*4.1. Material*

In this study, we used a commercial material—Aksolagen membrane—provided by the Akses Swiss company (Zug, Switzerland). Aksolagen membrane is a specially treated GSCM of *Dosidicus gigas*. The squid mantle consists of several layers (Figure 15), with the

central muscle layer surrounded by two collagenous membranes (outer and inner tunics); the GSCM represents the outer tunic of the mantle.

**Figure 15.** The *D. gigas* mantle and its inner structure.

We also studied the structure of the collagenous membrane of another squid species, a small squid *B. magister.* A frozen squid *B. magister* was purchased in a supermarket, thawed, and the collagenous membrane (outer tunic) was mechanically separated from the muscle layer of the mantle.

The thickness of the GSCM of *D. gigas* measured with a micrometer (a 5–10 N load) was 50 ± 5 μm, and the thickness of the *B. magister* membrane was 25 ± 5 μm.

### *4.2. Amino Acid Analysis*

To study the GSCM composition, we conducted the amino acid analysis. The analysis was performed by ion-exchange chromatography, with the post column derivatization, using an L-8800 amino acid analyzer (Hitachi, Ltd., Tokyo, Japan) with a steel Hitachi Ion-Exchange Column 2622SC(PH) (Hitachi, Ltd., Tokyo, Japan) 4.6 × 80 mm. The column temperature was 57 ◦C, the flow rate was 0.4 mL/min, the charge volume was 50 μL, and the elution regime involved a stepwise gradient of eluents A (AAA PH-1 Buffer— AN0-8706 Merck Hitachi, Tokyo, Japan), B (AAA PH-2 Buffer—AN0-8707 Merck Hitachi, Tokyo, Japan), C (AAA PH-3 Buffer—AN0-8708 Merck Hitachi, Tokyo, Japan), D (AAA PH-4 Buffer—AN0-8709 Merck Hitachi, Tokyo, Japan), and E (0.2 M NaOH solution). As a calibration mixture, standard concentrated amino acid mixtures in ampoules were used (Amino Acid Standard Sigma Aldrich, St. Louis, MI, USA).

To prepare the studied solution, a dry sample was placed in a molybdenum glass ampoule, and 0.3 mL of a freshly prepared hydrolyzing mixture (concentrated hydrochloric and trifluoroacetic acids in a 2:1 ratio with the addition of 0.1% β-mercaptoethanol Sigma Aldrich, St. Louis, MI, USA) was added. The sample was frozen, and the ampoule was evacuated and sealed. The hydrolysis was conducted at 155 ◦C for 1 h. After the hydrolysis, the ampoule was cooled, opened, and the content was quantitatively transferred (0.1 mL of water twice) in a plastic 1.5 mL tube, then the hydrolyzing mixture was completely removed with a CentriVap vacuum concentrator (Labconco corporation, Kansas, MO, USA) at 50 ◦C. The residual acids were removed by repeating twice the procedure of evaporation of small water portions (0.1 mL) added to the dry residue at 50 ◦C. Then, 0.1 N HCl was added to the dry residue, the mixture was centrifugated, and 0.1 N HCl was added to the supernatant in a 10:1 ratio.

### *4.3. Collagen Molecular Weight Estimation (SDS-PAGE)*

Following the collagen extraction, the protein concentration was evaluated by the gravimetric analysis. The sample was ≈100-fold concentrated by ultrafiltration on a Microcon Centrifugal filter unit with a 10 kDa molecular cut-off (MRCPRT010, Millipore, Burlington, MA, USA) to obtain the final collagen at 10 mg/mL. Collagen from GSCM

and Type I collagen from the cattle dermis were isolated using a protocol described in [82], while Type II collagen was isolated from the tracheal cartilage by a protocol described in [83] omitting the use of pepsin. An amount of 10 μg of the proteins were diluted with an SDS-loading buffer supplemented with 100 mM DTT (20710, SERVA, Heidelberg, Germany) and heated at 95 ◦C for 5 min. The samples were resolved by denaturing polyacrylamide gel electrophoresis in 5% stacking and 8% separating gel using a Mini-PROTEAN Electrophoresis System (Bio-Rad, Hercules, CA, USA). The electrophoresis running conditions were as follows: at 15 mA, until samples reached the separating gel, then at 30 mA until the front reached 0.5 cm from the gel edge. The gel was stained with Coomassie Blue R-250 (35051, SERVA, Heidelberg, Germany) and scanned with a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). The molecular weights of the visual bands were calculated in the ImageLab software against the molecular weight standards (Spectra Multicolor High Range Protein Ladder, SM1851, Fermentas, Waltham, MA, USA).
