## 2 Results and Discussion
### Characterization of Gelatin from the Bones of Skipjack Tuna (STB-G)
#### 2.1.4 Fourier transform infrared (FTIR) Spectrum of STB-G  
As shown in Figure 2, STB-G and STB-C exhibited similar FTIR spectral profiles. The spectrum of STB-G had major peaks associated with amide bands I (1631.9 cm\({}^{-1}\)), II (1546.5 cm\({}^{-1}\)), III (1242.6 cm\({}^{-1}\)), A (3354.2 cm\({}^{-1}\)), and B (2932.8 cm\({}^{-1}\)), which were similar to those of gelatins from the skins of seabass [30], golden carp [2], and brownbanded bamboo shark and blacktip shark [31].  
Figure 1: SDS-PAGE patterns of STB-G and STB-C analyzed by 7.5% separating gel and 4% stacking gel. Lane 1. Type I collagen from bones of skipjack tuna (STB-C); Lane 2. Gelatin from the bones of skipjack tuna (STB-G); Lane 3. Protein marker.  
Amide I, II, and III bands are closely related to the triple helical structure of gelatin and collagen, resulting from C=O stretching, N-H bending, and C-H stretching, respectively [5]. Amide I bands occurring in the range of 1600-1700 cm\({}^{-1}\) are primarily associated with C=O stretching vibration along the polypeptide backbone or a hydrogen bond coupled with COO-, and a reduction of the molecular order will result in a peak shift to a lower wavenumber [2; 14]. [PERSON] et al. (2005) reported that the absorption wavenumber of the amide I band at 1634.7 cm\({}^{-1}\) represented gelatin with a distinctive coiled conformation [32]. The lower wavenumber of STB-G (1631.9 cm\({}^{-1}\)), compared to that of STB-C (1642.1 cm\({}^{-1}\)), indicated that C=O stretching vibration was reduced and partial leopeptides were degraded by heating during the preparation process [21]. The amide II band with wavenumbers ranging from 1500 to 1600 cm\({}^{-1}\) specifies the number of N-H groups involved in hydrogen bonding with the adjacent \(\alpha\)-chain; therefore, a lower wavenumber of the amide II band indicates both increased hydrogen bonding by N-H groups, and a higher structure order [5; 14]. The wavenumbers of STB-G and STB-C were found to be 1546.5 and 1541.6 cm\({}^{-1}\), respectively. The present data indicated that there was less hydrogen bonding in STB-G than that in STB-C. Amide III absorption, which is associated with the triple helix structure of collagen and is involved in C-N stretching and N-H in plane bending from amide linkages, is normally very weak in FTIR. It also arises from wagging vibrations of CH\({}_{2}\) groups from the glycine backbone and proline side-chains [26]. In this study, the amide III bands of STB-G and STB-C were located at wavenumbers of 1242.6 cm\({}^{-1}\) and 1240.2 cm\({}^{-1}\), respectively. The higher amplitude was suggested to be due to the conversion of the \(\alpha\)-helix structure to random coils upon heating. The changes were linked to the denaturation of collagen to gelatin with a loss in the triple helical structure [2].  
Normally, stretching vibration of free N-H groups occurs within the range of 3400-3440 cm\({}^{-1}\)[21]. The wavenumber shifts to a lower frequency when the N-H group of the peptide is involved in hydrogen bonding [14]. Figure 2 shows that the amide A wavenumbers of STB-C (3329.5 cm\({}^{-1}\)) and STB-G (3354.2 cm\({}^{-1}\)) indicate that some N-H groups in STB-G and STB-C contributed to the formation of hydrogen bonds, and the degree of hydrogen bonding in STB-C was more than that of STB-G. The amide B band is related to asymmetric stretch vibrations of -NH\({}_{3}^{+}\) and =C-H, and the shift of amide B to a higher wavenumber is associated with an increase in free NH-NH\({}_{3}^{+}\) groups from both lysine residues and the \(N\)-terminus [5; 14]. The wavenumbers of the amide B band of STB-C and STB-G were found at 2936.1 cm\({}^{-1}\) and 2932.8 cm\({}^{-1}\), respectively, indicating STB-C had fewer free -NH\({}_{3}^{+}\) groups than STB-G.