for further analysis.

#### *4.5. Electrophoresis*

#### 4.5.1. SDS-PAGE

All used reagents were purchased from Fluka (SDS-PAGE Kit, Thermo fisher Scientific, Waltam, MA, USA). Collagen batches were analysed after one month in DS through a fast dialysis (2 step at 1:10,000) and at the end of the second month in DS after the extensive dialysis above described. Samples were mixed 1:1 with loading buffer, boiled for 5 min at 100 ◦C and run in 1.5 mm 7.5% or 15% gels in a MiniProtean system (Biorad, Hercules, CA, USA).

Coomassie R-250 staining: gel was stained according to Laemmli [65]. Alcian blue staining: gels previously stained with Coomassie were treated with 12.5% thrichloroacetic acid for 30 min.; after 4 washes in dH2O gels were treated with 1% sodium metaperiodate in 3% acetic acid for 1 h. After numerous washes (until the discarded water was not reacting with 1% silver nitrate) gels were immersed in 0.5% alcian blue in 3% acetic acid for 4 h; excess of alcian blue was removed with extensive washes in 7% acetic acid.
