*2.3. FTIR Analysis*

The FTIR spectra of ASC and PSC are exhibited in Figure 1. Each peak in the FTIR spectrums corresponds to the vibration of functional groups in the molecule [55]. The secondary structure of collagen is closely related to different types of hydrogen bonds [56]. By analyzing the FTIR spectrum of ASC and PSC, the different effects of the two extraction methods on the secondary structure of collagen were obtained. At room temperature, ASC and PSC mainly exhibited five absorption peaks at 3323 cm−1, 2931 cm−1, 1677 cm−1, 1546 cm−1, and 1242 cm−1, corresponding to amide A, amide B, amide I, amide II, and amide III, respectively.

The wavenumber of the free N–H stretching vibration was located in the range of 3400–3440 cm<sup>−</sup>1, and the wavenumber of amide A was measured at 3323 cm−1, indicating that the N–H stretching vibration and the hydrogen bonding are combined [51]. The amide A absorption peak of PSC at 3323.20 cm−<sup>1</sup> is slightly higher than ASC at the wavenumber of 3327.06 cm−1, indicating that more N–H groups in the ASC are hydrogen-bonded, which suggested that the PSC is slightly weaker than

ASC in structural stability. Both ASC and PSC have a weak absorption peak at the amide B band at 2931.67 cm−1, indicating the asymmetric stretching vibration of –CH2 [57]. Studies have shown that the amide I, amide II, and amide III bands are related to the triple-helix structure of collagen [58]. The amide I bands were attributed to C=O stretching vibration, and the amide I absorption bands of ASC and PSC appeared at 1677.99 cm−<sup>1</sup> and 1654.78 cm−1, respectively. The red shift of C=O stretching vibration may be caused by the use of the pepsin-degraded part of the telopeptides during the preparation process. The telopeptides plays an important role in the triple-helix structure of collagen, which was attributed to the covalent aldol cross-linking and the collagen fiber formation. Excision of the telopeptides does not completely destroy the natural collagen structure [59], but it leads to an incomplete collagen protein structure and increased solubility [60], which explained why the PSC yields are higher than those of the ASC. From the consideration of biomedical materials applications, much attention should be paid to the effects of the telopeptides on the immunogenicity of collagen. It has been reported that the immunogenicity of collagen's telopeptides was considered the most important factor of the collagen-induced immune response [59]. Previous studies have also suggested that the peptides located at the center of the triple helix of pepsin-treated collagen (from skin of bovine) are the major antigenic sites that cause human immune responses [61].

**Figure 1.** Fourier transform infrared spectroscopy (FTIR) of ASC and PSC from Nile tilapia skin.

Amide II bands produced by N–H bending vibrations and C–N stretching vibrations are usually located in the range of 1550 to 1600 cm<sup>−</sup>1. Research has shown that the red shift of the amide II peak is related to the hydrogen bond increase of the N–H group [39]. The amide II absorption bands of ASC and PSC were detected at wavenumbers of 1546.84 cm−<sup>1</sup> and 1551.66 cm−1, respectively. The result indicates that there are more hydrogen bonds between the peptide chains in ASC than PSC.

The amide III bands represent the combination of the C–N stretching vibration and the N–H bending vibration [56]. The amide III absorption bands of ASC and PSC appeared at 1242.10 cm−<sup>1</sup> and 1240.17 cm−<sup>1</sup> respectively, which is consistent with previous studies [41].
