*2.7. Enzymatic Degradation Procedure*

CH (solution A): 75 mg of sample were dissolved in 10 mL of PBS and stirred to obtain a completely solubilized solution. HA (solution B): 62 mg of HA sample were dissolved in 5 mL of PBS and stirred for 16 h. HA/Ch complex (solution C): 62 mg HA were dissolved in 5 mL of solution A. The final concentration of the polysaccharide (HA/Ch) was about 20 mg/mL (7.5 mg/mL CH + 12.5 mg/mL HA). For enzyme solution preparations, about 15 mg of hyaluronidase were dissolved in about 1.5 mL of deionized water and stirred for 1 h (10 mg/mL). Lysozyme: 75 mg of lysozyme was dissolved in about 1 mL of deionized H2O and stirred for 1 h (75 mg/mL). The solutions were stirred at 38 ◦C in an oil bath for about 2 h, before adding the enzyme solution. Different aliquots of solutions were collected at different times (15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 24 h) and heated at 100 ◦C for 5 min, using a thermo-shaker, (BioSan, Riga, Latvia) to denature the enzyme. The solutions were diluted to different concentrations for HP-SEC-TDA analysis. The Ch solutions were diluted to 2 mg/mL with the mobile phase, whereas the solutions containing HA or HA/Ch were diluted to 0.5 mg/mL (calculated on the total amount of polysaccharide in solution) with the mobile phase. Before the HP-SEC-TDA analysis, all solutions were filtrated to remove the precipitated enzyme (LLG-Syringe filter, CA pore size 0.20 µm, Ø 13 mm).
