3.3. Natural Cross-Linking Degree Determination

The degree of natural R. pulmo collagen cross-linking was determined by ninhydrin assay which could measure the percentage of free amino groups remaining in the collagen before and after cross-linking [38]. In the ninhydrin assay, 1 mg/mL of collagen type I from rat tail (RTC; Corning, Turin, Italy), was used as control because of its ability to form a gel stable at 37 °C, pH 7.0. The same gelling protocol has been used for 1 mg/mL of jellyfish collagen (JFC). A 150 µL volume of each sample was mixed with 600 µL of ninhydrin solution and 30 µL TIN(II) chloride; the reaction was developed at 100 °C for 15 min in thermomixer at 20 g, then cooled in an ice-water bath. An equal volume (200 µL) of each solution was added into 1 mL 50% isopropanol and the optical absorbance at 570 nm (Abs 570) was measured by a spectrophotometer (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). The amount of free amino groups is related to the value of Abs 570; glycine at various known concentrations was used to create a standard curve (glycine concentration vs. absorbance). The degree of cross-linking of the sample was then calculated using the following equation:

where NH₂o is the free NH₂ concentration in non-cross-linked samples and NH₂c is the free NH₂ concentration in cross-linked samples.