*4.7. Amino Acid Analysis*

The amino acid profile analysis was performed at TAMU Protein Chemistry Lab (College Station, TX, USA). Finely-ground ICC samples (30–40 mg) were used for liquid HCl (6 N) hydrolysis. Hydrolyzed proteins were derivatized pre-column with o-phthalaldehyde and 9-fluoromethyl-chloroformate prior to separation and quantitation by reverse phase HPLC. The component amino acids were then separated by HPLC (Agilent 1260), detected by UV (Agilent G1365D) or fluorometry (Agilent G1321B), and quantitated. All system control and data analysis was performed by Agilent Chemstation software. Values are the means of two independent experiments that did not differ by more than 2.9%.

### *4.8. Titration*

Samples (70 mg) of freeze-dried material (SF-InSC) were dispersed in 7 mL distilled-deionized water by ultrasonication (GeneralSonic GS3) [26]. One sample was titrated with 0.1 N NaOH and the other with 0.1 N HCl. After each titrant addition, the suspension was stirred for 10 min at room temperature and subsequently the pH was recorded (Jenway 3310 pHmeter). A blank sample without collagen was titrated under the same conditions. The resulting pHs versus the amount of NaOH and HCl were plotted within the pH range from 2 to 12.
