*4.9. Differential Scanning Calorimetry*

ICCs were initially dispersed in 0.01 M EDTA, pH 8.0 and the resulting suspension remained under stirring overnight at 8 ◦C. The collagens were collected after centrifugation at 13,000 rpm for 15 min. Subsequently, a 1% SDS (*w*/*v*) solution was used for the removal of entangled glycoconjugates [46]. Finally, the collagens were collected after centrifugation, washed exhaustively with distilled-deionized water, and lyophilized.

TMDSC measurements were performed by employing a MDSC 2920 calorimeter (TA Instruments, New Castle, DE, USA) under nitrogen flow (20 mL/min), using a heating rate of 2 ◦C/min, a temperature modulation amplitude of 0.318 ◦C every 60 s, and an empty pan as a reference. In such experiments the linear heating rate is superimposed by a sinusoidal temperature variation and it is, thus, possible to separate the total signal (corresponding to that of a conventional DSC) into two different components, corresponding to the reversible and the irreversible heat flows. The TMDSC profiles were obtained only on heating. Heat and temperature calibrations were performed by using indium as a standard. The enthalpic content (Δ*H*) of each transition was calculated from the area under each peak, while the transition temperature was taken at the center of each transition. For each experiment ~2 mg of lyophilized collagen, weighted with an accuracy of ± 0.01 mg, was hydrated with distilled-deionized water at a collagen/water ratio of 1:20 (*w*/*w*) and placed in sealed aluminum pans. The samples were then kept at 4 ◦C for 48 h before analysis.

#### *4.10. Circular Dichroism Spectroscopy*

The molecular conformation and denaturation temperature (Td) of ICCs, dissolved in distilled-deionized water to a concentration of 0.1 mg/mL, were assessed by CD spectroscopy using a Jasco J-715 circular dichroism spectropolarimeter equipped with a Peltier-type temperature control system (Jasco PTC-348Wi). CD spectra were recorded at 20 ◦C using a 0.1-cm path length quartz cell at 190–250 nm with a step size of 0.5 nm and a band width of 1.0 nm. Experiments were run in triplicate, and 10 scans for each spectrum were signal-averaged.

To determine the Td, the rotatory angle at a fixed wavelength of 221 nm, [*θ*]221, was recorded with heating from 15 to 50 ◦C at a rate of 1 ◦C/min. The collagen concentration was adjusted to 0.1 mg/mL and the temperature was controlled. The Td was determined as the midpoint temperature between native-folded and completely unfolded forms. The mean molecular ellipticity (*θ*) was calculated using the equation [*θ*] = 10−<sup>3</sup> *θ* M/LC (expressed in deg cm2 dmol<sup>−</sup>1), where *θ* is the measured ellipticity in degrees, L is the path length in mm, C is the concentration in mg/mL, and M is the average residue molecular weight of collagen equal to 91.2 [74].
